Processing methods for human amniotic membrane as scaffold for tissue engineering with mesenchymal stromal human cells.

Tissue engineering is an interdisciplinary field that applies the principles of engineering and life sciences toward the development of biological substitutes that restore, maintain, or improve tissue function. The aims of this work were to compare chemically and physically processed human Amniotic Membranes (hAM) and analyze the cytocompatibility and proliferation rate (PR) of two primary human mesenchymal stromal cell lines, from different sources and donor conditions seeded over these scaffolds. The evaluated hAM processes were: cold shock to obtain a frozen amniotic membrane (FEAM) with remaining dead epithelial cells, denudation of hAM with trypsin for 20/10 min (DEAM20/10) or treatment with sodium dodecyl sulfate to decellularized hAM (DAM). All samples were sterilized with gamma radiation. The selection of the treated hAM to then generate composites was performed by scanning and transmission electron microscopy and characterization by X-ray diffraction, selecting DEAM10 and FEAM as scaffolds for cell seeding. Two sources of primary human stromal cells were used, both developed by our researchers, human Dental Pulp Stem Cells (hDPSC) from living donors and human Mesenchymal Stromal Cells (hMSC) from bone marrow isolated from brain dead donors. This last line of cells conveys a novel source of human cells that, to our knowledge, have not been tested as part of this type of construct. We developed four in vitro constructs without cytotoxicity signs and with different PR depending on the scaffolds and cells. hDPSC and hMSC grew over both FEAM and DEAM10, but DEAM10 allowed higher PR.

Synthesis and characterization of a bovine collagen: GAG scaffold with Uruguayan raw material for tissue engineering.

Tissue engineering (TE) and regenerative medicine offer strategies to improve damaged tissues by using scaffolds and cells. The use of collagen-based biomaterials in the field of TE has been intensively growing over the past decades. Mesenchymal stromal cells (MSCs) and dental pulp stem cells (DPSCs) are promising cell candidates for development of clinical composites. In this study, we proposed the development of a bovine collagen type I: chondroitin-6-sulphate (CG) scaffold, obtained from Uruguayan raw material (certified as free bovine spongiform encephalopathy), with CG crosslinking enhancement using different gamma radiation doses. Structural, biomechanical and chemical characteristics of the scaffolds were assessed by Scanning Electron Microscopy, axial tensile tests, FT-IR and Raman Spectroscopy, respectively. Once we selected the most appropriate scaffold for future use as a TE product, we studied the behavior of MSCs and DPSCs cultured on the scaffold by cytotoxicity, proliferation and differentiation assays. Among the diverse porous scaffolds obtained, the one with the most adequate properties was the one exposed to 15 kGy of gamma radiation. This radiation dose contributed to the crosslinking of molecules, to the formation of new bonds and/or to the reorganization of the collagen fibers. The selected scaffold was non-cytotoxic for the tested cells and a suitable substrate for cell proliferation. Furthermore, the scaffold allowed MSCs differentiation to osteogenic, chondrogenic, and adipogenic lineages. Thus, this work shows a promising approach to the synthesis of a collagen-scaffold suitable for TE.

Analysis of the Quality of Clinical Trials Published in Spanish-Language Dermatology Journals Between 1997 and 2012.

INTRODUCTION: The value of randomized clinical trials (RCTs) undertaken to identify an association between an intervention and an outcome is determined by their quality and scientific rigor. OBJECTIVE: To assess the methodological quality of RCTs published in Spanish-language dermatology journals. METHODS: By way of a systematic manual search, we identified all the RCTs in journals published in Spain and Latin America between 1997 (the year in which the CONSORT statement was published) and 2012. Risk of bias was evaluated for each RCT by assessing the following domains: randomization sequence generation, allocation concealment, blinding of patients and those assessing outcomes, missing data, and patient follow-up. Source of funding and conflict of interest statements, if any, were recorded for each study. RESULTS: The search identified 70 RCTs published in 21 journals. Most of the RCTs had a high risk of bias, primarily because of gaps in the reporting of important methodological aspects. The source of funding was reported in only 15 studies. DISCUSSION AND CONCLUSIONS: In spite of the considerable number of Spanish and Latin American journals, few RCTs have been published in the 15 years analyzed. Most of the RCTs published had serious defects in that the authors omitted methodological information essential to any evaluation of the quality of the trial and failed to report sources of funding or possible conflicts of interest for the authors involved. Authors of experimental clinical research in dermatology published in Spain and Latin America need to substantially improve both the design of their trials and the reporting of results.

Identifying randomized clinical trials in Spanish-language dermatology journals.

INTRODUCTION: The necessary foundation for good clinical practice lies in knowledge derived from clinical research. Evidence from randomized clinical trials (RCTs) is the pillar on which decisions about therapy are based. OBJECTIVE: To search exhaustively and rigorously to identify RCTs in dermatology journals published in Spanish. METHODS: We located dermatology journals through the following search engines and indexes: PubMed, LILACS, SciELO, Periódica, Latindex, Índice Médico Español, C-17, IBECS, EMBASE, and IMBIOMED. We also sought information through dermatology associations and dermatologists in countries where Spanish was the usual language of publication, and we searched the Internet (Google). Afterwards we searched the journals electronically and manually to identify RCTs in all available volumes and issues, checking from the year publication started through 2012. RESULTS: Of 28 journals identified, we included 21 in the search. We found a total of 144 RCTs published since 1969; 78 (54%) were in Latin American journals and 66 (46%) were in Spanish journals. The most frequent disease contexts for RCTs in Spanish journals were psoriasis, mycoses, and acne vulgaris. In Latin American journals, the most frequent disease contexts were common warts, mycoses, acne vulgaris, and skin ulcers on the lower limbs. Manual searches identified more RCTs than electronic searches. CONCLUSIONS: Manual searches found a larger number of RCTs. Relatively fewer RCTs are published in Spanish and Latin American journals than in English-language journals. Internet facilitated access to full texts published by many journals; however, free open access to these texts is still unavailable and a large number of journal issues are still not posted online.

Functional interactions between dentate gyrus, striatum and anterior thalamic nuclei on spatial memory retrieval.

The standard model of memory system consolidation supports the temporal reorganization of brain circuits underlying long-term memory storage, including interactions between the dorsal hippocampus and extra-hippocampal structures. In addition, several brain regions have been suggested to be involved in the retrieval of spatial memory. In particular, several authors reported a possible role of the ventral portion of the hippocampus together with the thalamus or the striatum in the persistence of this type of memory. Accordingly, the present study aimed to evaluate the contribution of different cortical and subcortical brain regions, and neural networks involved in spatial memory retrieval. For this purpose, we used cytochrome c oxidase quantitative histochemistry as a reliable method to measure brain oxidative metabolism. Animals were trained in a hidden platform task and tested for memory retention immediately after the last training session; one week after completing the task, they were also tested in a memory retrieval probe. Results showed that retrieval of the previously learned task was associated with increased levels of oxidative metabolism in the prefrontal cortex, the dorsal and ventral striatum, the anterodorsal thalamic nucleus and the dentate gyrus of the dorsal and ventral hippocampus. The analysis of functional interactions between brain regions suggest that the dorsal and ventral dentate gyrus could be involved in spatial memory retrieval. In addition, the results highlight the key role of the extended hippocampal system, thalamus and striatum in this process. Our study agrees with previous ones reporting interactions between the dorsal hippocampus and the prefrontal cortex during spatial memory retrieval. Furthermore, novel activation patterns of brain networks involving the aforementioned regions were found. These functional brain networks could underlie spatial memory retrieval evaluated in the Morris water maze task.

Healthcare quality indicators of peripheral artery disease based on systematic reviews.

OBJECTIVES: Peripheral artery disease (PAD) is a major health problem whose clinical management includes multiple options regarding risk factor control, diagnosis, and medical and surgical treatment. The aim was to generate indicators based on systematic reviews to evaluate the quality of healthcare provided in PAD. METHODS: Electronic searches were run for systematic reviews in The Cochrane Library (Issue 6, 2011), MEDLINE, EMBASE, and other databases (up to June 2011). Conclusive systematic reviews of high methodological quality were selected to formulate clinical recommendations. Indicators were derived from clinical recommendations with moderate to very high strength of evidence as assessed by the GRADE system. RESULTS: From 1,804 reviews initially identified, 29 conclusive and high-quality systematic reviews were selected and nine clinical recommendations were formulated with a moderate to very high strength of recommendation. Six indicators were finally generated: four on pharmacological interventions, antiplatelet agents, naftidrofuryl, cilostazol, and statins; and two lifestyle interventions, exercise and tobacco cessation. No indicators were derived for diagnostic tests or surgical techniques. Most indicators targeted patients with intermittent claudication. CONCLUSIONS: These quality indicators will help clinicians to assess the appropriateness of healthcare provided in PAD. The development of evidence-based indicators in PAD is limited by the lack of methodological quality of the research in this disease, the inconclusiveness of the evidence on diagnostic and surgical techniques, and the dynamic nature of the vascular diseases field.

A role for dorsal and ventral hippocampus in response learning.

The hippocampus and the striatum have been traditionally considered as part of different and independent memory systems despite growing evidence supporting that both brain regions may even compete for behavioral control in particular learning tasks. In this regard, it has been reported that the hippocampus could be necessary for the use of idiothetic cues in several types of spatial learning tasks. Accordingly, the ventral striatum receives strong anatomical projections from the hippocampus, suggesting a participation of both regions in goal-directed behavior. Our work examined the role of the dorsal and ventral hippocampus on a response learning task. Cytochrome c oxidase (C.O.) quantitative histochemistry was used as an index of brain oxidative metabolism. In addition, determination of C.O. subunit I levels in the hippocampus by western blot analysis was performed to assess the contribution of this subunit to overall C.O. activity. Increased brain oxidative metabolism was found in most of the studied hippocampal subregions when experimental group was compared with a swim control group. However, no differences were found in the amount of C.O. subunit I expressed in the hippocampus by western blot analysis. Our results support that both the dorsal and ventral hippocampus are associated with the use of response strategies during response learning.

Different brain networks underlying the acquisition and expression of contextual fear conditioning: a metabolic mapping study.

The specific brain regions and circuits involved in the acquisition and expression of contextual fear conditioning are still a matter of debate. To address this issue, regional changes in brain metabolic capacity were mapped during the acquisition and expression of contextual fear conditioning using cytochrome oxidase (CO) quantitative histochemistry. In comparison with a group briefly exposed to a conditioning chamber, rats that received a series of randomly presented footshocks in the same conditioning chamber (fear acquisition group) showed increased CO activity in anxiety-related brain regions like the ventral periaqueductal gray, the ventral hippocampus, the lateral habenula, the mammillary bodies, and the laterodorsal thalamic nucleus. Another group received randomly presented footshocks, and it was re-exposed to the same conditioning chamber one week later (fear expression group). The conditioned group had significantly higher CO activity as compared with the matched control group in the following brain regions: the ventral periaqueductal gray, the central and lateral nuclei of the amygdala, and the bed nucleus of the stria terminalis. In addition, analysis of functional brain networks using interregional CO activity correlations revealed different patterns of functional connectivity between fear acquisition and fear expression groups. In particular, a network comprising the ventral hippocampus and amygdala nuclei was found in the fear acquisition group, whereas a closed reciprocal dorsal hippocampal network was detected in the fear expression group. These results suggest that contextual fear acquisition and expression differ as regards to the brain networks involved, although they share common brain regions involved in fear, anxiety, and defensive behavior.

Functional interaction between the dorsal hippocampus and the striatum in visual discrimination learning.

The hippocampus and the striatum have traditionally been considered as part of different and independent memory systems. However, there is evidence that supports a functional interaction between the hippocampus and the dorsal striatum at least in particular learning tasks. Here, we evaluated the functional contribution of both brain regions in a visual discrimination learning task using cytochrome c oxidase (CO) quantitative histochemistry. Compared with other brain metabolic mapping techniques, CO activity reflects steady-state neuronal energy demand. Rats were trained for 6 days in a water T-maze to find a hidden escape platform associated with an intramaze visual cue. A control group of animals swam for an equivalent amount of time compared as the trained group but without any escape platform available. After finishing the behavioral task, CO activity was measured in subdivisions of the dorsal hippocampus and the dorsal striatum in both groups. Results show significantly higher CO activity in the CA1 area and the dentate gyrus of the dorsal hippocampus in the trained rats compared with the control group. In addition, a significant negative functional cross-correlation between area CA1 of the dorsal hippocampus and the anterodorsal striatum was found. Our results support current theories on competitive interaction of different memory systems during visual discrimination learning.

Spatial learning of the water maze: progression of brain circuits mapped with cytochrome oxidase histochemistry.

The progression of brain circuits involved in spatial learning tasks is still a matter of debate. In addition, the participation of individual regions at different stages of spatial learning remains a controversial issue. In order to address these questions, we used quantitative cytochrome oxidase histochemistry as a metabolic brain mapping method applied to rats (Rattus norvegicus) trained in a water maze for 1, 3 or 5 days of training. Sustained changes throughout training were found in the lateral septal nucleus and anteroventral thalamic nucleus. As compared to naïve or habituation groups, rats with 1 day of training in the spatial learning task showed involvement of the lateral mammillary nucleus, basolateral amygdala and anterodorsal thalamic nucleus. By 5 days of training, there were mean changes in the hippocampal CA3 field and the prefrontal cortex. The regions involved and their pattern of network interactions changed progressively over days of training. At 1-day there was an open serial network of pairwise correlations. At 3-days there was a more closed reciprocal network of intercorrelations. At 5-days there were three separate parallel networks. In addition, brain-behavior correlations showed that CA1 and CA3 hippocampal fields together with the parietal cortex are related to the mastery of the spatial learning task. The present study extends previous findings on the progressive contribution of neural networks to spatial learning.

Temperature oscillations of magnetization observed in nanofluid ferromagnetic graphite.

We report on unusual magnetic properties observed for nanofluid room temperature ferromagnetic graphite (with an average particle size of [Formula: see text] nm). More precisely, the measured magnetization exhibits a low temperature anomaly (attributed to the manifestation of finite size effects below the quantum temperature [Formula: see text]) as well as pronounced temperature oscillations above T = 50 K (attributed to manifestation of the hard-sphere type of pair correlations between ferromagnetic particles in the nanofluid).

Changes in brain oxidative metabolism induced by water maze training.

Although the hippocampus has been shown to be essential for spatial memory, the contribution of associated brain regions is not well established. Wistar rats were trained to find a hidden escape platform in the water maze during eight days. Following training, the oxidative metabolism in different brain regions was evaluated using cytochrome oxidase histochemistry. Metabolic activations were found in the prelimbic cortex, cornu ammonis (CA) 1 subfield of the dorsal hippocampus and the anterior thalamic nuclei, relative to yoked swim controls and naïve rats. In addition, many cross-correlations in brain metabolism were observed among the latter regions. These results support the implication of a hippocampal-prefrontal-thalamic system to spatial memory in rats.

Fístulas postlaringectomía: variables que favorecen su desarrollo / Posttlaringectomy fistula: factors that favour its development

Antecedentes: A pesar del gran número de autores que se refieren a esta complicación, no hay aún hoy acuerdo sobre causas. Objetivo: Reconocer los factores que favorecen su desarrollo. Lugar de aplicación: Hospital público de atención terciaria de pacientes oncológicos. Diseño: Prospectivo no randomizado. Población: 100 enfermos, 93 por ciento masculinos, edad media 59,13 años, 98 por ciento portadores de carcinoma epidermoide sometidos a laringectomía total. Método: Análisis uni y multivariado de 21 variables potencialmente favorecedoras de fístulas postoperatorias. Resultados: Ninguna de las vriables resultó estadísticamente significativa. La radioterapia previa se comportó como el factor de mayor probabilidad del evento. La incidencia de fístulas alcanzó el 55 por ciento de los operados. Su momento de aparición tuvo una media de 9 días. El 76,3 por ciento cerró espontáneamente. Las peores consecuencias del evento resultaron la prolongación en el uso 3,6 veces mayor a la media de la sonda de alimentación, la necesidad de nuevos tiempos quirúrgicos en 13 pacientes y la rehabilitación 2 meses después que el resto. Conclusiones: La dimensión de la muestra resultó insuficiente para desarrollar el modelo predictivo del fenómeno

Evaluation of two experimental models of hepatic encephalopathy in rats.

The serious neuropsychological repercussions of hepatic encephalopathy have led to the creation of several experimental models in order to better understand the pathogenesis of the disease. In the present investigation, two possible causes of hepatic encephalopathy, cholestasis and portal hypertension, were chosen to study the behavioral impairments caused by the disease using an object recognition task. This working memory test is based on a paradigm of spontaneous delayed non-matching to sample and was performed 60 days after surgery. Male Wistar rats (225-250 g) were divided into three groups: two experimental groups, microsurgical cholestasis (N = 20) and extrahepatic portal hypertension (N = 20), and a control group (N = 20). A mild alteration of the recognition memory occurred in rats with cholestasis compared to control rats and portal hypertensive rats. The latter group showed the poorest performance on the basis of the behavioral indexes tested. In particular, only the control group spent significantly more time exploring novel objects compared to familiar ones (P < 0.001). In addition, the portal hypertension group spent the shortest time exploring both the novel and familiar objects (P < 0.001). These results suggest that the existence of portosystemic collateral circulation per se may be responsible for subclinical encephalopathy.

Evaluation of two experimental models of hepatic encephalopathy in rats

The serious neuropsychological repercussions of hepatic encephalopathy have led to the creation of several experimental models in order to better understand the pathogenesis of the disease. In the present investigation, two possible causes of hepatic encephalopathy, cholestasis and portal hypertension, were chosen to study the behavioral impairments caused by the disease using an object recognition task. This working memory test is based on a paradigm of spontaneous delayed non-matching to sample and was performed 60 days after surgery. Male Wistar rats (225-250 g) were divided into three groups: two experimental groups, microsurgical cholestasis (N = 20) and extrahepatic portal hypertension (N = 20), and a control group (N = 20). A mild alteration of the recognition memory occurred in rats with cholestasis compared to control rats and portal hypertensive rats. The latter group showed the poorest performance on the basis of the behavioral indexes tested. In particular, only the control group spent significantly more time exploring novel objects compared to familiar ones (P < 0.001). In addition, the portal hypertension group spent the shortest time exploring both the novel and familiar objects (P < 0.001). These results suggest that the existence of portosystemic collateral circulation per se may be responsible for subclinical encephalopathy.

Influence of gonadal steroids on the glial fibrillary acidic protein-immunoreactive astrocyte population in young rat hippocampus.

It is known that expression of glial fibrillary acidic protein (GFAP) as an astrocyte-specific marker can be regulated by levels of circulating gonadal steroids during postnatal development. In addition, astrocytes play an important role in the physiology of the hippocampus, a brain region considered sexually dimorphic at the neuronal level in rodents. To evaluate the contribution of glial cells to gender-related differences in the hippocampus, we estimated the number of GFAP-immunoreactive (GFAP-IR) astrocytes in the hippocampus (CA1 and CA3 areas, dorsal and ventral regions) of male and female rats aged 30 days. Groups of 30-day-old masculinized females (TP-females; injected with testosterone propionate at birth) and feminized males (FLU-males, castrated and treated with flutamide, an androgen receptor antagonist) were included to assess the effects of gonadal hormones on these hippocampal astrocytes. Using the optical fractionator method, the total number of GFAP-IR cells found in CA1 and CA3 areas was significantly higher in males compared to that in age-matched females. This numerical pattern was reversed in TP-females and FLU-males in both hippocampal areas. In addition, more GFAP-IR cells were found in dorsal hippocampus than in the ventral region in the CA1 area from all experimental groups, whereas this result was found in the CA3 area from males and TP-females. Our results suggest an essential contribution of gonadal hormones to gender differences found in the astrocyte population of the rat hippocampus during development.

Brain differences in newborn rats predisposed to helpless and depressive behavior.

Inborn brain differences in metabolic capacity were mapped in congenitally helpless rats, a genetically selected strain predisposed to show helpless and depressive behavior. There are a number of brain regions showing abnormal metabolism in adult congenitally helpless rats. Some of these alterations may be innate while others may be due to environmental factors, such as maternal care and postnatal stress. To identify which brain structures show innate differences, brains of newborn rats from congenitally helpless and non-helpless strains were compared using cytochrome oxidase histochemistry, an endogenous marker of regional metabolic capacity. A smaller subset of regions affected in adults showed significantly less metabolic activity in the newborn brains, including paraventricular hypothalamus, habenula, hippocampus, subiculum, lateral septal nucleus, anterior cingulate cortex, infralimbic cortex, and medial orbitofrontal cortex. A covariance analysis further revealed a striking reduction of functional connectivity in the congenitally helpless brain, including a complete decoupling of limbic forebrain regions from midbrain/diencephalic regions. This pattern of brain metabolism suggests that helplessness vulnerability is linked to altered functioning of limbic networks that are key to controlling the hypothalamic-pituitary-adrenal axis. This implies that vulnerable animals have innate deficits in brain systems that would normally allow them to cope with stress, predisposing them in this manner to more readily develop helpless and depressive behaviors.

Quantitative histochemical assessment of oxidative metabolism in the subfornical organ after partial aortic ligature in rats.

Circumventricular organs are considered to be involved in hydromineral homeostatic responses. In this study we used quantitative histochemistry of cytochrome oxidase to evaluate the oxidative metabolic activity of the subfornical organ of rats with a partial aortic occlusion. These rats showed a significant increase in water intake from the second day after the ligature, while natriophilia was already significant on the first day. Greater levels of cytochrome oxidase activity were found in subfornical organs of partial aortic ligated rats when compared with control, providing further evidence for the involvement of this circumventricular structure in fluid homeostasis at least in this hyperdipsic, hypernatriophilic, hyperreninemic and hypertensive experimental model.

Hippocampal AgNOR activity after chronic alcohol consumption and alcohol deprivation in rats.

Chronic alcohol consumption induces morphological changes in the central nervous system and withdrawal does not reverse these changes. It is well known that the hippocampal formation is one of the brain regions most sensitive to prolonged alcohol ingestion. The aim of our study was to evaluate the transcriptional neuronal activity by measuring the argyrophilic nucleolar organizer regions (AgNORs) in the dentate gyrus, CA3, and CA1 hippocampal areas from adult male rats receiving chronic administration of ethanol (ALC) and after withdrawal (WDL). The parameters evaluated were the number and area of AgNORs, together with the area of nucleus and the proportion between AgNOR and nuclear areas (ratio). The animals from ALC and WDL groups showed a reduction in the number of AgNOR per cell as compared to the control group. CA3 was the hippocampal area most affected by chronic alcohol intake. No improvement was observed in animals after withdrawal. Our data support the idea that the chronic intake of alcohol decreases protein synthesis in hippocampal neurons at an early age. This decrease may explain the memory impairment showed by rats receiving chronic treatment with alcohol because, both in humans and rats, it is associated with a reduction in the number of cholinergic neurons in the basal forebrain that would in turn affect the hippocampal function.

Total laryngectomy: pre- and intrasurgical variables of infection risk.

Postoperative infection has influence on costs, quality of life, and outcome of the disease. It is suspected that post-total laryngectomy infections have increased in frequency and seriousness, because of the failure of the preservation protocol or the previous radiotherapy, making rescue surgery necessary. The objective of this study was to develop a predictive model of infection based on the pre- and intrasurgical variables considered risky. One hundred fifty five patients with E III-IV laryngeal cancer, with 24.8:1 male to female ratio (mean age, 58 years) who underwent total laryngectomy were evaluated for uni- and multivariate analysis of age, sex, histological grade, primary or recurrent disease, tobacco, alcohol, diabetes, tuberculosis/chronic emphysema, red and white cell counts, erythrosedimentation rate (ESR), albumin, chemotherapy, neck radiotherapy and/or previous surgery, confinement days, type and time of surgery, which were factors in the infection event. A predictive model of infection was developed and included albuminemia (<3.5 g%), >1 liter of alcohol daily, and exclusive surgery of the primary. The sensitivity was 90.5% and the specificity 68%. The variance reached 29.6%. The causes of infection were multiple, having analyzed only 30% of them. However, the resulting model was classified correctly in 83.2% of cases. A careful preoperative assessment, an adjusted planning of the surgery, an appropriate use of antibiotics, and a meticulous operative technique are needed to prevent infection.