Histologic Study of Acute Vocal Fold Wound Healing After Corticosteroid Injection in a Rabbit Model

Objectives: Injectable corticosteroids have been used in phonosurgery to prevent scarring of the vocal fold because of their effects of wound healing, and to ensure better voice quality. We histologically evaluated the effects of dexamethasone sodium phosphate infiltration on acute vocal fold wound healing in rabbits 3 and 7 days after surgically induced injury by quantification of the inflammatory reaction and collagen deposition. Methods: A standardized surgical incision was made in the vocal folds of 12 rabbits, and 0.1 mL dexamethasone sodium phosphate (4 mg/mL) was injected into the left vocal fold. The right vocal fold was not injected and served as the control. The larynges were collected 3 and 7 days after surgery. For histologic analysis, the vocal folds were stained with hematoxylin-eosin for quantification of the inflammatory response and with picrosirius red for qunatification of collagen depostion. Results: There was no quantitative difference in the inflammatory response between vocal folds injected with the corticosteroid and control vocal folds. However, the rate of collage deposition was significantly lower in the corticosteroid-treated group at 3 and 7 days after injury (p = 0.002). Conclusions: The present results suggest that dexamethasone reduces collagen depostion during acute vocal fold wound healing.

Metalloproteinase inhibition ameliorates hypertension and prevents vascular dysfunction and remodeling in renovascular hypertensive rats

Altered activity of matrix metalloproteinases (MMPs) is implicated in the vascular remodeling of hypertension. We examined whether increased MMP-2 expression/activity plays a role in the vascular remodeling and dysfunction found in the two-kidney, one-clip (2K-1C) hypertension. Sham operated or 2K-1C hypertension rats were treated with doxycycline 30 mg/(kg day) (or vehicle). Systolic blood pressure was monitored weekly. After 8 weeks of treatment, aortic rings were isolated to assess endothelium-dependent and independent relaxations. Quantitative morphometry of structural changes, collagen, and elastin contents in the aortic wall were studied in hematoxylin/eosin, Sirius Red, and Orceine stained aortic sections, respectively. Aortic MMP-2 levels were determined by gelatin zymography and aortic MMP-2 proteolytic activity was measured using DQ gelatin as the substrate after MMP-2 was captured by a specific antibody and immobilized on a microplate. Aortic MMP-2/tissue inhibitor of metalloprotemases (TIMP)-2 mRNA levels were determined by real time RT-PCR. Doxycycline attenuated 2K-1C hypertension (215 +/- 8 mmHg versus 167 +/- 13 mmHg in 2K-1C rats and 2K-1C + doxy rats, respectively; P < 0.01) and prevented the 35% reduction in endothelium-dependent vasorelaxation found in 2K-1C rats. Doxycycline prevented the increases in media thickness, and was associated with lower media/lumen and cross-sectional areas (all P<0.01). Doxycycline also prevented excessive collagen and elastin deposition in the vascular wall. Increased MMP-2 and Pro-MMP-2 levels and MMP-2 activity were found in the aortas of 2K-1C rats (all P<0.05). A 21-fold increase (P<0.001) in the ratio of MMP-2/TIMP-2 mRNA expression was found in the 2K-1C group, whereas this ratio remained unaltered in 2K-1C+doxy rats. Our results suggest that MMP-2 plays a role in 2K-1C hypertension and its structural and functional vascular changes, which were attenuated by doxycycline. (C) 2007 Elsevier Ireland Ltd. All rights reserved.

Proteomic analysis of low- to high-grade astrocytomas reveals an alteration of the expression level of raf kinase inhibitor protein and nucleophosmin

Proteomic approaches have been useful for the identification of aberrantly expressed proteins in complex diseases such as cancer. These proteins are not only potential disease biomarkers, but also targets for therapy. The aim of this study was to identify differentially expressed proteins in diffuse astrocytoma grade II, anaplastic astrocytoma grade III and glioblastoma multiforme grade IV in human tumor samples and in non-neoplastic brain tissue as control using 2-DE and MS. Tumor and control brain tissue dissection was guided by histological hematoxylin/eosin tissue sections to provide more than 90% of tumor cells and astrocytes. Six proteins were detected as up-regulated in higher grade astrocytomas and the most important finding was nucleophosmin (NPM) (p < 0.05), whereas four proteins were down-regulated, among them raf kinase inhibitor protein (RKIP) (p < 0.05). We report here for the first time the alteration of NPM and RKIP expression in brain cancer. Our focus on these proteins was due to the fact that they are involved in the PI3K/AKT/mTOR and RAS/RAF/MAPK pathways, known for their contribution to the development and progression of gliomas. The proteomic data for NPM and RKIP were confirmed by Western blot, quantitative real-time PCR and immunohistochemistry. Due to the participation of NPM and RKIP in uncontrolled proliferation and evasion of apoptosis, these proteins are likely targets for drug development.

Analysis of placenta vascularization in patients with uterine altered artery Doppler flow velocity exams

Background: One of the frequent questions in obstetric practice is to determine placental vascular changes that may account for abnormal Doppler flow velocity alterations in maternal uterine vessels from women and fetuses without pregnancy pathology. Methods: A retrospective morphometric study was realized using 27 placentas from patients submitted for Doppler flow velocity exam during pregnancy. The placentas were morphologically examined using hematoxylin-eosin staining. Measurements of villi were made with the use of a video camera coupled to a common light microscope and a computer with automatic image analyzing software. Results: Of the 27 placentas, 13 (48%) were of patients showing unaltered Doppler and 14 (52%) showing altered Doppler. The number of stem villi vessels was significantly larger in the placentas of patients with Doppler exam alterations (P = 0.003). This group also presented greater stem villi vessel thickness, although without significant difference. The number of intermediary and terminal villi vessels was greater in the placentas of patients with altered Doppler exams (P < 0.001), and a greater terminal villi area was observed in these cases (P < 0.001). Conclusion: The morphological proof that uterine artery Doppler flow velocity exam alterations are associated with placental vascular alterations demonstrates the importance of this exam during prenatal care, even in the absence of maternal-fetal alterations.

Neuroprotective effects of oral lamotrigine administration on rabbit retinas after pars plana vitrectomy and silicone oil injection

Purpose: To investigate potential retinal neuroprotective effects of oral lamotrigine in rabbits after pars plana vitrectomy (PPV) and intravitreal silicone oil injection (SOI). Methods: Twelve New Zealand rabbits (weight, 2.0-2.5 kg) underwent PPV with SOI on the right eye. For 30 days postoperatively, 6 rabbits received a daily oral dose of lamotrigine (25 mg/kg), and 6 rabbits received a daily oral dose of water. The animals were killed 30 days after surgery. All retinas were processed histologically, immunostained using glial fibrillary acidic protein (GFAP), and analyzed by fluorescence microscopy. Retina sections from all groups were analyzed by TUNEL for the presence of apoptosis and stained with hematoxylin-eosin for morphologic analysis and retina cell density measurements in each layer using a Zeiss Axiophot microscope and KS 400 software. Results: Retinas from water-operated eyes showed a significant decrease in cell density associated with cell death compared with retinas from water-control eyes; cell density was reduced by 56% in the outer nuclear layer (ONL), 49% in the inner nuclear layer (INL), and 64% in the ganglion cell layer (GCL). Lamotrigine-operated retinas showed a reduction in cell death when compared with water-operated retinas; cell death was reduced by 52% in the ONL, 25% in the INL, and 56% in the GCL. Water-operated retinas showed TUNEL-positive cells and GFAP immunofluorescence throughout Muller cell processes; lamotrigine-operated retinas showed no TUNEL-positive cells and decreased GFAP staining when compared with water-operated retinas. Conclusions: PPV with SOI was associated with apoptosis of retinal cells and activation of glial cells in rabbit eyes. Oral lamotrigine administration provided protection against these effects.

Biocompatibility evaluation of alendronate paste in rat`s subcutaneous tissue

Alendronate is a known inhibitor of root resorption and the development of alendronate paste would enhance its utilization as intracanal medication. Therefore, this study aimed to investigate the biocompatibility of experimental alendronate paste in subcutaneous tissue of rats, for utilization in teeth susceptible to root resorption. The study was conducted on 15 male rats, weighing similar to 180-200 grams. The rats` dorsal regions were submitted to one incision on the median region and, laterally to the incision, the subcutaneous tissue was raised and gently dissected for introduction of two tubes, in each rat. The tubes were sealed at one end with gutta-percha and taken as control. The tubes were filled with experimental alendronate paste. The animals were killed at 7, 15 and 45 days after surgery and the specimens were processed in laboratory. The histological sections were stained with hematoxylin-eosin and analyzed by light microscopy. Scores were assigned to the in. ammatory process and statistically compared by the Tukey test (P < 0.05). Alendronate paste promoted severe inflammation process at 7 days, with statistically significant difference compared to the control (P < 0.05%). However, at 15 days, there was a regression of in. ammation and the presence of connective tissue with collagen fibers, fibroblasts and blood vessels was observed. After 45 days, it was observed the presence of well-organized connective tissue, with collagen fibers and fibroblasts, and few in. ammatory cells. No statistical difference was observed between the control and experimental paste at 15 and 45 days. The experimental alendronate paste was considered biocompatible with subcutaneous tissue of rat.

Comparative study on antioxidant effects and vascular matrix metalloproteinase-2 downregulation by dihydropyridines in renovascular hypertension

The vascular remodeling associated with hypertension involves oxidative stress and enhanced matrix metalloproteinases (MMPs) expression/activity, especially MMP-2. While previous work showed that lercanidipine, a third-generation dihydropyridine calcium channel blocker (CCB), attenuated the oxidative stress and increased MMP-2 expression/activity in two-kidney, one-clip (2K1C) hypertension, no previous study has examined whether first- or second-generation dihydropyridines produce similar effects. We compared the effects of nifedipine, nimodipine, and amlodipine on 2K1C hypertension-induced changes in systolic blood pressure (SBP), vascular remodeling, oxidative stress, and MMPs levels/activity. Sham-operated and 2K1C rats were treated with water, nifedipine 10 mg/kg/day, nimodipine 15 mg/kg/day, or amlodipine 10 mg/kg/day by gavage, starting 3 weeks after hypertension was induced. SBP was monitored weekly. After 6 weeks of treatment, quantitative morphometry of structural changes in the aortic wall was studied in hematoxylin/eosin-stained sections. Aortic and systemic reactive oxygen species levels were measured by using dihydroethidine and thiobarbituric acid-reactive substances (TBARs), respectively. Aortic MMP-2 levels and activity were determined by gelatin zymography, in situ zymography, and immunofluorescence. Nifedipine, nimodipine, or amlodipine attenuated the increases in SBP in hypertensive rats by approximately 17% (P<0.05) and prevented vascular hypertrophy (P<0.05). These CCBs blunted 2K1C-induced increases in vascular oxidative stress and plasma TBARs concentrations (P<0.05). All dihydropyridines attenuated the increases in aortic MMP-2 levels and activity associated with 2K1C hypertension. These findings suggest lack of superiority of one particular dihydropyridine, at least with respect to antioxidant effects, MMPs downregulation, and inhibition of vascular remodeling in hypertension.

GreenFix®: avaliação da morfologia em tecido mamário

Na última década têm desenvolvido fixadores para substituição do formol que é tóxico para o homem. O principal objectivo foi avaliar microscopicamente a histomorfologia e as características tintoriais de tecido mamário fixado em GreenFix®, durante 24 e 72 horas, comparativamente ao fixado em formol, através da coloração de Hematoxilina-Eosina. Uma análise global da histomorfologia revelou existir uma diferença estatisticamente significativa entre a fixação pelo GreenFix® e pelo formol (p=0,050), tendo-se registado uma melhoria da coloração e detalhe nuclear nos tecidos fixados com GreenFix® durante 24 (p=0,007) ou 72 horas(p=0,024). O GreenFix® é um potencial substituto do formol na rotina histológica.

Subcutaneous hyalohyphomycosis caused by Acremonium recifei: case report

We present a case of subcutaneous hyalohyphomycosis due to Acremonium recifei, a species whose habitat is probably the soil, first identified in 1934 by Arêa Leão and Lobo in a case of podal eumycetoma with white-yellowish grains and initially named Cephalosporium recifei. A white immunocompetent female patient from the state of Bahia, Brazil, with a history of traumatic injury to the right hand is reported. The lesion was painless, with edema, inflammation and the presence of fistulae. Seropurulent secretion with the absence of grains was present. Histopathological examination of material stained with hematoxylin-eosin showed hyaline septate hyphae. A culture was positive for Acremonium recifei. Treatment with itraconazole, 200 mg/day, for two months led to a favorable course and cure of the process. We report for the first time in the literature a case of subcutaneous hyalohyphomycosis due to Acremonium recifei in a immunocompetent woman. Treatment with itraconazole 200 mg/day, for two months, resulted in cure.

Intestinal spirochetosis: first cases reported in Brazil and the use of immunohistochemistry as an aid in histopathological diagnosis

Colonization of the colon and rectum by intestinal spirochetes is detected for the first time in Brazil in 4 of 282 (1.41%) patients who had undergone sigmoidoscopy and/or colonoscopy with a histopathological diagnosis of chronic non specific-colitis. This frequency is probably understimated, since surgically obtained specimens were not considered in the present study. Histopathological diagnosis was performed using routine stains like hematoxylin-eosin which showed the typical, of 3-µm thick hematoxyphilic fringe on the brush border of the surface epithelium, and by silver stains like the Warthin-Starry stain. Immunohistochemical procedures using two, polyclonal, primary antibodies, one against Treponema pallidum and the other against Leptospira interrogans serovar copenhageni serogroup Icterohaemorrhagiae cross-reacted with spirochetal antigen/s producing a marked contrast of the fringe over the colonic epithelium, preserving the spiral-shaped morphology of the parasite. In one case with marked diarrhea, immunohistochemistry detected spirochetal antigen/s within a cell in an intestinal crypt, thus demonstrating that the infection can be more widely disseminated than suspected using routine stains. Immunohistochemical procedures, thus, greatly facilitate the histological diagnosis of intestinal spirochetosis and may contribute to a better understanding of the pathogenesis of the disease. Transmission and scanning electron microscopy performed in one case showed that the spirochete closely resembled the species designated as Brachyspira aalborgi.

An immunofluorescence test for diagnosis of ophthalmic herpes in a mouse corneal model

Herpes simplex virus type 1 (HSV-1) ophthalmic disease is the most common cause of corneal blindness in humans world-wide. Current culture techniques for HSV take several days and commercially available HSV laboratory based diagnostic techniques vary in sensitivity. Our study was conducted to evaluate the use of a quicker and simpler method to herpes ophthalmic diagnosis. Corneal smears were made by firm imprints of infected mouse eyes to glass slides, after smears were fixated with cold acetone, and an indirect immunofluorescence (IIF) method was performed using monoclonal antibodies in a murine model of ophthalmic herpes. Eye swabs from infected mice were inoculated in Vero cells for virus isolation. Cytology and histology of the eye were also performed, using hematoxylin-eosin routine. Mouse eyes were examined by slit-lamp biomicroscopy for evidence of herpetic disease at various times postinoculation. We made a comparative evaluation of sensitivity, specificity and speed of methods for laboratory detection of HSV. Our results indicate that this IIF method is quick, sensitive, specific and can be useful in the diagnosis of ophthalmic herpes as demonstrated in an animal model.

Immunoperoxidase technique using an anti-Leishmania (L.) chagasi hyperimmune serum in the diagnosis of culture-confirmed American tegumentary leishmaniasis

The present study reports the production of the rabbit anti-Leishmania (L.) chagasi hyperimmune serum, the standardization of the immunohistochemistry (IHC) technique and the evaluation of its employment in cutaneous leishmaniasis (CL) lesions diagnosed by Leishmania sp. culture isolation. Thirty fragments of active CL lesions were examined as well as 10 fragments of cutaneous mycosis lesions as control group. IHC proved more sensitive in detecting amastigotes than conventional hematoxylin-eosin (HE) stained slides: the former was positive in 24 (80%) biopsies whereas the latter, in 16 (53%) (p = 0.028). The reaction stained different fungus species causing cutaneous mycosis. Besides, positive reaction was noticed in mononuclear and endothelial cells. Nevertheless, this finding was present in the control group biopsies. It is concluded that IHC showed good sensitivity in detecting amastigotes.

Morphological changes in the digestive system of 93 human immunodeficiency virus positive patients: an autopsy study

Involvement of the digestive system in patients with acquired immunodeficiency syndrome (AIDS) is frequent and many changes in these patients are diagnosed only at autopsy. There are few studies of autopsy with detailed analysis of this system and only one was conducted in Brazil. We evaluated each segment of the digestive system in 93 consecutive autopsies of patients infected with human immunodeficiency virus (HIV) and the importance of these lesions to death. Of these, 90 (96.8%) patients had AIDS. We reviewed medical records, autopsy reports and histological sections from tongue to rectum stained with hematoxylin-eosin. When necessary, we analyzed special stains and immunohistochemistry to investigate infections. There was damage to the digestive system in 73 (78.5%) cases. The most common infections were candidiasis (42%), cytomegalovirus (29%), histoplasmosis (11.8%), toxoplasmosis (9.7%) and mycobacterial infection (9.7%). Malignancies were rare, present in four (4.3%) cases (two Kaposi's sarcoma, one adenocarcinoma and one metastatic embryonal carcinoma). All segments showed lesions: tongue (48.6%), esophagus (44.8%), stomach (44.7%), colon (43.2%) and small intestine (28.9%). The lesions found were immediate cause of death in five (5.4%) cases. In another 36 (38.7%) cases the basic disease was systemic and also compromised the digestive system.

BLOOD VESSELS IN GANGLIA IN HUMAN ESOPHAGUS MIGHT EXPLAIN THE HIGHER FREQUENCY OF MEGAESOPHAGUS COMPARED WITH MEGACOLON

This study aimed to determine the existence of blood vessels within ganglia of the myenteric plexus of the human esophagus and colon. At necropsy, 15 stillborns, newborns and children up to two years of age, with no gastrointestinal disorders, were examined. Rings of the esophagus and colon were analyzed and then fixed in formalin and processed for paraffin. Histological sections were stained by hematoxylin-eosin, Giemsa and immunohistochemistry for the characterization of endothelial cells, using antibodies for anti-factor VIII and CD31. Blood vessels were identified within the ganglia of the myenteric plexus of the esophagus, and no blood vessels were found in any ganglia of the colon. It was concluded that the ganglia of the myenteric plexus of the esophagus are vascularized, while the ganglia of the colon are avascular. Vascularization within the esophageal ganglia could facilitate the entrance of infectious agents, as well as the development of inflammatory responses (ganglionitis) and denervation, as found in Chagas disease and idiopathic achalasia. This could explain the higher frequency of megaesophagus compared with megacolon.

Role of Notch signaling on the differentiation of early lymphoid progenitors cells: a view throughout the development of the embryonic chicken thymus and spleen

Dissertação para a obtenção do grau de Mestre em Genética Molecular e Biomedicina