Picrosirius Red F3BA Research Articles

The effects of valproic acid on skin healing: experimental study in rats.

ABSTRACTPurpose:To recognize the effects of valproic acid (VPA), an epigenetic drug, on the skin healing process.Methods:Sixty male Wistar rats were divided into two groups: the experiment treated with VPA (100 mg/kg/day); and the control, with 0.9% sodium chloride by gavage. Skin healing was studied in three moments (the third, the seventh, and the 14th day), evaluating the parameters: inflammatory reaction and its intensity (anti-LCA), angiogenesis (anti-CD34), collagen I and III (anti-collagen I, anti-collagen III and Picrosirius-red F3BA) and myofibroblasts (anti-alpha-AMS).Results:The inflammatory reaction was acute or sub-acute in both groups on the third day. On the seventh and the 14th day, chronic predominated in the control (p=0.006), and sub-acute in the experiment (p=0.020). There was a greater number of leukocytes in the group treated only on the third day (p=0.036). The number of vessels was lower in the treated group at the three times (p3=0.002, p7<0.001, and p14=0.027). Myofibroblasts were rare in the third day and moderate quantity in the remaining periods. Collagen I density was higher in the control at the three times (p<0.001) and collagen III in the treated group (p<0.001).Conclusions:VPA led to a more intense inflammatory reaction, decreased angiogenesis and collagen deposition, especially type I collagen.

IS THERE NEURAL AND FUNCTIONAL RECOVERY AFTER CLIP REMOVAL IN CERVICAL EXPERIMENTAL SYMPATHECTOMY?

ABSTRACT Background:The surgical treatment of hyperhidrosis by thoracic sympathectomy has brought, in addition to symptomatic relief for many, its main adverse effect: compensatory or reflex sweating. The clipping technique in place of the sympathetic nerve section gave rise to the hope of reversibility, but the positive results showed to be quite divergent, evidencing the academic deficiency regarding the study of this phenomenon. Aim:To observe micro and macroscopic damage caused by the polymer clip on sympathetic nerve of rabbits seven days after their clipping and the findings after three weeks of clip removal. Method:In this experimental study, 20 rabbits were divided into two groups of 10, group 1 (clipping) and group 2 (de-clipping). The right cervical sympathetic nerve of all animals was clamped with polymeric clip, and in group 2 the nerve was unclipped seven days later. Group 1 rabbits were induced to death on the 7th postoperative day, and group 2 on the 21st after removal of the polymer clip. Macroscopic variables were: clip appearance, presence of discontinuity lesion, infection and adhesions around the nerve. H&E was used in the evaluation of the phases and degree of the inflammatory process and presence of necrosis, and picrosirius red F3BA for quantification of collagen. Results:The cervical sympathetic nerve was intact, without necrosis or infection in all animals of the experiment; there were adhesions in both groups, being minimal in eight animals of each group and moderate or intense in two; the clip was completely closed in all animals at the 7th postoperative day; the inflammatory process shown was chronic, with monomorphonuclear predominance. There was no significant difference between groups regarding the intensity the inflammatory process, but the amount of collagen type I and type III was significantly higher in group 2. Conclusions:The injury caused by the polymer clip on the sympathetic nerve may be reversible, allowing functional return in the areas involved in the simulated cervical sympathectomy. Clipping of the cervical sympathetic nerve using a polymer clip does not cause discontinuity injury.

Devising tissue ingrowth metrics: a contribution to the computational characterization of engineered soft tissue healing

The paradigm shift brought about by the expansion of tissue engineering and regenerative medicine away from the use of biomaterials, currently questions the value of histopathologic methods in the evaluation of biological changes. To date, the available tools of evaluation are not fully consistent and satisfactory for these advanced therapies. We have developed a new, simple and inexpensive quantitative digital approach that provides key metrics for structural and compositional characterization of the regenerated tissues. For example, metrics provide the tissue ingrowth rate (TIR) which integrates two separate indicators; the cell ingrowth rate (CIR) and the total collagen content (TCC) as featured in the equation, TIR% = CIR% + TCC%. Moreover a subset of quantitative indicators describing the directional organization of the collagen (relating structure and mechanical function of tissues), the ratio of collagen I to collagen III (remodeling quality) and the optical anisotropy property of the collagen (maturity indicator) was automatically assessed as well. Using an image analyzer, all metrics were extracted from only two serial sections stained with either Feulgen & Rossenbeck (cell specific) or Picrosirius Red F3BA (collagen specific). To validate this new procedure, three-dimensional (3D) scaffolds were intraperitoneally implanted in healthy and in diabetic rats. It was hypothesized that quantitatively, the healing tissue would be significantly delayed and of poor quality in diabetic rats in comparison to healthy rats. In addition, a chemically modified 3D scaffold was similarly implanted in a third group of healthy rats with the assumption that modulation of the ingrown tissue would be quantitatively present in comparison to the 3D scaffold-healthy group. After 21 days of implantation, both hypotheses were verified by use of this novel computerized approach. When the two methods were run in parallel, the quantitative results revealed fine details and differences not detected by the semi-quantitative assessment, demonstrating the importance of quantitative analysis in the performance evaluation of soft tissue healing. This automated and supervised method reduced operator dependency and proved to be simple, sensitive, cost-effective and time-effective. It supports objective therapeutic comparisons and helps to elucidate regeneration and the dynamics of a functional tissue.

Genetically Programmed Biases in Th1 and Th2 Immune Responses Modulate Atherogenesis

Atherosclerotic lesions contain T lymphocytes, which orchestrate adaptive immunity and regulate many innate immune pathways. This study examined the influence of Th1 and Th2 helper cell subsets on atherogenesis in two ApoE(-/-) mouse strains, C57BL/6 and BALB/c, which display opposite T-cell subset polarizations. ApoE(-/-) BL/6 mice showed predominant Th1-like immune responses on polyclonal stimulation of splenic CD4(+) T cells and had IgG2a antibodies to oxidized low-density lipoprotein (a disease-related antigen) whereas ApoE(-/-) BALB/c mice displayed predominant Th2 responses by CD4(+) T cells and an IgG1 isotype response toward oxidized low-density lipoprotein. ApoE(-/-) BL/6 and BALB/c mice consumed a high-cholesterol diet for 10, 16, and 24 weeks with equivalent cholesterolemic responses. The Th1-slanted BL/6 mice developed significantly more atherosclerosis in the aortic root and abdominal aorta at all time points compared with BALB/c mice, supporting a proatherogenic role for Th1 response. Progression of atherosclerosis was associated with increased levels of interleukin (IL)-6 in mouse serum and CD4(+) T-cell culture supernatants and increased levels of the acute-phase protein, serum amyloid A (SAA). Both IL-6 and SAA levels rose significantly in ApoE(-/-) BL/6 mice compared with BALB/c mice. The circulating cytokine milieu (IL-6) and acute phase reactants such as SAA may reflect alterations in the Th1/Th2 balance. The results presented here illustrate how genetically determined modifiers of both immune and inflammatory responses can modulate atherogenesis independently of lipid levels.

Effect of Pantoprazole Administered Subcutaneously on the Healing of Sutured Gastric Incisions in Rats

Background: The properties of proton pump inhibitors most investigated are related to peptic diseases and upper gastrointestinal bleeding, but their influence on the healing of sutured gastric incisions has not been assessed. In the present study we evaluated the effect of subcutaneously administered pantoprazole on the healing of sutured gastric incisions in rats. Methods: Sixty rats were divided into a pantoprazole group and a control group. All rats were submitted to gastric suture in the gastric body and in the gastric fundus and had their gastric pH measured. The pantoprazole group received 20 mg/kg pantoprazole and the control group received 0.9% isotonic NaCl, both subcutaneously t.i.d. Healing analysis was carried out using collagen picrosirius red F3BA staining, and breaking strength was measured on the 4th and 7th postoperative days in all groups. Results: Gastric pH was higher in the pantoprazole group. In the fundus, the pantoprazole group had a higher measurement of breaking strength and a higher proportion of type-I over type-III collagen on the 7th postoperative day. In the body, the pantoprazole group had a higher proportion of type-I over type-III collagen on the 4th and 7th postoperative days. Conclusions: Pantoprazole given subcutaneously promoted a reduction in gastric acid secretion and was associated with improved healing of the sutured gastric incision in the fundus (squamous epithelium) of rats. These findings suggest that pantoprazole has healing properties in sutured gastric incisions with potential benefits in gastric surgery.

Síntese da parede abdominal: avaliação de dois tipos de sutura contínua em ratos

OBJETIVO: Comparar a sutura simples contínua e a sutura contínua em oito vertical no fechamento da parede abdominal de ratos. MÉTODOS: Quarenta e oito ratos machos Wistar, foram submetidos a laparotomia padronizada e fechamento da parede abdominal com sutura simples contínua (n=24) e sutura contínua em oito-vertical (n=24), com fio de polipropileno. No 7° e 14° pós-operatório foram submetidos a eutanásia 12 animais de cada grupo e deles retirados a camada músculo-aponevrótica da parede abdominal envolvendo a cicatriz operatória e preparados para exames histológico e imunohistoquímico. Os segmentos levados ao exame histológico foram corados por Hematoxilina-eosina sendo feita observação qualitativa do processo cicatricial e Picrosirius red F3BA, para avaliação quantitativa do colágeno. Também foram estudadas as porcentagens de macrófagos na linha de sutura por imunohistoquímica. Para a quantificação de macrófagos e fibras colágenas foi utilizado avaliação histológica por digitalização de imagem, baseados nos princípios de espectrofotometria. Os dados encontrados foram analisados estatisticamente pelos testes qui-quadrado, exato de Fisher e Mann-Whitney (p&lt; 0,05). RESULTADOS: A análise qualitativa, nos parâmetros necrose, fibrose, neovascularização, presença de abscesso, reação de corpo estranho e coaptação das bordas de sutura, não mostrou dados significantes nos dois grupos aos 7 ou 14 dias. A porcentagem de fibras colágenas foi significantemente maior, apenas no 7° dia, na sutura contínua em oito-vertical. A porcentagem de macrófagos mostrou-se significantemente maior na sutura simples contínua no 7º. dia. CONCLUSÃO : No 7° dia de pós-operatório a parede abdominal suturada em oito vertical apresenta significantemente, maior quantidade de fibras colágenas e menor quantidade de macrófagos do que a suturada por técnica contínua. Aos 14 dias de observação as suturas mostraram-se morfologicamente semelhantes.

Fluorescence spectroscopic imaging to detect changes in collagen and elastin following laser tissue welding.

A study was performed to evaluate the use of native fluorescence imaging to detect in situ molecular changes. There is no ideal noninvasive method to monitor molecular changes in a local region at a laser weld joint without removing a section of tissue for histological examination. Two sections of animal skin were welded together border to border using a Ti:sapphire laser beam (800 nm). Fluorescence imaging was performed on the cross section of the welded site at specific emission wavelengths (lambda c) for collagen at 380 nm and for elastin at 450 nm using excitation wavelengths (lambda e) of 340 nm, and 380 nm, respectively. A reduction of the collagen and elastin emission was observed in the fluorescence images of the welded region. These results were confirmed with histology using picrosirius red F3BA under polarized light and orcein stains. Optical spectroscopic imaging offers a new noninvasive detecting method for microscopic evaluation of laser tissue welding.

Laser irradiative tissue probed in situ by collagen 380-nm fluorescence imaging.

There is no ideal method to detect molecular changes in situ of laser-irradiated tissue without removing a section of tissue for histologic examination. A new method is reported to evaluate laser-induced thermal tissue damage in situ by using 380-nm native fluorescence imaging under 340-nm excitation. Native fluorescence imaging was performed on laser irradiated bovine tendon tissue and compared with light illuminated photography and histology with picrosirius red F3BA stain. The results indicate that a decrease in collagen fluorescence at 380 nm was observed in laser-induced thermally damaged tissues. The injured region of tissue defined by its fluorescence image coincided with an area defined by photography and histology.

Laser irradiative tissue probed in situ by collagen 380‐nm fluorescence imaging

Background and Objective There is no ideal method to detect molecular changes in situ of laser-irradiated tissue without removing a section of tissue for histologic examination. A new method is reported to evaluate laser-induced thermal tissue damage in situ by using 380-nm native fluorescence imaging under 340-nm excitation. Study Design/Materials and Methods Native fluorescence imaging was performed on laser irradiated bovine tendon tissue and compared with light illuminated photography and histology with picrosirius red F3BA stain. Results and Conclusion The results indicate that a decrease in collagen fluorescence at 380 nm was observed in laser-induced thermally damaged tissues. The injured region of tissue defined by its fluorescence image coincided with an area defined by photography and histology. Lasers Surg. Med. 27:158–164, 2000. © 2000 Wiley-Liss, Inc.

Biliary structures lead to tumour recurrences after laser-induced interstitial thermotherapy.

Thermal diffusion during laser-induced interstitial thermotherapy (LITT) has not yet been fully investigated in heterogeneous tissue architecture such as liver. LITT was performed on rabbit liver tumours to analyse the role of biliary structures in thermal diffusion. Twenty-four VX2 tumours were grafted onto 12 rabbit livers. The animals were randomly separated into two groups when tumour size reached 8 mm. Thermotherapy was performed by delivering the 830-nm output of a diode laser to the centre of the tumour with a 300-,microm fibre. Irradiation conditions were 1.5 W over 900 sec. On day 7 or 14, the tumours were removed and stained with haematoxylin-eosin and picrosirius red F3BA (PR). Thermal damage was evaluated by PR and electron microscopic examinations. Among the treated tumours, recurrences were found both at the periphery (one on day 7, seven on day 14) and within the treated area (two on day 7, two on day 14). All recurrences were located in the vicinity of the biliary structures, which are frequently spared from thermal injury. Biliary ducts lead to a heat sink, thereby facilitating tumour recurrences.

EFEITO DO DICLOFENACO DE SÓDIO NA CICATRIZAÇÃO DA PAREDE ABDOMINAL DE RATOS

O objetivo deste trabalho foi avaliar o efeito do diclofenaco de sódio na cicatrização da parede abdominal de ratos. Foram utilizados 40 (quarenta) ratos machos Wistar, submetidos a laparotomia com técnica padronizada, distribuidos em dois grupos, um grupo controle e outro onde se administrou o diclofenaco de sódio - 3 mg/kg/dia, via intramuscular durante 4 (quatro) dias consecutivos. Ao sétimo e ao décimo quarto dias de pós operatório, respectivamente, fora realizada a eutanásia, retirando-se a camada músculo-facial abdominal envolvendo a cicatriz operatória para a realização do estudo histológico. Os segmentos foram corados por Hematoxilina-Eosina e Picrosirius Red F3BA, sendo feita observação qualitativa do processo cicatricial e quantitativa do colágeno. Os resultados encontrados foram analisados estatisticamente. Concluiu-se que a cicatriz da parede abdominal de rato tratado com o diclofenaco de sódio apresenta menor quantidade de fibras colágenas no 7&lt;FONT FACE="Symbol"&gt;°&lt;/font&gt; e 14&lt;FONT FACE="Symbol"&gt;°&lt;/font&gt; dias de pós-operatório, quando comparado a animal do grupo controle.

Combined Microwave Stain for Plastic Embedded Sections of Nerve and Connective Tissue

A staining method for glycol methacrylate embedded tissue sections utilizing microwave radiation is reported. Following the Bodian silver stain, tissue sections were exposed to resorcin-fuchsin and picro-Sirius red F3BA techniques. Microwaving reduced staining time from 20 to less than 6 hours. Neurofibril and nerve fibers stained purple-black, elastic fibers were blue-black to black and collagenous connective tissue was red. Cytoplasm, in general, stained graygreen. (The J Histotechnol 15:113, 1992)

Combined Microwave Stain for Plastic Embedded Sections of Nerve and Connective Tissue

A staining method for glycol methacrylate embedded tissue sections utilizing microwave radiation is reported. Following the Bodian silver stain, tissue sections were exposed to resorcin-fuchsin and picro-Sirius red F3BA techniques. Microwaving reduced staining time from 20 to less than 6 hours. Neurofibril and nerve fibers stained purple-black, elastic fibers were blue-black to black and collagenous connective tissue was red. Cytoplasm, in general, stained graygreen. (The J Histotechnol 15:113, 1992)

The histological measurement of laser-induced thermal damage in vascular tissue using the stain picrosirius red F3BA

A histological technique for the measurement of laser-induced thermal tissue damage is described using the stain picrosirius red F3BA. This stain enhances the birefringence of normal collagen when viewed in polarized light. Areas of (thermally) denatured collagen, however, have no optical activity and can be measured directly by optical micrometry. The technique has been applied to experimental studies on both laser angioplasty and laser vascular anastomosis.

Are picro-dye reactions for collagens quantitative? Chemical and histochemical considerations.

Previous studies of picro-dye reactions demonstrated wide variations in the binding of different dyes. Picro-Sirius Red F3BA was recommended because it colors all collagens intensely and is suitable for polarization microscopy. Recent publications on quantitative uses of this stain were surprising. To obtain further information on the chemical mechanisms of dye binding by proteins, 94 sulfonated azo dyes were tested under the conditions of the picro-Sirius Red F3BA reaction. Reaction patterns varied widely, from failure to compete successfully with picrate ions for binding sites to strong coloration of all tissue structures. Only a few dyes stained collagen, reticulum fibers and basement membranes intensely and selectively. The reactivity of dyes was determined by their molecular configuration and the nature and position of substituents. Correlation with physico-chemical data showed that dye binding is due to non-ionic interactions, i.e. van der Waals and dispersion forces and hydrophobic bonding. Coulomb forces do not impart affinity - increasing sulfonation actually decreases dye uptake - but draw dyes within reach of non-ionic sites. Bound dyes form aggregates with additional dye ions; the aggregation number can range from 2 to many powers of 10. Clearly, dye binding by proteins is not stoichiometric.

Light microscopic distinction of collagens in hepatic cirrhosis.

Histochemical studies by Sesta et al. (1965) demonstrated in hepatic cirrhosis an embryonic-type collagen that differs from reticulum fibers. Grimaud and coworkers observed type IV collagen in cirrhotic lesions. To determine whether or not this collagen can be visualized by light microscopy, sections fixed in Carnoy-type solutions were treated with the periodic acid -Na bisulfite - resorcin-fuchsin (PBRF) reaction for basement membranes. Reticulum and coarse (Type I) collagen fibers were visualized with picro-Sirius Red F3BA. In normal livers, basement membranes occurred only around bile ducts and blood vessels. In hepatic cirrhosis basement membrane-like material extended from septa into nodules. The reaction patterns were similar to immunofluorescence pictures of Type IV collagen. The ratios of different collagens varied widely and were apparently determined by type and of lesions and other factors. For further studies, improved light microscopic reactions are needed, especially for simultaneous demonstration of embryonic and basement membrane-type collagens in contrasting colors.

Histochemical observations on Pneumocystis carinii: selective demonstration of honeycomb forms.

Histochemical investigations of pulmonary lesions indicated selective coloration of membranes of honeycomb stages of Pneumocystis carinii by the periodic acid--sodium bisulfite--resorcin-fuchsin reaction for basement membranes; mucus, fibrin and other deposits in respiratory pathways did not react. These membranes were colored selectively also by the picro-Sirius Red F3BA method for collagens; fungi in tissues from patients with candidiasis remained unstained. For simultaneous demonstration of honeycomb and cyst forms of Pneumocystis carinii, sections were prestained with Grocott's modification of Gomori's methenamine-silver nitrate technic and then treated with the periodic acid-Schiff (PAS) or picro-Sirius Red F3BA reaction. In contrast to other Gram-positive microorganisms, cysts of Pneumocystis carinii were immediately decolorized by acetone-ether mixtures; this indicates differences in the mode of dye binding. Frequently, only one stage of Pneumocystis carinii was found in a given area. Hence a combination of reactions showing different stages is recommended for studies of small tissue samples.

Selective Staining of Human Dermal Collagen: II. The Use of Picrosirius Red F3BA with Polarization Microscopy

Selective Staining of Human Dermal Collagen: II. The Use of Picrosirius Red F3BA with Polarization Microscopy