Weigert's Stain Research Articles

Basic Histological Techniques (MicroTechniques) for Staining of Animal Tissue

Background: Animal tissues when removed from the body must be well preserved in life-like manner as possible. Tissues must be further processed in graded alcohol and other essential chemicals to make them ready for studies under microscope.
 Aim/Objectives: To highlight the processes of handling animal tissues from the time they were removed from the body to the point they were processed, stained and made ready for microscopically diagnosis.
 Methodology: Standard and academic search engines were utilized and histological text consulted to review essential protocols for histological handling and processing of animal tissues and how they are stained. Basic staining techniques were included while advanced techniques were excluded.
 Results and Recommendation: Staining techniques such as Haematoxylin and Eosin, Weigert stain, Trichrome stain, Silver stain, Periodic acid Schiff and Wright stains were highlighted as with their staining appearance on animal tissues. Reaction of tissue components following application of dyes is given in Table 1.
 It is recommended that standard protocols be followed during staining of animal tissues for histological studies.

Characterization of a decellularized rat larynx: comparison between microscopy techniques

BackgroundNo effective method has yet been developed to efficiently reconstruct the larynx and restore its function. Decellularization has recently been tested for this purpose with very promising results. The goal of decellularization is to remove cells leaving an intact scaffold made of an extracellular matrix (ECM). Although the use of hematoxylin/eosin and Masson trichrome stains is widely accepted to highlight tissue structure, the methods based on evaluation of collagen and elastin are considered highly variable. The aim of this study was to develop a whole organ decellularization protocol and compare the qualitative and quantitative efficiency of some microscopy techniques for collagen and elastin detection in paraffin-embedded tissues. MethodsH&E, Masson Trichrome and DAPI staining as well as DNA quantification were used to evaluate decellularization efficiency. Van Gieson stain, Picrosirius Red stain (PRS) and multiphoton laser scanning microscopy (MPM) were carried out for collagen detection and quantitative assessment. Polarized PRS was used to investigate collagen network, and Weigert stain and MPM were used to detect and estimate elastin content. ResultsThe decellularization process removed the cellular components without affecting glycosaminoglycan, collagen and elastin content. Concerning collagen quantification, Van Gieson stain underestimated collagen content, while PRS, apparently less fading, did not reach reliable results when used as quantitative method. MPM effectively quantified collagen content. Collagen fibers were visualized much better under polarized light microscopy, allowing to underline that decellularization process affects the homogeneity of 3D collagen network. Concerning elastin detection, Weigert stain and MPM produced overlapping results. ConclusionsAn efficient protocol to decellularize the whole larynx was developed, allowing the removal of cells without affecting ECM integrity. The results supported the use of non-polarized PRS to highlight collagen, even the thin fibers, second harmonic generation for major fibrillar collagens and polarized PRS for 3D collagen network. Concerning elastin, Weigert stain and MPM showed similar results, thus the use of MPM, rather than that of the Weigert stain, may be suitable to avoid the additional time and costs of a histological staining.

Histological analysis of different types of port-wine stains to guide clinical decision making: A retrospective study.

Port-wine stains are defined as congenital benign vascular lesions. The treatment of port-wine stains remains a challenge, worldwide. This study aimed to analyze the histological characteristics in different types of port-wine stains and provide guidance for clinical decision-making. Biopsies were from the hospital from 2015 to 2021. H&E staining, Immunofluorescence staining, Masson's trichrome staining and Weigert staining were performed on the tissues. A total of 35 port-wine stains patients were included in the study of four distinct types, namely red port-wine stains (11 cases), purple port-wine stains (seven cases), hypertrophic port-wine stains (nine cases) and nodular port-wine stains (eight cases). The mean vessel diameter of the different types was 38.7 ± 5.9 μm, 93.5 ± 9.7 μm, 155.6 ± 21.8 μm and 155.6 ± 29.54 μm, respectively. Mean vessel depth was 396.4 ± 31 μm, 944.2 ± 105.4 μm, 2,971 ± 161.3 μm and 3,594 ± 364.6 μm, respectively. The vessels in red port-wine stains, purple port-wine stains and hypertrophic port-wine stains were mainly composed of capillary and venous malformations, whereas those in nodular port-wine stains were venous or arteriovenous malformations. The main limitation of the current study was the small number of patients. As the disease progresses, vessel diameters become larger, the vessel wall becomes thicker and vessels were found in a greater depth. A treatment plan should be scientifically formulated keeping in mind the histological characteristics of port-wine stains.

Acute Kidney Injury in a Patient with Trisomy 21.

1. Hannah S. Kim, MD* 2. Francesca Costigliolo, MD† 3. Serena Bagnasco, MD† 4. Jeffrey Fadrowski, MD, MHS* 5. Rebecca L. Ruebner, MD, MSCE* 1. *Division of Pediatric Nephrology and 2. †Division of Pathology, Johns Hopkins University, Baltimore, MD A 5-year-old former 34-week girl with trisomy 21, hypothyroidism, and obstructive sleep apnea presents to the emergency department with a chief complaint of fever. She initially presented to her pediatrician 3 weeks earlier with sore throat and was diagnosed as having group A streptococcal pharyngitis. She completed 10 days of amoxicillin with resolution of sore throat. Now for the past 3 days she has had fever, chest pain, decreased enteral intake, and emesis. Review of systems is negative for diarrhea, gross hematuria, dysuria, polyuria, polydipsia, edema, weight loss, rash, arthralgia, arthritis, oral ulcers, epistaxis, and hemoptysis. She has had normal growth. She takes levothyroxine and took several doses of ibuprofen for fever over the past several weeks. There is no family history of kidney disease. She had no recent travel or sick contacts. On physical examination her weight is 45.2 lb (20.5 kg) (65th percentile); height, 46.1 in (117 cm) (62% percentile); temperature, 100.8°F (38.2°C); heart rate, 110 beats/min; respiratory rate, 24 breaths/min; oxygen saturation, 100% on room air; and blood pressure, 92/42 mm Hg (systolic 31st and diastolic 9th percentiles for age and sex). There is no pharyngeal injection or exudate. She appears well-hydrated, including moist mucous membranes. Lungs are clear to auscultation. Her abdomen is soft, nontender, and nondistended. There is no edema, and she has brisk capillary refill. She has no rash or joint swelling, erythema, or warmth. Laboratory evaluation includes a white blood cell count of 15,310/µL (15.31×109/L) with 83.6% neutrophils and 0.8% bands; hemoglobin …

Employing optical microscopy technique to determine the tissue action of Hamamelis virginianaL. (Hamamelidaceae), Maytenus ilicifoliaMart. ex Reissek (Celastraceae) and Casearia sylvestris Sw. (Salicaceae) tinctures on skin-fixed histological sections of Wistar rats

Objective: In this study, we aimed to determine the action of the tinctures of Hamamelis virginiana, Maytenus ilicifolia, and Casearia sylvestris on tissues. For this, we investigated the histological sections of fixed skin tissue of Wistar rats for the changes in collagen and elastic fibers, epithelial cells, conjunctive cells, epidermal attachments, pigments, and granules using the optical microscopy technique. Since in the literature and published articles, the use of in vivo models, such as Wistar rats, is predominant to evaluate the healing action of herbal medicines. Methods: The tinctures of Hamamelis virginiana, Maytenus ilicifolia, and Casearia sylvestris, and ethanol 70% (blank) were used. The tinctures were obtained at 10% (w/v) through percolation using 70% ethanol (v/v) as the extraction liquid. This study was conducted in duplicate for each tincture and different incubation times (4, 24, and 48 h) at 37 °C in an oven. The slides used in this study (Wistar rat skin) were previously processed at the histology laboratory since the waste material was donated to conduct this experiment. The Research Ethics Committee approved the use of animals of the Life Sciences Center of the Pontifical Catholic University of Campinas (PUC-CAMPINAS) under the protocol approval number 367/08. Before initiating the staining process, the slides were removed from the oven, and the tincture deposited on the slides was removed. Weigert staining was performed subsequently. For semi-quantitative analysis, the histological sections were carefully observed, and the number of collagen and elastic fibers was evaluated based on the following scale: (+) normal fiber presence, (−) slight decrease, (− −) moderate decrease, and (− − −) intense decrease. The images were digitally captured to obtain the results using a photomicroscope. Results: The degradation of collagen fibers was best evidenced upon using Hamamelis virginiana tincture, which is concordant with the existing reports in the literature on its healing action via the precipitation of dermal proteins. Maytenus ilicifolia and Casearia sylvestris tinctures exhibited low proteolytic capacity as they only caused degradation of elastic fibers that are more delicate and very different from collagen in their constitution. Therefore, the application of the latter two as healing agents (which acts through precipitating proteins) is limited. Conclusions: This experimental histological model using the optical microscopy technique exhibits the advantage of agility and high efficiency as a simple and powerful approach.

Therapeutic effect of concentrated growth factor preparation on skin photoaging in a mouse model.

ObjectiveTo establish a nude mouse model of photoaging and study the therapeutic effect of a concentrated growth factor preparation (CGF) on skin photoaging.MethodsCGF was prepared from blood from Sprague–Dawley rats. A skin photoaging nude mouse model was developed using UV irradiation combined with the photosensitizer, 8-methoxypsoralen. Mice were divided randomly into seven groups (n = 6 per group): normal control, photoaging, mock treatment, saline treatment, CGF treatment, Filoca 135HA treatment, and plasma skin regeneration system irradiation (the latter two were positive controls). Body weight and skin appearance were observed and pathological changes were determined by hematoxylin and eosin staining. Fiber elasticity was evaluated by Weigert staining. Expression levels of proliferating cell nuclear antigen (PCNA) and matrix metalloproteinase 1 (MMP1) were determined by immunohistochemistry.ResultsA mouse model with typical features of photoaging skin was successfully developed. CGF significantly improved the skin appearance, wrinkle scores, pathological changes, and fiber elasticity, and increased PCNA and decreased MMP1 expression levels in photoaging mice, comparable to the two positive controls.ConclusionCGF can improve the symptoms of skin photoaging in mice, suggesting that it may have applications in the treatment of skin aging in humans.

Therapeutic effects and modulatory mechanism of Alpiniae oxyphyllae Fructus in chronic intermittent hypoxia induced enuresis in rats.

The objective of this study was to explore the effect of Alpiniae oxyphyllae Fructus (AOF) on a rat model of chronic intermittent hypoxia (CIH)-induced enuresis. Findings of this study may help identify therapeutic targets in children with nocturnal enuresis (NE). Female rats were randomly divided into a control group (saline gavage, 4 weeks of normal air), CIH group (saline gavage, 4 weeks of CIH), and AOF group (AOF gavage, 4 weeks of CIH). The variables measured in this study included water intake, urine output, bladder leak point pressure (BLPP), malondialdehyde (MDA) levels, and superoxide dismutase (SOD) activity. The expression levels of the purinergic P2X3 receptor, muscarinic M3 receptor, and ß3-adrenergic receptor (ß3-AR) in the bladder were also measured. The bladder was subjected to haematoxylin and eosin (HE) and Weigert staining, and histological changes were observed under a light microscope to evaluate the morphological changes in the bladder in each group. Compared with the control group, urine output was increased, and the BLPP was decreased in the CIH group, but AOF administration decreased urine output and increased BLPP. In addition, the serum MDA level increased and the SOD activity decreased in the CIH group compared with the control group. Administration of AOF decreased the MDA level and increased the SOD activity. Additionally, compared with the control group, HE and Weigert staining in the CIH group showed that the bladder detrusor muscle bundles were disordered and loose, some muscle bundles were broken, the content of collagen fibres in the gap was reduced, and the gap was significantly widened. However, following the administration of AOF, the bladder detrusor muscle bundles were neatly arranged, and the content of collagen fibres in the gap was increased. Furthermore, compared with the control group, the purinergic P2X3 receptor and muscarinic M3 receptor were expressed at higher levels, and ß3-AR was expressed at lower levels in the CIH group, but AOF administration decreased the expression of the purinergic P2X3 receptor and muscarinic M3 receptor and increased the expression of the ß3-AR. AOF improves enuresis by inhibiting oxidative stress and regulating the expression of the purinergic P2X3 receptor, muscarinic M3 receptor, and ß3 adrenergic receptor.

Effect of intracellular uptake of nanoparticle-encapsulated trehalose on the hemocompatibility of allogeneic valves in the VS83 vitrification protocol.

Trehalose is a disaccharide molecule consisting of two molecules of glucose. Industrially, trehalose is derived from corn starch and utilized as a drug. This study aims to examine whether the integration of nanoparticle-encapsulated trehalose to the Ice-Free Cryopreservation (IFC) method for preserving heart valves has better cell viability, benefits to protect the extracellular matrix (ECM), and reduce immune response after storage. For the experiment to be carried out, we obtained materials, and the procedures were carried out in the following manner. The initial step was the preparation of hydroxyapatite nanoparticles, followed by precipitation to acquire Apatite colloidal suspensions. Animals were obtained, and their tissue isolation and grouping were done ethically. All samples were then divided into four groups, Control group, Conventional Frozen Cryopreservation (CFC) group, IFC group, and IFC + T (IFC with the addition of 0.2 M nanoparticle-encapsulated Trehalose) group. Histological analysis was carried out via H&E staining, ECM components were stained with Modified Weigert staining, and the Gomori Ammonia method was used to stain reticular fibers. Alamar Blue assay was utilized to assess cell viability. Hemocompatibility was evaluated, and samples were processed for immunohistochemistry (TNFα and IL-10). Hemocompatibility was quantified using Terminal Complement Complex (TCC) and Neutrophil elastase (NE) as an indicator. The results of the H&E staining revealed less formation of extracellular ice crystals and intracellular vacuoles in the IFC + T group compared with all other groups. The CFC group’s cell viability showed better viability than the IFC group, but the highest viability was exhibited in the IFC + T group (70.96 ± 2.53, P < 0.0001, n = 6). In immunohistochemistry, TNFα levels were lowest in both IFC and IFC + T group, and IL-10 expression had significantly reduced in IFC and IFC + T group. The results suggested that the nanoparticle encapsulated trehalose did not show significant hemocompatibility issues on the cryopreserved heart valves.

An intra-amniotic injection of mesenchymal stem cells promotes lung maturity in a rat congenital diaphragmatic hernia model.

We aimed to evaluate the effect of human mesenchymal stem cells (hMSCs) on congenital diaphragmatic hernia (CDH) by intra-amniotic injection in a rat CDH model. Nitrofen (100mg) was administered to pregnant rats at E9.5. hMSCs (1.0 × 106) or PBS was injected into each amniotic cavity at E18, and fetuses were harvested at E21. The fetal lungs were classified into normal, CDH, and CDH-hMSCs groups. To determine the lung maturity, we assessed the alveolar histological structure by H&E and Weigert staining and the alveolar arteries by Elastica Van Gieson (EVG) staining. TTF-1, a marker of type II alveolar epithelial cells, was also evaluated by immunohistochemical staining and real-time reverse transcription polymerase chain reaction. The survival rate after intra-amniotic injection was 72.1%. The CDH-hMSCs group had significantly more alveoli and secondary septa than the CDH group (p < 0.05). The CDH-hMSCs group had larger air spaces and thinner alveolar walls than the CDH group (p < 0.05). The medial and adventitial thickness of the pulmonary artery in the CDH-hMSCs group were significantly better (p < 0.001), and there were significantly fewer TTF-1-positive cells than in the CDH group (p < 0.001). These results suggest that intra-amniotic injection of hMSCs has therapeutic potential for CDH.

Platelet Rich Plasma Enhancement of Skin Regeneration in an ex-vivo Human Experimental Model.

This study reports on the development of an original, ex-vivo wounded skin culture protocol using autologous Platelet Rich Plasma (PRP) and enriched Dulbecco's Modified Eagle's Medium (DMEM). Human skin samples obtained from specimens harvested during reduction mammoplasty procedures, were injured in their central portion—to create a standard wound—and cultured under three different conditions:– enriched DMEM with saline solution in the central wound (control)– enriched DMEM with the same medium in the central wound– enriched DMEM plus 2.5% autologous PRP, with the same PRP added medium in the central wound.Morphological analysis was carried out at 0 h (T0) and on days 1, 3, 5 and 10 (T1-T3-T5-T10) using Hematoxylin and Eosin; Masson's trichrome staining; Weigert staining and Ki-67 staining to identify the skin histological features in the different experimental conditions. The combination of DMEM and PRP allowed a favorable modulation of the epithelial cells and fibroblasts proliferation, and a relevant anti-inflammatory action. PRP also demonstrated an inhibitory effect on both the collagen and elastic fibers' de-structuration and a favorable modulation of the re-organization of these fibers. The step by step histological and immune-histo-chemical regenerative effects of PRP on human skin wound repair and regeneration process was observed over a period of 10 days.

Hawthorn Polyphenol Extract Inhibits UVB-Induced Skin Photoaging by Regulating MMP Expression and Type I Procollagen Production in Mice.

Ultraviolet (UV) B radiation can cause skin aging by increasing matrix metalloproteinase (MMP) production and collagen degradation, leading to the formation of wrinkles. This study investigated whether hawthorn polyphenol extract (HPE) protects against UVB-induced skin photoaging using HaCaT human keratinocytes, normal human dermal fibroblasts (HDFs), and mice. Analysis of the phenol composition of HPE by high-performance liquid chromatography-mass spectrometry showed that chlorogenic acid (13.5%), procyanidin B2 (19.2%), and epicatechin (18.8%) collectively accounted for 51.4% of total phenol content and represent the active ingredients of hawthorn fruit. A cell viability assay revealed that HPE treatment promoted cell proliferation in HaCaT cells and HDFs. On the other hand, MMP-1 and type I procollagen production was decreased and increased, respectively, in UVB-exposed cells treated with HPE as compared with those without treatment, as determined by enzyme-linked immunosorbent assay. Hematoxylin and eosin and Weigert staining of dermal tissue specimens from mice demonstrated that HPE also reversed UVB-induced epidermal thickening and dermal damage. The increase in production of reactive oxygen species and decrease in antioxidant enzyme activity as well as the increase in nuclear factor-κB activation and mitogen-activated protein kinase phosphorylation induced by UVB irradiation were reversed by HPE (100 or 300 mg/kg body weight), which also suppressed MMP expression and stimulated the production of type I procollagen in the dorsal skin of UVB-irradiated mice. These results suggest that HPE is a natural product that can prevent UVB radiation-induced skin photoaging.

Stereological and Morphometric Study of Type 3 Collagen Formation in the Cutaneous Wounds of Diabetic Mice Treated with Mesenchymal Stem Cells

Introduction: Wound healing is a progressive, essential and complex physiological process that occurs as a restorative response after a tissue injury. It involves three phases: inflammation, proliferation and maturation. Exogenous, endogenous and pathological factors may interfere in the cicatricial process in humans and animals by altering the balance between the synthesis, degradation and remodelling of collagen and elastic fibres. Diabetes mellitus is a progressive metabolic disease that alters elastogenesis and collagenesis and induces delays in the healing process. Scientific evidence suggests that mesenchymal stem cells modulate the cicatricial response. Thus the objective of this work was to perform stereological and morphometric analysis to determine the formation of dermal fibres in cutaneous fragments of a murine model of diabetes mellitus.Materials, Methods &amp; Results: Histological sections were obtained from the cutaneous wounds of diabetic mice. The cutaneous wounds were previously treated with autogenous mesenchymal stem cells, physiological solution or polyurethane membrane. The histological sections were subsequently processed and stained for type 1 and 3 collagen fibres and elastic fibres using Picrosirius Red and Weigert staining, respectively. Histological sections stained with Picrosirius Red presented three types of birefringence under polarised light microscopy that corresponded to red colours for type 1 collagen and green and yellow colours for type 3 collagen. Weigert staining presented three colours for histological structures under white light microscopy that corresponded to black colours for elastic fibres, variations in colour from pink to purple for other structures and dermal attachments. The elastic fibres, represented by a black colour, presented in a heterogeneous form and were either identified as thin, punctiform or rectangular fibres or as elastic agglomerates. A greater volume of elastic fibres was observed in the superficial dermis than in the deep dermis, arranged irregularly. These fibres were organised longitudinally to the dermo-epidermal junction and surrounding the blood vessels and hair follicles. The images obtained were evaluated using the Cavalieri principle of stereology to obtain quantitative data in three-dimensions (3D), represented by the volume of the dermal fibres, and by the colour segmentation method. The K-means clustering plug-in in Image J® was used to quantify the area of the dermal fibres in the cutaneous wounds after the proposed dermatological treatments. A total of 90 images were obtained and analysed. No statistically significant differences (P &gt; 0.01) were observed in the volume or area of type 1 collagen fibres between the treatment groups. Significant differences (P &lt; 0.01) were only identified for the volumes and areas of type 3 collagen, with treated animals also presenting lower mean values for the volume and area of elastic fibres compared to the control group.Discussion: The preponderance of type 3 immature collagen in the cutaneous wounds of animals treated with stem cells indicates active collagenase and greater fibroblastic activity, which is probably induced by stem cells. Diametrically, the identification of lower levels of elastic fibres in the cutaneous fragments treated with stem cells suggests that cell therapy does not contribute satisfactorily to elastogenesis. Previous reports suggested that mesenchymal stem cells may decrease elastin synthesis, and such a situation may have occurred in this study. The autologous mesenchymal stem cells increased the formation of collagen fibres in diabetic mice at the detriment of the formation of elastic fibres, thus suggesting active early collagen in the first 2 weeks of the cicatricial process.

Topographic and microscopic anatomical description of the emissary sinus of foramen ovale in adult humans

ObjectiveAlthough the Emissary Sinus of Foramen Ovale (ESFO) was first described by Trolard in 1868, its definition remains confused and neglected in the medical literature. This structure represents a vein, two veins, a venous plexus, or a dural sinus? Does it really exist? To understand this topic, this work aimed to describe the anatomy, topography, and microscopic features of the ESFO, precisely characterizing its structure, routes and anatomical correlations. Patients and methodsESFO from the skull’s base of adults were dissected into fifty anatomical blocks and evaluated using Hematoxylin and Eosin, Picro-sirius red and Weigert staining, and by Scanning Electron Microscopy (SEM). ResultsESFO was always present between cavernous sinus and pterygoid plexus on both antimeres, its inferior route passing through the foramen ovale and/or sphenoidal emissary foramen (foramen of Vesalius), anterior to the mandibular branch of trigeminal nerve. Its microscopic arrangement resembled what was found on transverse sinus, that is composed by layers of collagen fibers oriented on transversal and longitudinal planes. It wasn’t possible to identify the media and adventitial tunica, features seen in veins, and the elastic layer was very thin near its lumen. SEM analysis showed that, like the transverse sinus, the ESFO was composed by parallel cells that presented a rhombus shape containing central rounded nuclei. ConclusionIn summary, the venous channel passing through the foramen ovale and/or sphenoidal emissary foramen (foramen of Vesalius) is a dural venous sinus constituted by dura mater layers and should be considered during surgical approaches near the foramen ovale in the middle cranial fossa.

Testosterone replacement maintains smooth muscle content in the corpus cavernosum of orchiectomized rats

ObjectiveTo evaluate the effects of testosterone (T) on the maintenance of corpus cavernosum (CC) structure and apoptosis. MethodsAnimals were divided into three groups: sham operation group (n = 8) underwent sham operation; Orchiectomized (Orchiec)+ oily vehicle group (n = 8) underwent bilateral orchiectomy and received a single dose of oily vehicle by intramuscular injection (i.m.) 30 days after orchiectomy; and Orchiec + T group (n = 8) underwent bilateral orchiectomy and received a single dose of T undecanoate 100 mg/kg i.m. 30 days after the surgery. Animals were euthanized 60 days after the beginning of the experiment with an anesthetic overdose of ketamine and xylazine. Blood samples and penile tissue were collected on euthanasia. Azan's trichrome staining was used to evaluate smooth muscle, Weigert's Fucsin-Resorcin staining was used to evaluate elastic fibers and Picrosirius red staining was used to evaluate collagen. Apoptosis was evaluated using TUNEL technique. ResultsT levels decreased in Orchiec + oily vehicle when compared to sham operation and Orchiec + T groups (p < 0.001). T deprivation reduced trabecular smooth muscle content and penile diameter and T replacement maintained both parameters (p = 0.005 and p = 0.001, respectively). No difference was observed in the content of sinusoidal space (p = 0.207), elastic fibers (p = 0.849), collagen (p = 0.216) and in apoptosis (p = 0.095). ConclusionNormal testosterone levels maintain CC smooth muscle content and do not influence elastic fibers, collagen content and apoptotic index. Further studies should be performed in order to investigate the mechanisms by which androgen mediates its effects on CC structure.

Ex vivo evaluation of the effect of photodynamic therapy on skin scars and striae distensae.

Skin scars and striae distensae (SD) are common dermal disorders with ill-defined treatment options. There is emerging clinical evidence for use of photodynamic therapy (PDT) in treating skin fibrosis. Therefore, the aim here was to investigate the effect of PDT on skin scars and SD in an exvivo model of human skin scarring. Photodynamic therapy, with 5ALA or MALA in addition to illumination with 40J/cm(2) of red light, was applied to striae alba, fine line, hypertrophic and keloid scars exvivo (n=18). General morphology was assessed by H&E, Herovici's and Weigert's differential staining. Apoptosis, proliferation, metalloproteinase 3 and tropoelastin expression were quantified immunohistochemically, and differential gene expression of proliferating cell nuclear antigen (PCNA), collagen (COL) type I and type III, matrix metalloproteinase 3 (MMP3) and tropoelastin (ELN) was assessed by real-time quantitative reverse transcription polymerase chain reaction. Apoptosis increased, which correlated with decreased proliferation and PCNA gene expression. Post-PDT, matrix components were found to be re-organised in both hypertrophic and keloid scars. COLI and COLIII gene expression levels decreased, whilst MMP3 and ELN increased significantly post-PDT compared to normal skin and untreated controls (P<0.05). However, no significant difference between 5ALA and MALA-PDT treatments was observed. Using our unique exvivo model, we show for the first time morphological and cellular effect of application of PDT, which correlates with the degree and severity of dermal fibrosis. In view of this, PDT may be ideal in targeting treatment of abnormal skin scarring.

Calcification of the Aortic Valve: the Arrival Point of a Complex Degenerative Process

The histology of the calcified aortic valve has been sequentially revisited in the light of modern concepts of cardiovascular pathology. Fifteen cases of calcified aortic valves, surgically removed for an acquired steno-insufficiency, have been investigated by histochemistry (Weigert's stain, Van Gieson's stain, Verhoeff's stain, Alcian blue / PAS stain). Following the lipidic infiltration of the endothelium, a diffuse proteolytic phenomenon, mainly related to aging, gives rise to tissue degeneration. The subsequent remodelling attempt is ineffective to maintain the valve function, as demonstrated by the limited presence of newly formed muscle-like cells. The arrival point of this degenerative process is represented by a marked calcification, involving the aortic leaflets and the annulus. Proteolysis, absence of an effective remodelling mechanism and calcification are so the fundamental sequential steps of the acquired aortic steno-insufficiency. At the basis of this complex pathological cascade of events, genetic and metabolic disorders play an important role. The conventional histological techniques permit to easily distinguish calcification of the aortic valve from endocarditis and rheumatic lesions and they allow to demonstrate pathogenetic mechanisms in common with thoracic aortic aneurysms.

Structural Study of Gubernaculum Testis in Fetuses with Prune Belly Syndrome

We compared and contrasted the structure of the gubernaculum testis in fetuses with prune belly syndrome and normal controls. We studied a total of 6 gubernacula from 3 male fetuses with prune belly syndrome and a total of 14 from 7 male fetuses without an anomaly. Gubernacular specimens were cut into 5 μm sections and stained with Masson trichrome to quantify connective tissue and smooth muscle cells, with Weigert stain to observe elastic fibers and with picrosirius red with polarization to observe collagen. Immunohistochemical analysis was done with tubulin to observe the nerves. Images were captured with a BX51 microscope and DP70 camera (Olympus®). Stereological analysis was done with Image-Pro and ImageJ (MediaCybernetics®) using a grid to determine volumetric density. Means were statistically compared with the Mann-Whitney test. All tests were 2-sided with p <0.05 considered statistically significant. Prune belly syndrome fetuses were at 17 to 31 weeks of gestation and control fetuses were at 12 to 35 weeks of gestation. Quantitative analysis showed no difference in the volumetric density of smooth muscle cells in prune belly syndrome vs control gubernacula (mean 15.70% vs 19%, p = 0.2321). Collagen fiber analysis revealed a predominance of green areas in prune belly syndrome gubernacula, suggesting collagen type III, and a predominance of red areas in control gubernacula, suggesting collagen type I. Elastic fibers were significantly smaller in prune belly syndrome gubernacula than in control gubernacula (mean 14.06% vs 24.6%, p = 0.0190). Quantitative analysis demonstrated no difference in the volumetric density of nerves in prune belly syndrome or control gubernacula (mean 5.200% vs 3.158%, p = 0.2302). The gubernaculum in fetuses with prune belly syndrome had altered concentrations of collagen and elastic fibers. These structural alterations could be one of the factors involved in cryptorchidism in prune belly syndrome.

Involvement of calcium-sensing receptors in hypoxia-induced vascular remodeling and pulmonary hypertension by promoting phenotypic modulation of small pulmonary arteries.

Phenotype modulation of pulmonary artery smooth muscle cells (PASMCs) plays an important role during hypoxia-induced vascular remodeling and pulmonary hypertension (PAH). We had previously shown that calcium-sensing receptor (CaSR) is expressed in rat PASMCs. However, little is known about the role of CaSR in phenotypic modulation of PASMCs in hypoxia-induced PAH as well as the underlying mechanisms. In this study, we investigated whether CaSR induces the proliferation of PASMCs in small pulmonary arteries from both rats and human with PAH. PAH was induced by exposing rats to hypoxia for 7-21 days. The mean pulmonary arterial pressure (mPAP), right ventricular hypertrophy index (RVI), the percentage of medial wall thickness to the external diameter (WT %), and cross-sectional total vessel wall area to the total area (WA %) of small pulmonary arteries were determined by hematoxylin and eosin (HE), masson trichrome and Weigert's staining. The protein expressions of matrix metalloproteinase (MMP)-2 and MMP-9, the tissue inhibitors of metalloproteinase (TIMP)-3, CaSR, proliferating cell nuclear antigen (PCNA), phosphorylated extracellular signal-regulated kinase (p-ERK), and smooth muscle cell (SMC) phenotype marker proteins in rat small pulmonary arteries, including calponin, SMα-actin (SMAα), and osteopontin (OPN), were analyzed by immunohistochemistry and Western blotting, respectively. In addition, immunohistochemistry was applied to paraffin-embedded human tissues from lungs of normal human and PAH patients with chronic heart failure (PAH/CHF). Compared with the control group, mPAP, RVI, WT % and WA % in PAH rats were gradually increased with the prolonged hypoxia. At the same time, the expressions of CaSR, MMP-2, MMP-9, TIMP-3, PCNA, OPN, and p-ERK were markedly increased, while the expressions of SMAα and calponin were significantly reduced in lung tissues or small pulmonary arteries of PAH rats. Neomycin (an agonist of CaSR) enhanced but NPS2390 (an antagonist of CaSR) weakened these hypoxic effects. We further found that the expression change of CaSR, PCNA, and SMC phenotypic marker proteins in PAH/CHF lungs was similar to those in PAH rats. Our data suggest that CaSR is involved in the pulmonary vascular remodeling and PAH by promoting phenotypic modulation of small pulmonary arteries.

Tissue reaction induced by implanted venous access ports in adult patients after infection of the implantation site

Implantable long-term central venous port systems (CVPS) are widely used as a permanent means of accessing the vascular system for intravenous delivery of drugs, parenteral nutrition, blood transfusion, and blood sampling. These systems allow easy and repetitive puncture without causing much damage to the vessels. However, the body foreign surface of CVPS induces an inflammatory response with varying intensity (depending on the implant materials) that leads to formation of a fibrous tissue capsule around the implant. This study was designed to investigate the influence of bacterial infection on the tissue reaction induced by implanted CVPS in adult patients. 20 patients (9 women, 11 men, 58 ± 14 yrs of age) were included in this study. These patients received explantation of a polysulfone based CVPS (ChemoSite™, Covidien, Mansfield, USA) due to port related infections (patients with bacterial infections at the implantation site: group A, 5 men, 1 women) or to other reasons such as termination of treatment, thrombosis, or CVPS dysfunction (patients without bacterial infections, group B, 6 men, 8 women) 299.9 ± 261.2 days after CVPS implantation. A sample of the encapsulating tissue covering the CVPS together with surrounding tissue (at least 1 × 1 cm2) was placed in a small container with fixing agent, a buffered neutral 4% formalin solution (pH 7). Histological sections of the samples were prepared for light microscopic analysis after paraffin embedding. Sections of 3 μm were cut and stained with haematoxylin and eosin, Weigert's elastic stain, and Heidenhain's azan stain. There was no difference in thickness, collagen and elastin content, or cell and capillary density of the fibrous capsule between both groups. Due to the wound healing reaction involving angiogenesis and fibroblast activation cell density and number of capillaries in the capsule tissue of all patients showed a positive correlation (r = 0.45, p < 0.05). However, the study demonstrated that at the end of the foreign body reaction the artificial tissue layer which covers the CVPS after implantation due to foreign body reaction shows only low reactivity towards infections.

Acquired Cutis Laxa With an Interstitial Granulomatous Reaction Associated With IgG Lambda Monoclonal Gammopathy

Acquired cutis laxa (ACL) is a rare connective tissue disorder that affects the skin elastic fibers, resulting in the loss of elasticity. In 50% of cases, this condition is associated with other diseases, particularly plasma-cell dyscrasias. This report describes a case of ACL with unusual clinical and histopathological characteristics. A 29-year-old man presented with diffuse erythematous plaques that had first appeared 5 months previously. Examination revealed multiple flaccid erythematous plaques on his trunk, neck, and skinfolds. Immunophenotyping of bone marrow aspirate revealed 7% of monoclonal plasma cells with lambda light chain expression. Skin biopsy histology revealed foci of interstitial granulomatous reaction. Weigert stain showed a loss of elastic fibers in the dermis, areas with thickened fibers and elastophagocytosis. Immunohistochemistry was positive for CD68. The cutaneous findings enabled an early diagnosis of IgG lambda monoclonal gammopathy to be made. Microscopic examination revealed an interstitial granulomatous reaction and severe alterations in the elastic fibers that varied in intensity in the different biopsies. Curiously, little has been mentioned in the literature regarding the presence of an interstitial granulomatous reaction in ACL. It is our belief that this reaction is secondary to the degenerative process of the elastic fibers.