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The primo vascular system (PVS) is observed in different parts of the body under different physiological and disease conditions. Previously, the PVS was not observed in the vagina. The vaginal samples of this study were collected from the female genitalia of healthy New Zealand white rabbits from the animal house, Faculty of Medicine, Assiut University. The vaginal samples were fixed in Bouin's solution. The sections were stained with hematoxylin and eosin and Crossmon's trichrome. Additionally, the sections were immunohistochemically stained with neuron-specific enolase (NSE) and vascular endothelial growth factor (VEGF). A primo node was observed on the lymph vessel of the vagina and has several characteristics that resemble those of the previously discovered primo nodes. The primo node in this study was surrounded by mesothelial cells that provide positive immunoreactivity to NSE and VEGF. Sinuses of different sizes, floating cells, telocyte-like cell, and primo microcells were observed as the main constituents of the primo node. Additionally, migratory cells were detected, which passed from the primo node to the enclosing lymph vessel.

The purpose of this study was to determine the efficacy of highly concentrated tricalcium phosphate (TCP) biocomposite screws on local bone formation in a rabbit model of osteoporosis induced by bilateral ovariohysterectomy (OHE). Fourteen 24-week-old female New Zealand rabbits (weight, 3-3.5 kg) were divided into two groups: 1) OHE and biodegradable poly(lactic-co-glycolic acid) (PLGA) without s-TCP plate or screw insertion (OHE/BSR) group; and 2) OHE and biocomposite PLGA with highly concentrated s-TCP plate and screw insertion (OHE/HCCP). Both groups underwent bilateral OHE and had two different types of screws and plates inserted at the proximal tibia. Bilateral tibiae were extracted at 25 weeks post-OHE. The extracted tibiae were scanned with ex-vivo micro-computed tomography (micro-CT). Parameters including bone mineral density (BMD), trabecular bone volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp) were evaluated after staining the tibial samples with hematoxylin and eosin (H&E) and Masson's trichrome. We then performed pathological assessments. Micro-CT images revealed improved new bone formation near the implant in the OHE/HCCP group with higher values of BMD, BV/TV, and Tb.N but lower values of Tb.Th and Tb.Sp than the OHE/BSR group. Analyses of H&E and Masson's trichrome staining showed better new bone formation around the implant in the OHE/HCCP group than in the OHE/BSR group. The use of highly concentrated TCP biocomposite screw and plate might improve local bone formation and facilitate osteoconductivity in an osteoporotic rabbit model. This article is protected by copyright. All rights reserved.

Background Arteriolosclerotic ulcers of Martorell are histologically characterized by hyaline arteriolosclerosis resulting in concentric occlusion of the arteriolar lumina. Although several authors have previously reported on hyaline changes in hypertensive arteriolopathies, so far, little information is available on the molecular composition of hyaline wall depositions. Objectives This study aimed at the molecular characterization of hyaline arteriolar deposits in patients with hypertensive arteriolopathy using confocal Raman spectroscopy. Methods Samples of patients diagnosed with arteriolosclerotic ulcers of Martorell were analysed using confocal Raman spectroscopy. The findings were correlated with histological analyses. Skin samples from healthy, non-hypertensive patients served as controls. Results Confocal Raman spectroscopy analysis revealed that subendothelial hyaline deposits in arteriolosclerotic ulcers are mainly composed of collagen and phospholipids, in particular phosphatidylcholine. The presence of collagen within hyaline deposits was confirmed by Masson's Trichrome and Picrosirius Red staining. Additionally, the presence of collagen could also be shown for hypertensive nephrosclerosis. Actin was markedly decreased in hyalinized compared to control vessels, corresponding to the loss of smooth muscle cells in the process of hyalinization. This was confirmed by immunofluorescence staining for α-smooth muscle actin and desmin. Conclusion The present findings suggest that arteriolar hyaline deposits in hypertensive arteriolopathy are mainly composed of collagen and phospholipids, in particular phosphatidylcholine. Together with the concurrent absence of actin, these findings suggest that potentially critical disease mechanisms involve pressure-induced vascular smooth muscle cell apoptosis with subsequent deposition of collagen.

To identify and describe histological and immunohistochemical criteria that may differentiate between 4 skin and lymph node lesions associated with Mycobacterium (M.) bovis and M. microti in a diagnostic 5 pathology setting. 6Materials and Methods 7Archived skin and lymph node biopsies of tuberculous lesions were stained with haematoxylin and 8 eosin, Ziehl-Neelsen and Masson’s Trichrome. Immunohistochemistry was performed to detect 9 expression of calprotectin, CD3 and Pax5. Samples were scored for histological parameters (i.e. 10 granulomas with central necrosis vs small granulomas without central necrosis, percentage necrosis, 11 and/or multinucleated giant cells), number of acid-fast bacilli (bacterial index) and lesion percentage of 12 fibrosis and positive immunohistochemical staining. 13Results 14Twenty-two samples were examined (M. bovis n = 11, M. microti n = 11). When controlling for age, 15 gender and tissue, feline M. bovis-associated lesions more often featured large multi-layered 16 granulomas with central necrosis. Conversely, this presentation was infrequent in feline M. microti-associated lesions, where small granulomas without central necrosis predominated. The presence of an outer fibrous capsule was variable in both groups, as was the bacterial index. There were no differences in intralesional expression of immunohistochemical markers.Clinical SignificanceDifferences in the histological appearance of skin and lymph node lesions may help to infer feline infection with either M. bovis or M. microti at an earlier stage when investigating these cases, informing clinicians of the potential zoonotic risk. Importantly, cases of tuberculosis can present with numerous acid-fast bacilli. This implies that a high bacterial index does not infer infection with non-zoonotic non-tuberculous mycobacteria.

Background Several experimental methods for evaluating dermal structures exist; however, most of these are not used in dermatology clinics because of cost and functional limitations. Objective To propose a simple, non-invasive method for dermal structure evaluation using a green light-emitting diode (LED) with cross-polarized light (CPL) imaging and compare the quality of the images taken using either green or white LED. Materials and methods Skin specimens were taken from fifteen cadavers. Images were captured using CPL photography with a green or white LED. The Commission International d'Eclairage L*a*b* (CIELAB) values were calculated for each image. The skin specimens were processed and stained with Masson's trichrome to visualize collagen fibers with major image scattering. The images were histologically analyzed, and correlational and regression analyses were performed to determine the relationship between the L* values and histologic measurements. Results The L* values for the green images were positively correlated with collagen fiber density, reticular collagen bundle diameter, and dermal thickness. They were effective for estimating dermal properties. The L* values for the white images were positively correlated with reticular collagen bundle diameter and dermal thickness. Correlational coefficients for white images were lower than those for green images. In regression analysis, green images showed a higher coefficient of determination (R2 ) for predicting reticular collagen bundle diameter than white images (0.1128 vs. 0.0827). Conclusion Cross-polarized light imaging with a green LED is a simple, non-invasive method for evaluating dermal structures. The use of a green LED was also more effective for image analysis.

The goldfish is a model organism showing great potential for research, particularly in comparative endocrinology concerning the neuroendocrine signalling and regulation of vertebrate reproduction. Furthermore, this teleost is increasingly stressed as a relevant alternative to more common fish model organisms, namely zebrafish. However, quality descriptions and illustrations of the complete goldfish gonadal histology are surprisingly scarce, but needed, to support research using this fish. Therefore, the main aim of this work is to describe in detail and adequately illustrate the goldfish oogenesis, from oogonia to late maturation, by applying routine stains (haematoxylin-eosin) and special procedures (periodic acid-Schiff and Goldner's trichrome). We hypothesized that the combined strategies would enable not only to observe the most general features but also to perceive some poorly described details of oocytes better. We describe the details of the following maturation stages: oogonia proliferation, chromatin-nucleolus, primary growth (one nucleolus step, multiple nucleoli step, perinucleolar step, cortical alveoli step) and secondary growth (early secondary growth step, late secondary growth step). Additionally, we report aspects of early and late follicular atresia. The study allowed comparisons with other species and showed that the Goldner's trichrome has the best discriminative power and should be the preferred stain, despite more time-consuming.

BACKGROUND Keloids and hypertrophic scars are challenging to both patients and physicians. They can be aesthetically disfiguring, functionally debilitating, and emotionally distressing. Lasers have introduced new mechanisms to improve scars both on aesthetic and symptomatic levels. AIM OF WORK Comparing the efficacy of fractional CO2 laser, long-pulsed Nd:YAG laser and their combination in the treatment of hypertrophic scars and keloids on clinical, histopathological, and biochemical basis. PATIENTS AND METHODS Thirty patients with hypertrophic scars and keloids were enrolled in the study. Three scars in each patient were randomly assigned to treatment modalities (i) Fractional CO2 , (ii) Nd:YAG laser, (iii) Combined CO2 and Nd:YAG lasers. For each treatment area four sessions, 4-6 weeks apart were performed. Clinical evaluation was done before and 1 month following last session using the Vancouver Scar Scale (VSS) and the Patient and Observer Scar Assessment Scale (POSAS). Routine hematoxylin and eosin, Masson's trichrome, and Orcein stains were used to evaluate the appearance and pattern of dermal collagen and elastic fibers. Image analysis was used to quantitatively assess the density of collagen and elastic fibers. Biochemical evaluation of tissue level of transforming growth factor-β I (TGF-β I) and TGF-β III was performed using enzyme-linked immunosorbent assay studies. RESULTS Both VSS and POSAS showed significant improvement following treatment with the three used modalities. Collagen fibers showed significant improvement as regards appearance and pattern while it was insignificant as regards density. Elastic fibers density improvement was only significant in fractional CO2 (treatment area A). Hypertrophic scars showed more significant improvement with fractional CO2 laser, while in keloids there was no significant difference between the three modalities regarding improvement. Level of TGF-β I showed significant reduction after treatment in all treatment modalities, while TGF-β III levels showed insignificant elevation in all treatment modalities. Side effects were significantly higher in treatment area C (combined treatment). CONCLUSION Long pulsed Nd:YAG laser is effective and safe treatment of hypertrophic scars and keloids. Fractional CO2 laser yields better improvement in hypertrophic scars, while in keloids both fractional CO2 and Nd:YAG lasers achieve comparable improvement. Combination in the same session did not add significant additional benefit and the side effects profile was higher. LIMITATIONS small sample size and short follow-up period. Lasers Surg. Med. © 2020 Wiley Periodicals, Inc.

The expressions of matrix metalloproteinases 2 and 9 have been described during the development, as an example in heart and tooth but not in the small intestine yet. In this context, this study aimed to evaluate the expressions of MMP-2 and MMP-9 in the small intestine of Wistar rats during intrauterine (IU) and postnatal (PN) life. Expressions were determined on the 15th and 18th days of IU life and the 3rd, 10th, 17th, 25th, and 32nd days of PN life. Intestinal samples obtained from six animals were submitted to zymography, immunohistochemistry, and staining with Masson's trichrome. The results showed that MMP-2 and MMP-9 were not expressed during IU life; however, after birth, MMP-9 was immunolocalized in the goblet and mast cells. In conclusion, our results showed that MMP-2 and MMP-9 were not expressed in absorptive epithelial cells during the IU period of the small intestine but after birth, MMP-9 was expressed in the goblet cells, and mast cells present in the lamina propria.

Despite the extensive literature regarding peripheral nerve stretch injuries, there are few studies that compare the nerve histology with the mechanical properties in humans. There is clinical evidence suggesting that the peroneal nerve is at greater risk for injury compared to the tibial nerve following total hip arthroplasty and hip trauma. We examined the two nerves from fresh human cadavers with or without controlled stretch. The mechanical properties, stiffness, and strain were compared with light microscopic preparations in longitudinal sections stained by the trichrome method for collagen and showing the effects of structural deformation. The tibial nerve had an average failure load 1.7× that for the peroneal nerve (P = 0.0001). Although the corresponding average stiffness showed a trend toward being larger (4.39 vs. 3.81 N/mm), the difference was not significant (P = 0.126). Histologically, the perineurium along with the underlying nerve fascicle was undulated in the control specimens and straightened out in the stretched specimens. Peroneal nerves went on to failure at lower loads and exhibited a wavy pattern on pathologic slides after failure, which shows that peroneal nerves fail mechanically before they can unfold. The tibial nerve has a biomechanical and histological advantage compared to the peroneal nerve during tensile testing, which could be the reason why it is less commonly damaged. We conclude that the perineurium is especially protective against deformation changes in human nerves relative to the respective nerve size and number of fascicles. Anat Rec, 302:2030-2039, 2019. © 2019 American Association for Anatomy.

Background Proper assessment of dermal collagen fibers by dermatologists and researchers is essential. Histologic evaluation methods have limitations. We present a simple method for measurement of collagen fibers in human skin using Masson's trichrome staining. Materials and methods Normal skin specimens from a cadaver were processed with Masson's trichrome, which can effectively stain collagen fibers blue with aniline dye. Optical photomicrographs of these slides were analyzed using ImageJ software. Color image processing, a histogram-based function of ImageJ for image segmentation, was performed with color moments thresholding technique. We selected blue areas by adjusting the blue channel to include specific values. The selected areas were highlighted and evaluated. We divided the image into layers of 0.09-mm2 areas from the top to bottom of the dermis. Each area was cropped and evaluated. Results Quantitative assessment yielded the quantitative size occupied by collagen fibers in an area of 0.09 mm2 . Calculation of the percentage in each area can be used to determine the density of collagen fibers. Conclusion Measurements obtained with our method can be applied to research on dermal collagen fibers. We present a convenient quantitative assessment method for the dermal constituents in Masson's trichrome-stained slides.

The purpose is to examine early wound healing through histological analysis by characterizing connective tissue distribution and organization in the treated periodontium following nonsurgical therapy. Periodontal disease is a multifactorial pathological process that leads to the loss of the surrounding periodontium. Traditional periodontal therapies have proven beneficial in halting the progression of disease. The aim of this study is to investigate early wound healing in periodontal patients following hand/ultrasonic instrumentation alone, erbium‐doped yttrium aluminum garnet laser instrumentation alone, or a combination of hand/ultrasonic instrumentation and Er:YAG laser instrumentation for the nonsurgical treatment of periodontitis by histologic evaluation. Twenty‐one patients were randomized to receive nonsurgical therapy for the treatment of chronic periodontitis with three modalities prior to surgical therapy. Baseline clinical measurements were obtained prior to treatment. Wound healing was assessed by obtaining an otherwise discarded tissue sample following nonsurgical therapy of the selected study site. Samples were obtained at 2 or 6 weeks following initial therapy with a step‐back incision and fixated for histological and immunohistochemical analysis. There were minimal between‐group differences in the amount of collagen distribution when analyzing the Mallory–Heidenhain Azan trichrome, Picrosirus Red stain, and proliferating cell nuclear antigen at both time points. Descriptive analysis of baseline measurements showed no differences in probing depth change, bleeding on probing, and clinical attachment level following initial therapy between the three treatment groups at 2 or 6 weeks. Each treatment modality was effective in treating moderate to severe chronic periodontitis; however, the results of this study are inconclusive regarding superiority of any one treatment approach from a histologic and immunohistochemical perspective. Based on this assessment, there was increased fibroblast proliferation and collagen maturation between the 2‐ and 6‐week time point after treatment in all treatment groups, with few apparent differences between treatment groups. This pilot study qualitatively evaluated early wound healing in periodontal patients following non surgical therapy with various treatment modalities. When comparing descriptive outcomes of Er:YAG laser therapy and hand/ultrasonic instrumentation there were minimal differences in collagen distribution and density between groups. The evaluated modalities were each effective treating periodontal patients with non surgical therapy.

While epiglottis is essentially a mammalian structure, studying its microstructure in any placental model will add an important information to the field of comparative anatomy and the related branches of biology. The aim of this study was to describe the structure of the epiglottis in dromedary camels using light and scanning electron microscopy (SEM), with reference to the possible functions. A total of 11 epiglottis cartilages from 11 larynges were used. The study revealed unusual, deeply situated glands just beneath the cartilage plate. They have unusually, wide surface-openings, while their ducts were partly located within the cartilage. This is presumed to be an adaptation to the need for rapid and efficient mucosal surface hydration in the arid conditions. The possible secretion transport mechanisms in these glands were also discussed. Furthermore, the SEM revealed for the first time, the presence of taste buds in camel epiglottis. However, in histological sections, visibility of taste buds was dependent upon the staining techniques. The taste buds were not seen with standard H& E stain, as they blended imperceptibly with the surrounding epithelium. Conversely, Mallory's trichrome showed contrasting colors, and taste buds were visible. In conclusion, camel epiglottis has an unusual structure, which may be correlated to environmental adaptation and important for the general health of upper respiratory tract in this species.

Knowledge of skeletal muscle adaptations is important to understand the functional deficits in cerebral palsy (CP). This study aimed to investigate the morphofunctional characteristics of skeletal muscle in a CP animal model. Initially, pregnant Wistar rats were injected intraperitoneally with saline or lipopolysaccharide over the last five days of pregnancy. The control group (n = 8) consisted of male pups born to females injected with saline. The CP group (n = 8) consisted of male pups born to females injected with lipopolysaccharide, which were submitted to perinatal anoxia [day of birth, postnatal day 0 (P0)] and sensorimotor restriction (P1‐P30). The open‐field test was undertaken on P29 and P45. On P48, the animals were weighed, and the plantaris muscle was collected and its weight and length were measured. Transverse sections were stained with haematoxylin‐eosin, NADH‐TR, Masson's trichrome and non‐specific esterase reaction for analysis. and transmission electron microscopy was performed. In the CP group, reductions were observed in mobility time, number of crossings and rearing frequency, body weight, muscle weight and length, and nucleus‐to‐fibre and capillary‐to‐fibre ratios. There was a statistically significant increase in the percentage area of the muscle section occupied by collagen; reduction in the area and increase in the number of type I muscle fibres; increase in myofibrillar disorganization and Z‐line disorganization and dissolution; and reduction in the area and largest and smallest diameters of neuromuscular junctions. Thus this animal model of CP produced morphofunctional alterations in skeletal muscle, that were associated with evidence of motor deficits as demonstrated by the open‐field test.

The corpus spongiosum reportedly occupies a larger proportion of the human glans penis than does the penile body, embedding the end of the corpus cavernosus (CC). However, anatomic descriptions about the fibrous structures of glans penis in the literature cause confusion during dissection and reconstructive surgery. Forty-five penises of formalin-embalmed cadavers were dissected sagittally along the course of the distal urethra and observed macroscopically. Dense connective tissues adjacent to the fossa navicularis and spongiosum parts of the glans were cropped, and underwent Masson's trichrome and Verhoeff-Van-Gieson staining. Most (55.5%) of the specimens had distinct fibrous bands toward the distal tips of the glans penis, which elongated from the tunica albuginea of the CC. They comprised longitudinal collagen bundles continuous to the outer longitudinal layer of the tunica albuginea covering the CC and were intermingled with sparse elastic fibres. This architecture either did not reach the distal end of the glans penis (35.5% of cases), or was obscure or dispersed in all directions (9.0% of cases). The structural dimorphism and the variations in the ratio of dense connective tissue components of the fibrous skeleton are considered to contribute to the varying degrees of flexibility, distensibility and rigidity of the human glans penis.

In this study, the protective effect of spirulina on corneal injury after formaldehyde (FA) exposure was assessed. Thirty adult male albino rats were divided into four groups. Group I: 12 rats were divided into two subgroups: I-a (negative control) and I-b (positive control). Group II (spirulina group): six rats received spirulina via an oral gavage feeding needle at a daily dose of 400 mg/kg b.w. Group III (FA exposure group): six rats were subjected to 10% FA inhalation for 2 h per day for 2 weeks (5 days per week). Group IV (FA exposure group treated with spirulina): six rats were exposed to 10% FA as in group III, with co-administration of spirulina as in group II. After 2 weeks, all the rats were sacrificed; the corneas were dissected and processed for paraffin sections. The sections were stained with hematoxylin and eosin (H&E), Masson's trichrome (MTC), or avidin-biotin peroxidase, and examined by light microscopy. The sections of rat cornea exposed to FA (Group III) showed disorganized and compressed epithelium with erosions. Subepithelial mononuclear cell infiltration and invasion of blood vessels were also evident. Stromal collagen fibers were disorganized and widely separated. All these changes were ameliorated by administration of spirulina (Group IV). Corneal thickness was nearly normal in Group IV, statistically significantly less than in Group III. It was concluded that spirulina protects against FA-induced corneal injury in rats. Clin. Anat. 31:830-837, 2018. © 2018 Wiley Periodicals, Inc.

Objective To analyse the immunohistochemical expression of ameloblastin in the bell stage of tooth germ and compare with ameloblastoma to determine the level of differentiation of tumour cells. Study design This study included eleven human tooth germs with four in the early and seven in the late bell stage, and six selected archival tissue samples of ameloblastomas were studied using haematoxylin and eosin, Masson's trichrome and ameloblastin. Results All eleven tooth germs reacted positively to ameloblastin with a characteristic inverted and sequential pattern of expression in the acellular zone of the dental papilla and enamel organ. Of the six cases of ameloblastoma, five cases showed a variable level of expression of ameloblastin in the tumour cells, whereas in one case, ameloblastin was negative in the tumour cells but positive in the stromal fibrous tissue collar. Conclusion Expression of ameloblastin in human tooth germ is related to differentiation and mineralization, and it correlates with the state of differentiation of the tumour cells in ameloblastoma.

Abdominal distention occurred at an incidence of 1% (15 from 1500 fish) in the population of 1-year-old bester (Huso huso × Acipenser ruthenus). Computed tomography (CT) images and radiographs showed a soft tissue mass compressed the posterior part of the swim bladder. Ultrasonography showed that the masses had different patterns. Internal examination revealed the abdominal cavities to be filled with large masses which appeared to encompass most of the visceral organs, including the swim bladder. The masses originated from the posterior kidney. Histologically, the masses were composed of mixtures of embryonal epithelial (tubules and glomeruli), blastema and mesenchymal tissues. The tubules showed cystic, papillary and tubular patterns. Tubules and glomeruloid structures were surrounded by proliferating blastema cells. The primitive mesenchyme was composed of loose streams and whorls of spindle to stellate cells with elongate nuclei. Histological findings in the skeletal muscles, hypoderm and spleen confirmed the metastatic tumour from the kidney in two cases. Immunohistochemically, neoplastic cells of the tubules and glomeruloid structures were positive for cytokeratin AE1/AE3. Sections stained with Masson's trichrome showed blue staining of the stroma. The histopathologic findings were consistent with nephroblastoma.

Objective The epiglottis may contribute to upper airway obstruction in approximately 10% of patients with obstructive sleep apnea. Clinical experience indicates that older patients may be more likely to have epiglottis-related obstruction. This study was designed to examine tissue characteristics of the hyoepiglottic ligament as a possible factor in epiglottis-related obstruction based on previous research suggesting that older adults have fewer collagen, elastin, and muscle fibers in the hyoepiglottic ligament. Methods This is a cross-sectional study of 25 human cadaver hyoepiglottic ligaments. Specimens were stained using Masson's trichrome and Picrosirius red for collagen fibers and with Verhoeff-Van Gieson for elastin fibers. Percentage of collagen and elastin fiber staining for each specimen was calculated and averaged over three regions of each ligament section. Regression analysis was used to determine the association between age, smoking history, and collagen and elastin composition of the hyoepiglottic ligament. Results The average age of the specimens was 68.4 ± 15.1 years (range 30-90 years). Increasing age was associated with a lower percentage of collagen and elastin fibers. When accounting for tobacco use, each 1-year increase in age was associated with a 0.53% decrease in Masson's trichrome staining (P = 0.004), a 0.35% decrease in Picrosirius red staining (P = 0.023), and a 0.33% decrease in Verhoeff-Van Gieson staining (P = 0.008). Conclusion Increasing age is associated with decreases in the collagen and elastin content of the hyoepiglottic ligament. Level of evidence NA. Laryngoscope, 128:1245-1248, 2018.

Objective Stress urinary incontinence (SUI) is a common disease condition in elderly women, suggesting that its etiology may be linked to aging. To investigate the hypothesis that urethral dysfunction and histopathological changes are possible contributors to SUI in elderly women, several parameters of urethral function, as well as histological parameters, were compared between young and aged rats. Methods Virgin female rats were examined at 3 different ages, namely 3, 12, and 24 months, corresponding to young, middle-aged, and aged rats, respectively. Urethral function was assessed by measuring the leak point pressure (LPP), pudendal nerve stimulation (PNS)-induced elevation in urethral pressure, and phenylephrine-induced increase in urethral perfusion pressure (UPP). Histopathological assessments were performed following hematoxylin and eosin (HE), Masson's trichrome, and immunofluorescence staining of urethral tissue. Results LPP of aged rats was significantly reduced compared to that of both young and middle-aged rats. PNS-induced elevation in urethral pressure in aged rats was also significantly lower than that in young rats. In contrast, there were no significant differences in the phenylephrine-induced increase in UPP between young and aged rats. Connective tissue area in the external urethral sphincter (EUS) layer was increased in aged rats, whereas the smooth muscle layer was histologically similar to that in young rats. The number of EUS fibers was significantly reduced in aged rats, whereas the cross-sectional area of EUS fibers increased from differed compared with young rats. Conclusion We have demonstrated age-related changes in EUS function and morphology in the rat urethra, which are considered to be etiological risk factors for SUI in humans.

Objectives/Hypothesis To characterize the histologic and biochemical properties of auricular and septal cartilage and analyze age-related changes in middle-aged to older adults. Study Design Cross-sectional study of auricular and septal cartilage from 33 fresh cadavers. Methods Auricular and septal cartilage specimens were stained using Safranin O for glycosaminoglycans, Verhoeff's stain for elastin, and Masson's trichrome for collagen. Percentage of tissue stained, cell density and size were quantified. Relationships between donor characteristics and histologic properties were evaluated using mixed model analyses. Results The average donor age was 75 years (standard deviation = 11 years; range, 55–93 years). In auricular cartilage, each 1-year increase in age was associated with a 0.97% decrease in glycosaminoglycans (P < .001) and a 0.98% decrease in elastin (P < .001). In septal cartilage, glycosaminoglycans decreased 2.4% per year (P < .001). Age did not affect collagen content significantly in auricular (P = .417) or septal cartilage (P = .284). Cell density and cell size declined with age in auricular (both P < .001) and septal cartilage (P = .044, P = .032, respectively). Compared to septal cartilage in patients of all ages, auricular cartilage had more glycosaminoglycans, less collagen, higher cell density, and smaller cells. Conclusions In auricular and septal cartilage, glycosaminoglycans, elastin, cell density, and cell size decrease significantly with age in patients over 55 years of age. Glycosaminoglycan content declines faster with age in septal cartilage than auricular cartilage. These age-related changes may affect biomechanical properties and tissue viability, and thereby have implications for graft choice in functional, aesthetic, and reconstructive nasal surgery. Level of Evidence NA. Laryngoscope, 127:E399–E407, 2017

This study investigates the extent of cell seeding as well as lumen patency in acellular Internal mammary artery (IMA) scaffolds that have been re-cellularized by omentum as a natural bioreactor. Sixteen Virgin female Wistar rats were selected for implantation of acellular scaffold in omentum. Following laparotomy omentum was retracted to the outside of the abdomen and the more vascularized portion of it was selected for the experiment. The scaffold was wrapped by omentum and placed between two layers of rectus muscles that have been previously dissected. Samples were taken from scaffolds at 2 and 3 months for histopathological evaluation. All the grafts were explanted at 2 and 3 months and the lumens were completely patent compared to the native scaffold. The histology of implanted IMA after 2 and 3 months showed progressive recellularization especially by smooth muscle cells over the media layer. CD 34 staining was positive adjacent to the grafts which showed well angiogenesis. Trichrome and Movat's Pentachrome staining showed normal collagen formation in the inner media layer. Promising results obtained from the introduced protocol for in vivo recellularization, including patent lumen and proper cell seeding, encourages application of the mentioned technique for experimental vascular graft application. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2685-2693, 2017.

Aim To evaluate the influence of the addition of microparticulate (micro) and nanoparticulate (nano) zirconium oxide (ZrO2) and niobium pentoxide (Nb2O5) to a calcium silicate-based cement (CS) on the subcutaneous healing process in rats compared with MTA Angelus™. Methodology In each rat, two polyethylene tubes filled with the following materials: (i) MTA; (ii) CS + ZrO2micro; (iii) CS + ZrO2nano; (iv) CS + Nb2O5micro or (v) CS + Nb2O5nano were implanted subcutaneously; empty polyethylene tubes were used in the Control group. After 7, 15, 30 and 60 days, the specimens (n = 5 per group in each period) were fixed and embedded in paraffin. Masson's trichrome sections were used to obtain the volume density of the inflammatory cells (VvIC) and fibroblasts (VvFb). The sections were also stained with Picrosirius-red to calculate the birefringent collagen content. Fibroblast growth factor-1 (FGF-1) was detected by immunohistochemistry, and the number of immunolabelled cells was obtained. The data were subjected to two-way anova followed by Tukey's test (P ≤ 0.05). Results At all periods, the VvIC was significantly lower (P

Summary Measurement of collagen bundle orientation in histopathological samples is a widely used and useful technique in many research and clinical scenarios. Fourier analysis is the preferred method for performing this measurement, but the most appropriate staining and microscopy technique remains unclear. Some authors advocate the use of Haematoxylin-Eosin (H&E) and confocal microscopy, but there are no studies comparing this technique with other classical collagen stainings. In our study, 46 human skin samples were collected, processed for histological analysis and stained with Masson's trichrome, Picrosirius red and H&E. Five microphotographs of the reticular dermis were taken with a 200× magnification with light microscopy, polarized microscopy and confocal microscopy, respectively. Two independent observers measured collagen bundle orientation with semiautomated Fourier analysis with the Image-Pro Plus 7.0 software and three independent observers performed a semiquantitative evaluation of the same parameter. The average orientation for each case was calculated with the values of the five pictures. We analyzed the interrater reliability, the consistency between Fourier analysis and average semiquantitative evaluation and the consistency between measurements in Masson's trichrome, Picrosirius red and H&E-confocal. Statistical analysis for reliability and agreement was performed with the SPSS 22.0 software and consisted of intraclass correlation coefficient (ICC), Bland-Altman plots and limits of agreement and coefficient of variation. Interrater reliability was almost perfect (ICC > 0.8) with all three histological and microscopy techniques and always superior in Fourier analysis than in average semiquantitative evaluation. Measurements were consistent between Fourier analysis by one observer and average semiquantitative evaluation by three observers, with an almost perfect agreement with Masson's trichrome and Picrosirius red techniques (ICC > 0.8) and a strong agreement with H&E-confocal (0.7 0.8), better with Fourier analysis than with semiquantitative evaluation (single and average). These results in nonpathological skin samples were also confirmed in a preliminary analysis in eight scleroderma skin samples. Our results show that Masson's trichrome and Picrosirius red are consistent with H&E-confocal for measuring collagen bundle orientation in histological samples and could thus be used indistinctly for this purpose. Fourier analysis is superior to average semiquantitative evaluation and should keep being used as the preferred method.

Chronic kidney disease (CKD) is a relevant disease in feline clinic. The tubulointerstitial damage, with collagen deposition and fibrosis, is an important result of this process. The aim of this study was to quantify and correlate the deposition of collagen and severity of interstitial fibrosis (IF) in the kidney from cats in different stages of CKD. Kidney fragments from 10 adult cats with CKD were analyzed and stained by Masson's trichrome (MT) and Picrosirius red (PSR) for circular polarized microscopy. Random quantitative analysis was performed on MT sections to classify the degree of IF, per field area, with and without circular polarization. Statistics correlations were performed by Spearman's (ρ; p

The hard palate and mid‐palatal suture are highly important for orthodontic treatment. In cases of transverse maxillary deficiency, palatal expansion is the treatment of choice. As nowadays a growing number of adult patients receive orthodontic treatment, an understanding of suture development throughout life is important to derive tailored orthodontic treatment techniques for each age group. Histological, histochemical and immunohistochemical stains (haematoxylin & eosin, Azan, Movat pentachrome, Masson–Goldner trichrome, Sirius Red, CD 31, osteopontin and TRAP) and histomorphometric analyses were re‐established to detect the structural conditions of the mid‐palatal suture in human cadavers of three different age groups (20–39, 40–59 and 60–80 years). The mid‐palatal suture of the selected age groups (total of n = 12; n = 4 in every group m = f) exhibited marked differences in sutural morphology and metabolism. A wide, interdigitated and well‐vascularized suture was observed in younger specimens compared with straighter and smaller sutures with fewer vessels and lower bone density in the 60–80 year group. The fibre composition within the sutural gap differed between the three age groups. Delicate fibres were found in the 20–39 year group, and a tightly interwoven 3D fibre‐network was observed in the 40–59 year group. Atrophy primarily characterized the fibres in the 60–80 year group. This evidence demonstrates differences between the evaluated groups. These results suggest that the staining methods used are suitable for the description and evaluation of the morphology and metabolism of mid‐palatal sutures. Further investigation is necessary to provide an in‐depth description of sutural maturation over a lifetime.

The selection of an appropriate demineralizing solution in pathology laboratories depends on several factors such as the preservation of cellularity, urgency of diagnostic and financial costs. The aim of this study was to test different decalcification bone procedures in order to establish the best value of these in formalin-fixed and paraffin-embedded samples. Femurs were removed from 13 adult male Wistar rats to obtain 130 bone disks randomly divided into five groups that were demineralized in different concentrations of nitric acid (Group I); formic acid (Group II); acetic acid (Group III); EDTA, pH7.4 (Group IV) and Morses solution (Group V). Serial, 3-μm-thick sections were obtained and stained with hematoxylin-eosin to calculate the percentage of osteocyte-occupied lacunae. The sections were also stained with Masson's trichrome in conjunction with picrosirius red under polarized light followed by a semi-quantitative analysis to verify the adjacent muscle-to-bone integrity and preservation of collagen fibres. The highest percentage of osteocyte-occupied lacunae was found with 10% acetic acid solution (95.64 ± 0.95%) and Group I (nitric acid) demanded the shorter time (0.8-5.7days). Of all solutions, 5% nitric acid incurred the lowest cost to achieve complete demineralization compared with other solutions (p

Aim To evaluate pulpal tissue response after dental bleaching in normal and alloxan-induced diabetic rats. Methodology Twenty-eight rats were divided into two groups of normoglycaemic and diabetic rats (n = 14). Diabetes mellitus (DM) was induced with alloxan. After DM confirmation, all rats were anaesthetized and dental bleaching was performed with 35% hydrogen peroxide (H2O2) on the right maxillary molars for 30 min. Left molars were used as controls. Bleaching resulted in four hemimaxillae groups: normoglycaemic (N), N-bleached (NBle), diabetic (D) and D-bleached (DBle). After 2 or 30 days, the animals were euthanized and the hemimaxillae were removed, processed for histopathological analysis and stained with haematoxylin–eosin (HE), Masson's trichrome (MT) and picrosirius red (PSR). Results obtained within animals (normoglycaemic or diabetic rats) were submitted to Wilcoxon or paired t-tests, and between animal (normoglycaemic and diabetic rats), to Mann–Whitney test or t-tests. Results At 2 days, the NBle group had a mild inflammatory infiltration in the pulpal tissue, whilst the DBle had severe inflammation or necrosis (P

Aged and adult populations have differences in the structural, biological, and healing properties of skin. Comparative studies of healing under the influence of retinoids in both these populations are very important and, to the best of our knowledge, have not been performed to date. The purpose of this study was to compare the activities of topical tretinoin in aged and adult animal models of wound healing by secondary intention. Male aged rats (24 months old, n = 7) and adult rats (6 months old, n = 8) were used. The rats were assigned to the following groups according to the dates on which wound samples were excised (day 14 or 21 after model creation): treated group, control group, and naive group. Topical application of tretinoin cream was used only on the proximal wound and was applied daily for 7 days. Wound healing areas were measured using metal calipers, and morphological analysis was performed. Slides were stained with Hematoxylin and Eosin, Masson's trichrome, and periodic acid-Schiff stains. Statistical analysis adopted a 5% coefficient for rejection of the null hypothesis. Although aged animals showed skin repair, complete reepithelialization was found on day 21 in some animals of both groups (treated and control). In aged rats, the wound area was significantly smaller in treated wounds than in untreated wounds, resulting in a larger scar area compared with the adult group. When treated wounds were compared, no differences were found between the wound areas in adult and aged rats. As expected, the collagen concentration was higher in normal skin from adult rats than in normal skin from aged animals, but there was no difference when aged skin was treated with tretinoin. These results indicate that tretinoin increases collagen synthesis in aged skin and returns the healing process to a normal state of skin healing.

Dawson’s clearing technique is widely used for the study of skeletal embryology, particularly at examining endochondral ossification in human developmental stages (Fig 1). While it has proven to be a useful tool for visualizing bony structures through cleared flesh, little is known about the histological changes suffered by both bony and muscular tissue. Our aim is to contrast cleared and formaldehyde fixated musculoskeletal tissue in fetal lower limbs at cellular and tissular levels. Two previously hard-fixated fetuses’ lower limbs were treated with 1) Dawson’s clearing method based on Alizarin Red staining and immersion in aqueous KOH at 1% weight/volume, until complete translucency was obtained and 2) immersion in 10% volume/volume formaldehyde. For both, transversal cuts of tarsus, metatarsus and phalanges were obtained and stained in Hematoxylin and Eosin (H&E), Periodic acid-Schiff (PAS) stain and Masson’s Trichrome (MT) stain. Samples were observed under a light microscope at 4×, 10× and 20×. Clear...

Vascular and cardiac reconstruction involves the use of biological patches to treat trauma and defects. An in vivo study was performed to determine the remodeling and biologic effects of novel nanostructured vascular patches with and without gold nanoparticles. Porcine vascular tissue was decellularized and conjugated with gold nanoparticles to evaluate if integration would occur while avoiding rupture and stenosis. Swine underwent a bilateral patch angioplasty of the carotid arteries with experimental patches on the right and control patches of bovine pericardium on the left. Animals were sacrificed after surgery and at 3 and 9 weeks. Ultrasound was performed during surgery, every 3 weeks, and before euthanasia. Endothelial regeneration was examined using Evans Blue dye and histology using Trichrome and H&E. There was a 100% success rate of implantation with 0% mortality. All patches were patent on ultrasound. At 3 weeks, experimental patches had regenerating endothelial cell growth and normal healing responses. At 9 weeks, the experimental patches demonstrated excellent integration. Histology demonstrated cellular in-growth into the experimental patches and no major immune reactions. This is one of the first studies to demonstrate the feasibility of nanomaterial-tissue patches for vascular and cardiac reconstruction.

Picasso, M.B.J., Mario, R., Barbeito, C.G. 2015. The skin structure of greater rhea (Rheidae, Palaeognathae). —Acta Zoologica (Stockholm) 00 :0 00–000. The aim of this study was to describe the histological structure of the skin of greater rhea (Rhea americana), a ratite bird native to South America. Skin samples were taken from three regions of the trunk (alar, dorsal and pelvic) in 14 specimens which ages ranged from 7 days to adulthood. Serial sections were obtained and subjected to different staining procedures (haematoxylin and eosin, orcein, Masson’s trichrome and Gomori), and a morphometric analysis was carried out on stained slides. In general ,b oth epidermis and dermis showed increased thickness of its layers with age. Some differences between regions can be detected both in epidermis and in dermis; for example in adults and 7-dayold birds, the stratum corneum of the alar region was thicker than of the dorsal region. In general, the skin of greater rhea was similar to that described in ratites and other birds (a thin epidermis compared to dermis, dermis with scarce elastic fibres, a slender and vascularized stratum superficiale, collagen fibres arranged in three directions). The scarcity of elastic fibres and the general cross-weaved arrangement of the collagen fibres in the dermis of the adult greater rhea provide strength and flexibility to the dermis, two important features in leather industry.

Background and Aim Traditional pathological scoring systems for liver fibrosis progression are predominantly based on the description of architectural changes with no consideration of the amount of collagen fiber deposition. Our purpose was to explore a true histological standard in accordance with the liver stiffness measured by point shear wave elastography (PSWE) in patients with chronic hepatitis B. Methods A total of 78 patients with liver neoplasms underwent liver stiffness measurements with PSWE as well as biochemical investigations within 3 days before partial hepatectomy. One tissue section of the liver specimens was stained with HE trichrome and evaluated traditionally with the Scheuer scoring system. The other tissue section was stained with picroSirius red and was evaluated according to the semiquantitative Chevallier et al. scoring system. In addition, this second tissue section was evaluated for the collagen proportionate area (CPA) with computer-assisted digital image analysis. The reproducibility of PSWE technology was explored through the intra-class correlation coefficient of a reliability analysis. Results The PSWE technology revealed good reproducibility in liver stiffness measurements, and the PSWE values increased with the pathological severity of liver fibrosis on both the Scheuer scoring system and the semiquantitative Chevallier et al. scoring system. PSWE values exhibited more reasonable relationships with CPA (r = 0.628, P = 0.00

In rodents, vaginal distention after delivery or experimental manipulation affects innervations as well as the amount of striated/smooth musculature or collagen in both the urethra and vagina. These changes are associated with modifications in excretory and reproductive processes. Although successive and consecutive vaginal deliveries (multiparity) affect the contractile and functional properties of the female lower urogenital tract (LUT), its impact on LUT morphometry, including persistency, has been barely studied. The caudal urethra (CU) and cranial (V1) and caudal (V2) pelvic vaginal regions were excised from young and adult nulliparous (YN and AN, respectively) and multiparous (YM and AM, respectively) rabbits. Tissues were histologically processed and stained with Masson's trichrome. The thickness of the tissue layers and areas covered by tissue components were measured and compared using two-way ANOVA followed by Student-Newmann-Keuls tests to determine statistical differences (P ≤ 0.05). Compared to YN, YM, and AN tissues showed a reduction in the thickness of the epithelium, as well as in areas covered by striated musculature, collagen, and blood vessels of the LUT. In comparison with YM, only some morphometric changes were recovered in the AM group. Our study shows that multiparity and age can be associated with epithelial and muscular atrophy of urethral and vaginal walls. The morphometry of the LUT between young and adult female rabbits varies with multiparity. These findings may help to better understand the effects of multiparity on young and adult females and its correlation with the development of pelvic dysfunctions. Anat Rec, 297:1963–1970, 2014. © 2014 Wiley Periodicals, Inc.

Objective To investigate the feasibility of a new approach for cystoplasty using autologous smooth muscle cell (SMC) sheet and scaffold-less bladder tissue engineering with the main focus on histological outcomes in a rabbit model. Materials and Methods In all, 24 rabbits were randomly divided into two groups. In the experimental group, SMCs were obtained from the bladder muscular layer, labelled with PKH-26, and seeded on temperature-responsive culture dishes. Contiguous cell sheets were noninvasively harvested by reducing the temperature and triple-layer cell-dense tissues were constructed. After partial detrusorectomy, the engineered tissue was transplanted onto the urothelial diverticulum. The control group underwent partial detrusorectomy followed by peritoneal fat coverage. At 2, 4, and 12 weeks the rabbits were humanely killed and haematoxylin and eosin, Masson's trichrome, cluster of differentiation 34 (CD34), CD31, CD3, CD68, α-smooth muscle actin (α-SMA), picrosirius red, and pentachrome staining were used to evaluate bladder reconstruction. Results At 2 weeks after SMC-sheet grafting, PKH-26 labelled SMCs were evident in the muscular layer. At 4 weeks, 79.1% of the cells in the muscular layer were PKH-positive cells. The portion of the muscular layer increased in the experimental group during the follow-up and was similar to normal bladder tissue after 12 weeks. α-SMA staining showed well organised muscle at 4 and 12 weeks. CD34+ endothelial progenitor cells and CD31+ microvessels increased continuously and peaked 4 and 12 weeks after grafting, respectively. Conclusion In the present study, we show that autologous SMC-sheet grafting has the potential for reliable bladder reconstruction and is technically feasible with a favourable evolution over the 12 weeks following implantation. Our findings could pave the way toward future bladder tissue engineering using the SMC-sheet technique.

Aims Aortic dissection (AD) is characterized by changes in the extracellular matrix, including fibrosis with collagen production. P54(nrb) /NonO is known to be involved in collagen formation. In this study, we examined whether AD is associated with abnormal P54(nrb) /NonO expression. Methods and results Aortic specimens and serum were obtained from 10 patients with AD and 10 controls. In-vitro cultures of vascular smooth muscle cells (VSMCs) and adventitial fibroblasts (AFs) were obtained from organ donors. P54(nrb) /NonO protein and mRNA levels were determined by Western blot, immunohistochemistry and quantitative real-time reverse transcription-polymerase chain reaction (quantitative real-time RT-PCR). To evaluate collagen expression, we stained tissue sections with Masson's trichrome. Serum concentration of TNF-α was determined by enzyme-linked immunosorbent assay (ELISA). Aortic P54(nrb) /NonO protein and mRNA were decreased in AD patients, compared with controls. Decreased P54(nrb) /NonO mRNA correlated significantly with increased collagen deposition and fibrosis in AD aortas. In VSMCs and AFs from normal human aortas, P54(nrb) /NonO was expressed strongly and localized to the nucleus. Conclusions Patients with AD exhibited significantly decreased expression of P54(nrb) /NonO. The significant correlation between P54(nrb) /NonO and collagen may point to novel thinking about collagen metabolism research in AD aorta.

This study investigated and compared the ultrastructural and chemical properties of representative biomaterials for ocular surface regeneration: a human amniotic membrane (AM) in a basal plate, a human AM in reflected chorion, a preserved AM, and a human corneo-scleral tissue. Assessments of the morphological differences in the extracellular matrices were evaluated by hematoxylin-eosin, Masson's trichrome (for total collagen), and picrosirius-red (for newly synthesized collagen) staining. Assessments of the changes in the molecular structures and chemical compositions of the biomaterials for ocular surface regeneration were evaluated by Raman spectroscopy. A placental AM (52 %) was a dense and thick collagenous structure compared to a reflected AM (23 %). The spectroscopy did not obtain any structural information for a preserved AM. The cornea group (100 %, control) and sclera group (104 %) showed the collagen lamellae and interfibrillar spacing, and a slight inflammatory reaction with more fibrous and granulomatous tissues. There was a formation of newly synthesized collagen in a placental AM, while there were few collagen components in a reflected AM. Human AM tissues showed consistent Raman spectra and the characteristic collagen bands, similar to the corneal and scleral tissues. Therefore, these findings suggest that human placental AM and reflected AM are structurally suitable for scleral and corneal surface regeneration, respectively, while human placental or preserved AM and reflected AM are molecularly and chemically suitable for corneal and scleral surface regeneration, respectively.

The demand for scaffolds comprised of natural materials such as collagen has increased in recent years. However, many scaffolds rely on chemical or physical modifications in order to comply with the necessary requirements for biomedical engineering. We evaluated the in vivo biocompatibility and biodegradation of a novel, thin, mechanically stable, and chemically non-crosslinked collagen cell carrier (CCC). CCC was implanted subcutaneously into 25 adult Lewis rats and biopsies were taken on days 7, 14, 21, 42, and 84 after surgery. For histological analysis, paraffin sections of implanted skin were immunolabeled for CD68 and stained by hematoxylin-eosin and Masson-Goldner's trichrome method. Macroscopic analysis of skin surface during wound healing process showed a normal physiological reaction. Biodegradation of CCC was completed 42 days after subcutaneous implantation. Histological evaluation revealed no evidence of encapsulation, scar formation, or long-term vascularization and inflammation. The collagen type I based biomaterial demonstrated a high in vivo biocompatibility, low irritability, complete resorption, and replacement by autologous tissue. The in vivo biocompatibility and degradation behavior encourage for further evaluation of CCC in surgical applications and regenerative medicine.

The purpose of this study was to investigate the morphological and histological change of vocal folds (VFs) after steroid injection in a rabbit model.Prospective animal study.Tertiary academic medical center.Twenty-four New Zealand white rabbits were used in this study. We randomly classified rabbits into the 3 groups and triamcinolone acetonide suspension was injected to the right VF with different concentrations. Left VF was injected with the same volume of phosphate-buffered saline as control. Endoscopic evaluation was performed to measure morphological changes. The larynges were collected for histological analysis, and the VFs were stained with hematoxylin-eosin for assessing inflammatory response, glandular atrophy, and muscular atrophy and with Masson's trichrome for assessing collagen deposition.In morphological assessment, there were no differences in VF mass reduction, mucosal atrophy, and granulation formation between both VFs. Histological assessments showed no significant difference in inflammatory response, glandular atrophy, and collagen deposition between both VFs. However, there was a difference in muscular atrophy and epithelial layer thinning in steroid injected right VFs. Muscular atrophy had been completely recovered over time, but mild epithelial thinning was continued until 12 weeks. The longer exposure time and larger dose did not increase the intensity of muscular atrophy or epithelial thinning.We demonstrated that the VF steroid injection resulted in no significant changes in morphology and histology of rabbit VF. However, steroid injection may induce several VF histological changes and these results are needed to be considered when treating humans.

Collagen-based dressings are of great interest in wound care. However, evidence supporting their mechanism of action is scanty. This work provides first results from a preclinical swine model of excisional wounds, elucidating the mechanism of action of a modified collagen gel (MCG) dressing. Following wounding, wound-edge tissue was collected at specific time intervals (3, 7, 14, and 21 days postwounding). On day 7, histological analysis showed significant increase in the length of rete ridges, suggesting improved biomechanical properties of the healing wound tissue. Rapid and transient mounting of inflammation is necessary for efficient healing. MCG significantly accelerated neutrophil and macrophage recruitment to the wound site on day 3 and day 7 with successful resolution of inflammation on day 21. MCG induced monocyte chemotactic protein-1 expression in neutrophil-like human promyelocytic leukemia-60 cells in vitro. In vivo, MCG-treated wound tissue displayed elevated vascular endothelial growth factor expression. Consistently, MCG-treated wounds displayed significantly higher abundance of endothelial cells with increased blood flow to the wound area indicating improved vascularization. This observation was explained by the finding that MCG enhanced proliferation of wound-site endothelial cells. In MCG-treated wound tissue, Masson's trichrome and picrosirius red staining showed higher abundance of collagen and increased collagen type I:III ratio. This work presents first evidence from a preclinical setting explaining how a collagen-based dressing may improve wound closure by targeting multiple key mechanisms. The current findings warrant additional studies to determine whether the responses to the MCG are different from other collagen-based products used in clinical setting.

Summary This study investigated the inflammatory effect of cryotherapy application on collagen matrix network in human infant sclera. Donor scleral tissues taken from three infant patients divided into five groups: control group, sham-treated group, and three cryotreated groups. In the cryotherapy groups, the sclera was treated for 5 s, 10 s, and 20 s with −80°C freezing by a cryosurgical system. The cryotreated reactions were examined using double histological analysis with hematoxylin-eosin and Masson's trichrome, and atomic force microscopy analysis to quantify the diameter and D-banding of collagen fibrils. The infant scleral tissues treated with cryotherapy showed a significantly increased collagen density associated with inflammatory response (p < 0.05), increased fibril diameter (p < 0.005) compared to the scleral tissues in the control group. The results directly suggest that the cryotherapy affects the morphology of scleral collagen. SCANNING 35:302-307, 2013. © 2012 Wiley Periodicals, Inc.

OBJECTIVES To histologically analyze the early angiogenesis-osteogenesis interplay in post-extraction sockets augmented with magnesium-enriched hydroxyapatite (Mg-enriched HA). MATERIAL AND METHODS Ten post-extraction sites underwent post-extraction ridge preservation procedure. According to randomization, sites were divided into two balanced groups and bone specimens were collected 2 or 4 months after surgery. Sections were stained with hematoxylin/eosin, Masson-Goldner trichrome, and tartrate-resistant acid phosphatase (TRAP), respectively. Furthermore, indirect immunohistochemistry was performed using alkaline phosphatase, CD34 and caveolin-1 antibodies. Mean values and standard deviations were calculated for each outcome variable. Data were then compared using one-way ANOVA test. P

Kim Y-T, Park J-C, Choi S-H, Cho K-S, Im G-I, Kim B-S, Kim C-S. The dynamic healing profile of human periodontal ligament stem cells: histological and immunohistochemical analysis using an ectopic transplantation model. J Periodont Res 2012; 47: 514–524. © 2012 John Wiley & Sons A/S Background and Objective: Human periodontal ligament stem cells (hPDLSCs) have been reported to play the pivotal role in periodontal regeneration. However, the dynamic cellular healing process initiated by hPDLSCs still remains to be elucidated. In the present study, the sequence of regeneration by hPDLSCs was assessed using histological and immunohistochemical observation in an ectopic transplantation model, which is a well-standardized assessment tool that excludes the innate healing factors from the animals. Material and Methods: Human periodontal ligament stem cells that were isolated and characterized from teeth (n = 12) extracted for the purpose of orthodontic treatment were transplanted with carriers into ectopic subcutaneous pouches in immunocompromised mice (n = 20). Animals were killed after several different healing periods: 3 d (n = 4), 1 (n = 4), 2 (n = 4), 4 (n = 4) and 8 wk (n = 4). Histological analysis for regenerated tissues formed by hPDLSCs was conducted using hematoxylin and eosin, Masson’s trichrome and picrosirius red staining. In addition, immunohistochemical staining was performed to observe the sequential expression of osteogenic/cementogenic and periodontal ligament tissue-specific markers associated with periodontal regeneration. Results: The whole healing process by transplanted hPDLSCs could be broadly divided into four distinctive phases. In the first phase, proliferated hPDLSCs migrated evenly all over the carrier, and collagenous tissues appeared in the form of amorphous collagen matrices. In the second phase, collagen fibers were well arranged among the carriers, and cementoid-like tissues were observed. In the third phase, the formation of mature collagen fibers, resembling Sharpey’s fibers, was associated with active mineralization of cementum-like tissues, and in the fourth phase, the maturation of cementum-like tissues was observed on carrier surfaces. Various osteogenic/cementogenic markers related to the regeneration processes were expressed in a well-orchestrated time order. Interestingly, well-organized cementum-like and periodontal ligament fiber-like tissues and cells with early and late osteogenic/cementogenic markers were frequently observed in the secluded area of carrier surfaces. We termed this area the cell-rich zone. Conclusion: The results from this study clearly demonstrated the sequential histological changes during periodontal tissue regeneration by hPDLSCs. Understanding of this process would potentially enable us to develop better cell-based treatment techniques.

The distribution and organisation of the intralingual lymphoid tissue was studied in sheep, goat and cattle. For each species, the tongues of two animals were harvested and divided in sample blocks extending over the total surface of the tongue. With 2.5 mm intervals, ten serial histological sections were made for conventional histological staining (haematoxylin-eosin, Van Gieson, Masson's trichrome) and immunohistochemical staining of lymphoid cells (anti-CD3, anti-CD21, anti-CD45). Lymphocytes were scattered in the subepithelial propria-submucosa and in the connective tissue cores of the lingual papillae. The connective tissue cores of fungiform papillae, including those located on the lingual apex, and vallate papillae showed relatively more lymphocytes than the propria-submucosa. Lymphoid cell aggregations were even more abundant beneath the grooves surrounding the vallate papillae in small ruminants. In cattle, a well-organised lingual tonsil was additionally found at the root of the tongue. CD3-positive lymphocytes were observed in all species examined. CD21-positive lymphocytes were numerous in the lymphoid nodules of the bovine lingual tonsil but very scarce in the ovine and caprine tongues. Therefore, the lymphoid cell aggregations in the tongues of small ruminants should not be referred to by the term 'lingual tonsil'.

The evaluation of clinical specimens for ova and parasites in the clinical laboratory can involve the use of direct macroscopic examination of the specimen and microscopic examination of fresh and preserved specimens, as well as culture for some parasitic organisms. These examinations necessitate the use of a variety of stains, reagents, and culture media, the most common of which are discussed in this chapter. Formalin has always been used in parasitology as an allpurpose preservative and in concentration procedures. The accompanying compound with the polyvinyl alcohol (PVA), specifically, mercuric chloride, zinc sulfate, or cupric sulfate, acts as the preservative and allows fixation of protozoan cysts and trophozoites for use with trichrome or iron hematoxylin stains for permanent smears. Specimens treated with Zn-PVA may also be stained with trichrome or iron hematoxylin stains for permanent smears. Specimens treated with Cu-PVA may also be stained with trichrome or iron hematoxylin stains for permanent smears. Examination of blood films for parasites includes the use of two common stains, the Giemsa stain and Wright's stain, both derivatives of the original Romanowsky stain. These stains are very similar, differing primarily in that no fixative is included in the Giemsa stain and the blood film must be fixed with absolute methanol prior to staining. In a basic staining procedure, thick films must be laked in distilled water or treated with saponin prior to performance of the staining procedure.

da Silva GF, Guerreiro-Tanomaru JM, Sasso-Cerri E, Tanomaru-Filho M, Cerri PS. Histological and histomorphometrical evaluation of furcation perforations filled with MTA, CPM and ZOE. International Endodontic Journal, 44, 100–110, 2011. Abstract Aim To evaluate the biological response of the periodontium adjacent to furcation perforations in rat molars filled with Endo-CPM-Sealer (CPM), MTA-Angelus (MTA) or zinc oxide–eugenol cement (ZOE). Methodology The pulp chamber floors of maxillary right first molar teeth were perforated and sealed with CPM, mineral trioxide aggregate (MTA) or ZOE; the left first molars, without any treatment, were used as controls (CG). After 7, 15, 30 and 60 days, fragments of maxilla were fixed, decalcified and embedded in paraffin. Sections were stained with H&E, Masson’s trichrome and submitted to tartrate-resistant acid phosphatase (TRAP) reaction, used as an osteoclast marker. The width of the periodontal space, the numerical density of inflammatory cells and the number of TRAP-positive osteoclasts in the bone surface were measured, and statistical analyses were performed using analysis of variance and Tukey test (P ≤ 0.05). Results In all experimental groups, the greatest number of inflammatory cells was observed at 7 days, especially in the ZOE group. In this group, the intense inflammatory process was related to a significant increase (P ≤ 0.05) in the number of osteoclasts and, thereby, in an increase in the width of the periodontal space. At 60 days, no significant differences in osteoclast numbers amongst CPM, MTA and CG groups occurred; the periodontal space was also significantly reduced in the experimental groups in comparison with the initial periods. However, in the ZOE group, the periodontal space was significantly larger (P ≤ 0.05) in comparison with MTA-based materials. Conclusions The periodontium adjacent to perforations filled with MTA and CPM exhibited clear evidence of re-establishment and thus better biocompatibility than ZOE.

Objective To determine the influence of allergic rhinitis (AR) on the expression of matrix metalloproteinase (MMP)-9, MMP-2, and tissue inhibitor-1 of metalloproteinase (TIMP-1) in nasal polyposis. Study Design A case-control study. Setting A tertiary referral center. Subjects and Methods The expression of MMP-9, MMP-2, and TIMP-1 was investigated in the nasal polyp tissue (NP) and maxillary sinus mucosa (MM) samples from 20 AR patients and 20 nonallergic patients undergoing endoscopic sinus surgery. Trichrome, periodic acid–Schiff, and hematoxylin-eosin staining were also performed and those expression levels were compared. Results Infiltration of eosinophils was shown more intensely in NP rather than in MM, especially in the presence of AR. In the NP of AR patients, increased expression of MMP-9, MMP-2, and TIMP-1 was observed more prominently than in that of the control group. In case of MM, however, there was no significant difference between AR patients and the control group. Conclusion The presence of AR may enhance the expression of MMP-9, MMP-2, and TIMP-1 associated with airway remodeling in nasal polyposis.

We performed an animal experiment with an emphasis on time-related histopathologic changes to evaluate the clinical feasibility of immunologically nontreated xenogenic vascular grafts. Bilateral porcine carotid arteries were harvested, and then, after short-term freezing at -70 degrees C, interposed into goats' carotid arteries. An antiplatelet agent was administered orally for 3 months postoperatively. The goats were randomly assigned to five periods of observation (1 week, and 1, 3, 6, and 12 months after implantation); two animals were observed at each of these times. Doppler ultrasonography was performed periodically during the observation period. At predetermined times, grafts were explanted and examined using hematoxylin and eosin, and Masson's trichrome stains. Immunohistochemical evaluations were conducted with T-lymphocyte indicator and von Willebrand factor. Two goats died prematurely, one from respiratory problems related to anesthesia and the other from pneumonia. A total of 16 grafts from the remaining eight animals were evaluated. Grafts were all patent except one at 3 months after transplantation. Histologically, xenogenic arterial grafts showed early endothelial cell loss at 1 week. This was followed by a progressive spread of recipient endothelial cells from the anastomotic site, and re-endothelialization was complete at 1 month. The degree of neointimal and medial thickening increased until 3 months, and then decreased. At 12 months, no additional growth of the intimal or medial layers was observed. Adventitial inflammation became severe at 3 months, but was reduced at 6-12 months. The proportions of CD3-positive T-lymphocytes among inflammatory cell infiltrations were very low. Fresh frozen xenogenic arterial grafts showed acceptable patency throughout the 12-month period and showed no evidence of being unduly influenced by rejection reactions.

This is the first reported case in which neoplastic cells not of granulocytic or monocytic origin have been shown to contain lysozyme. A highly differentiated metastasizing adenocarcinoma of the jejunum is presented. Areas of the tumour contained cells resembling Paneth cells, that is, they contained cytoplasmic granules which 1) stained red with Masson's trichrome and 2) were shown to contain lysozyme by an immunoperoxidase technique. No argentaffin cells could be identified within the tumour. Staining of mucosubstance revealed large intestine-type sulfated glycoproteins in the tumour tissue. The presence of lysozyme-containing neoplastic Paneth cells suggests that the tumour: 1) originated from the mucosal crypts, and 2) had a high degree of cellular differentiation.

Ground-glass hepatocytes resembling those seen in HBsAg carriers on hematoxylin and eosin and on trichrome stained sections, but giving a negative reaction to orcein and a positive one to PAS, were found in liver biopsy specimens from nine asymptomatic former alcoholics who were on treatment with cyanamide, in one of four who had been treated with cyanamide several months before the liver biopsy procedure, in none of 15 treated with disulfiram, and in one of eight who had apparently not received aversive drugs. Portal and periportal inflammatory changes and fibrosis were more frequently observed in biopsy specimens containing PAS-positive ground-glass hepatocytes than in those without, but cirrhosis was found with a similar frequency. It is concluded that periportal PAS-positive ground-glass hepatocytes are a histological marker of cyanamide treatment.