Search

Purpose Histological analysis of human carotid atherosclerotic plaques is critical in understanding atherosclerosis biology and developing effective plaque prevention and treatment for ischemic stroke. However, the histological staining process is laborious, tedious, variable, and destructive to the highly valuable atheroma tissue obtained from patients. Procedures We proposed a deep learning-based method to simultaneously transfer bright-field microscopic images of unlabeled tissue sections into equivalent multiple sections of the same samples that are virtually stained. Using a pix2pix model, we trained a generative adversarial neural network to achieve image-to-images translation of multiple stains, including hematoxylin and eosin (H&E), picrosirius red (PSR), and Verhoeff van Gieson (EVG) stains. Results The quantification of evaluation metrics indicated that the proposed approach achieved the best performance in comparison with other state-of-the-art methods. Further blind evaluation by board-certified pathologists demonstrated that the multiple virtual stains have high consistency with standard histological stains. The proposed approach also indicated that the generated histopathological features of atherosclerotic plaques, such as the necrotic core, neovascularization, cholesterol crystals, collagen, and elastic fibers, are optimally matched with those of standard histological stains. Conclusions The proposed approach allows for the virtual staining of unlabeled human carotid plaque tissue images with multiple types of stains. In addition, it identifies the histopathological features of atherosclerotic plaques in the same tissue sample, which could facilitate the development of personalized prevention and other interventional treatments for carotid atherosclerosis.

595-nm pulsed dye laser and fractional CO2 laser have been demonstrated effective to treat hypertrophic scar. The underlying mechanism may involve transforming growth factor-beta1 (TGFβ1) and proliferating cell nuclear antigen (PCNA), but remains to be clarified. Our study was performed to investigate how 595-nm pulsed dye laser combined with fractional CO2 laser treats hypertrophic scars in a rabbit model through regulating the expression of TGFβ1 and PCNA. Twenty-four New Zealand white rabbits were randomly divided into control group, pulsed dye laser group, fractional CO2 laser group, and pulsed dye laser + fractional CO2 laser (combination) group. Surgical wounds were made and allowed to grow into hypertrophic scars at day 28. Next, 595-nm pulsed dye laser (fluence: 15 J/cm2; square: 7 mm; pulse duration: 10 ms) was used in pulsed dye laser and combination group, while fractional CO2 laser (combo mode, deep energy: 12.5 mJ; super energy: 90 mJ) in fractional CO2 laser and combination groups, once every 4 weeks for 3 times. The appearance and thickness of hypertrophic scar samples were measured with hematoxylin-eosin and Van Gieson’s straining. The expressions of TGFβ1 and PCNA were evaluated by immunohistochemical and western blot analysis. A significant improvement was noted in the thickness, size, hardness, and histopathology of hypertrophic scar samples after laser treatment, especially in combination group. Scar Elevation Index (SEI), fiber density (NA), and collagen fiber content (AA) decreased most significantly in combination group (2.10 ± 0.14; 2506 ± 383.00; 22.98 ± 2.80%) compared to 595-nm pulsed dye laser group (3.35 ± 0.28; 4857 ± 209.40; 42.83 ± 1.71%) and fractional CO2 laser group (2.60 ± 0.25; 3995 ± 224.20; 38.33 ± 3.01%) (P

Artesunate (ART) is the derivative of artemisinin isolated from the traditional Chinese medicine qinghao. Although several studies reported the efficiency of artesunate in the treatment of malaria, inhibiting fibroblasts and collagen synthesis, the association between artesunate and scar formation is unclear. The research was designed to study the significance of artesunate (ART) on the expression of transforming growth factor (TGF-β1) and small mother against decapentaplegic (SMAD3) in rabbit's ear hypertrophic scar model. Twenty-four New Zealand white rabbits were randomly divided into six groups: control group, matrix group, low-concentration artesunate group (0.48%), medium-concentration artesunate group (0.96%), high-concentration artesunate group (1.92%) and silicone gel group. Punched defects were established on each rabbit’s ear which resulted in a hypertrophic scar. On the 28th day, topical artesunate creams were applied twice a day except on the control group. On the 56th day, scar samples were collected for histopathology and immunoassay. Hematoxylin and eosin staining, Van Gieson staining, immunohistochemistry and Western blot analysis were done. Amongst the six groups, findings showed that the medium-concentration artesunate group (0.92%) efficiently decreased hypertrophic scar formation and significantly reduced fibroblasts and collagen synthesis. The results had also shown a decrease in the expression of transforming growth factor (TGF-β1) and declined small signal mother against decapentaplegic (Smad3). The overall study shows efficacy and mechanism of artesunate. It concluded that the medium concentration of artesunate (0.92%) could be an effective therapeutic agent for hypertrophic scars.

The dynamic variation of renin-angiotensin system (RAS) in silicosis remains unclear. Seventy Wistar rats were divided into 7 groups including control group, silicosis groups (inhaling SiO2 for 2, 4, 8, 16 and 24 weeks, respectively) and Captopril (Cap) group. Rat lung primary fibroblasts were divided into control group, SiO2-stimulated group (0, 0.5, 1, 3, 6, 12, 24 and 48 h) and Cap group. The silicotic nodules were formed and collagens were deposited gradually in silicosis group observed by haematoxylin and eosin (HE) staining and Van Gieson (VG) staining. Cap relieved the lung fibrosis and collagen deposition. Immunohistochemistry indicated the positive expression of α-smooth muscle actin (α-SMA) was increased gradually in silicotic rat lung tissue. Western blotting revealed the expression of collagen type I (Col I) and α-SMA was up-regulated in silicotic rat lung tissue and fibroblasts stimulated by SiO2. Cap decreased the expression of Col I and α-SMA in silicotic rat lung tissue and fibroblasts stimulated by SiO2. Western blotting also demonstrated the expression of angiotensin-converting enzyme (ACE) and angiotensin II type 1 receptor (AT1) was increased, and the expression of ACE2 and Mas was decreased gradually in silicotic rat lung tissue and fibroblasts stimulated by SiO2. ELISA showed the serum levels of ACE and angiotensin II (Ang II) were also increased and ACE2 and Ang (1-7) were decreased in the silicosis group. Treatment with Cap decreased the expression levels of ACE, Ang II and AT1, and increased the expression levels of ACE2, Ang (1-7) and Mas. These findings suggested that an imbalance between ACE-Ang II-AT1 axis and ACE2-Ang (1-7)-Mas axis may participate in the development of silicosis.

Hematoxylin and eosin (H&E) stain is one of the most common specimen staining methods in pathology diagnosis due to the capability to show the morphological structure of tissue. However, the appearance of the specific component, i.e., elastic fibers might not be recognized easily because have similar color and pattern with ones of collagen fibers. To distinguish these two components, Verhoeff’s Van Gieson (EVG) staining method is commonly used. Nevertheless, procedures of EVG stain are more complex and expensive than H&E stain. In this study, we investigate the possibility to distinguish elastic and collagen fibers from H&E stained images by applying spectral image analysis based on hyperspectral images. With experiments, we measure the transmittance spectral of 61-band H&E stained hyperspectral image, which are converted into absorbance spectral of hematoxylin, eosin, and red blood cell. As many as 3000 sampling pixels both from RGB and hyperspectral images of HE stained specimens were trained using Linear Discriminant Analysis (LDA) to get a discriminant function to classify elastic and collagen components in H&E RGB and H&E hyperspectral images. We conducted verification based on leave-one-out cross-validation of six data sets for evaluation. The verification result both visually and quantitatively compared to EVG stained image shows that the usage of hyperspectral images performs better than RGB images.

Cartilage grafting is a common procedure in nasal reconstruction or dorsal augmentation. Costal cartilage is usually preferred to conchal or septal cartilage when a larger amount of graft is required, although this cartilage type is often complicated by postoperative warping. To overcome this complication, we suggest the use of multilayer costal grafts. This study aims to compare single-layer and multilayer costal grafts concerning angle of warpage, cartilage viability, and architectural changes in the animal model. Twelve costal grafts were harvested from a fresh Landrace pig cadaver; six were shaped in single-layer grafts and six into a multilayer structure, both having the same thickness. These grafts were implanted in subcutaneous dorsal skin pockets of six athymic nude rats. After 8 weeks, the animals were euthanized and the grafts removed. Pictures of the cartilages were acquired. The samples were processed and stained for histological (hematoxylin and eosin, Alcian blue, Von Kossa, Masson-Goldner trichrome, and Verhoeff/Van Gieson stains) and immunohistochemical (GFAP) analyses. A higher grade of deformation was observed in the single-layer group compared to the multilayer one (5.2° vs 0.3°). Multilayer cartilage showed significantly higher chondrocyte density (70.4 vs 44.9 cells per field at 40×) and the lowest loss of nuclei in chondrocyte lacunae percentage (0.03 vs 3.2 %). Single-layer grafts also showed sporadic inflammatory cell infiltration, not found in multilayer ones. Multilayer grafts showed a decreased degree of warping as well as an increased viability and should be considered as a valuable alternative in nasal reconstruction. Level of evidence: Not ratable

The aim of this study was to investigate whether a surface coating with graphene could enhance the surface bioactivation of titanium alloys (Ti6Al4V) to further accelerate in vivo osteogenesis and osseointegration at the implant surface. In this study, a New Zealand white rabbit femoral condyle defect model was established. After 4, 12 and 24 weeks, biomechanical testing, micro-computed tomography (Micro-CT) analyses and histological observations were performed. At the highest push-out forces during the test, microstructure parameters, such as the bone volume/total volume fraction (BV/TV) and mineral apposition rate (MAR), of the new bone were significantly higher in the graphene-coated Ti6Al4V group (G-Ti6Al4V) than in the Ti6Al4V group (P 6Al4V group had more new bone formation than the Ti6Al4V group, and the G-Ti6Al4V group showed a closer fit between the bone and implant. In conclusion, graphene might be a novel type of nano-coating material for enhancing the surface biological activity of Ti-based alloy materials and may further promote in vivo osteogenesis and osseointegration.

We investigated the effect of calcitonin (CT) on lumbar intervertebral disk degeneration (LIDD) in rats with ovariectomy-induced osteopenia. CT protected ovariectomized rats from LIDD by, at least in part, modifying extracellular matrix metabolism of the disks and preserving the microarchitecture and biomechanical properties of adjacent vertebrae. The present study aimed to investigate the effect of CT on lumbar vertebral bone mineral density and intervertebral disk degeneration in ovariectomized (OVX) rats. We first subjected 50 3-month-old female rats to either OVX (n = 30) or sham (n = 20). Twelve weeks later, ten OVX and ten sham rats were necropsied. The remaining OVX rats began to receive either saline vehicle (OVX + V, n = 10), or salmon CT (OVX + CT, 16 IU/kg/2 days, n = 10). After 12 weeks of treatment, necropsy was conducted and bone mineral density was determined in L3–4 and L5–6 vertebrae. The microstructure and biomechanical properties of L3 vertebrae were detected by micro-computed tomography and compression test, respectively. L5–6 was also used to measure intervertebral disk height and observe intervertebral disk histological changes by Van Gieson staining and histological scores, as well as immunohistochemistry (IHC) analysis of matrix metalloprotease (MMP)-1, MMP-13, and collagen II expression. At 12 weeks post-OVX, OVX rats had lower BV/TV and Tb.N and higher intervertebral disk histological score than sham rats. After 24 weeks, OVX + CT rats had higher BMD, BV/TV, Tb.N, and bone biomechanical strength values than OVX + V rats. Histological analysis showed OVX + CT rats had significantly lower disk degeneration scores than OVX + V rats. IHC analysis revealed CT treatment decreased expression of MMP-1 and MMP-13 and increased expression of collagen II compared with OVX + V rats. Our data demonstrate that CT-treated OVX rats display less intervertebral disk degeneration and favorable changes in intervertebral disk metabolism, associated with higher trabecular bone mass, better trabecular microarchitecture, and better biomechanical strength when compared to vehicle-treated OVX rats.

Elastofibroma is a rare, benign, fibrous tumor formed by the proliferation of characteristic elastic fibers that commonly occurs between the lower margin of the scapula and the ribcage. We undertook a histochemical, immunohistochemical, and ultrastructural study of an elastofibroma dorsi beneath the right scapula of a 77-year-old woman. Tumor cells comprised collagen fiber bundles, numerous elastic fibers, and spindle cells resembling fibroblasts. The elastic and collagen fibers in the tumor were stained positively with Elastic van Gieson and Masson trichrome staining, respectively. Immunostaining showed that the fibroblasts were strongly positive for CD34, positive for vimentin, and weakly positive for α-smooth muscle actin. Ultrastructural observations revealed elastin and microfibrils between numerous irregularly arranged collagen fiber bundles. Signs suggestive of elastin deposition were also evident in the tangled collagen fibers themselves. The fibroblasts contained a large amount of rough endoplasmic reticulum and were surrounded on the outside of cells by microfibrils and collagen fibers. Although fibroblasts may produce large quantities of elastin, microfibrils, and collagen, our findings suggested that the deposition of elastin on collagen fibers may be involved in the formation of abnormal elastic fibers.

This study evaluated five vessels as potential candidates for coronary artery bypass grafting at the posterior interventricular artery (PIVA) blockage site. We used light microscopy and digital image analysis of H&E and Van Gieson’s-stained slides to investigate luminal diameter, arterial wall thickness, and relative muscularity and elasticity of candidate vessels. Results from our sample indicate that the inferior epigastric artery (IEA) may be the preferred graft to the PIVA based on overall similarities in these measurements. Other arteries provided a favorable match based on a single measurement, but the IEA agreed most consistently with the PIVA. When choosing a vessel to bypass blockage at the PIVA, cardiothoracic surgeons should be aware of the many favorable features of the IEA.

Wound healing consists of re-epithelialization, contraction and formation of granulation and scar tissue. Amino acids from proteins are involved in these events, but their exact roles are not well understood. The present study was undertaken to investigate the anti-inflammatory effects of some amino acids from two molluscs, Mytilus galloprovincialis (L.) (Mediterranean mussel) and Rapana venosa (hard shell-clam) employed in induced skin burn injuries in Wistar rats. The treatment was evaluated in terms of essential amino acids composition which rendered the extracts very efficient in healing skin burns. The healing process was examined by periodic acid Schiff's, Verhoeff's Van Gieson and immunohistochemistry stains for collagen IV, CD 34 and CD 117 antibodies. According to the obtained results, as expressed by histological studies, the most abundant blood vessels, collagen fibres, basal and stem cells were found only for treated animals with amino acids from Rapana venosa extracts. The rich composition of amino acids from the two molluscs merits consideration as therapeutic agents in the treatment of skin burns.

Specific sites of atherosclerotic processes due to hemodynamic changes and resultant stress, including how these normal anatomical structures become problematic in certain individuals, have yet to be acknowledged. One of these areas of the cardiovascular system occurs at the sinutubular junction (SJ), causing altercation in an otherwise normal flow status. The anatomy of the SJ was examined in 100 adult human hearts during the gross anatomy course at St George's University, during the years 2006-2007. All hearts were examined in situ, using a General Electric model 3200S ultrasound machine with a 5 MHz linear probe. The aforementioned cadavers were also examined using a Stryker laparoscopic unit. Serial transverse histological sections were made through the SJ perpendicular to its axis, and stained with eosin-hematoxylin, van Gieson, Masson trichrome, and Orcein methods. In addition, an immunohistochemical analysis was performed for the detection of positive smooth muscle cells stained areas. During gross and endoscopic examination we were able to identify the SJ in all adult heart specimens. Neonatal and fetal hearts did not exhibit any gross evident SJ; however, a SJ was evident histologically. Ultrasonographically we were able to identify the SJ in all adult heart specimens examined, and a sinutubular ridge in 62%. A significant association was present between the thickness of the ridge and the age of the specimens. The SJ was found to exhibit atherosclerotic changes and plaque formation in an age-related manner. In older subjects, the SJ was marked with local calcification and hemorrhages. In contrast, the SJ of neonatal hearts appeared to have intimal thickening with focal fragmentation and absent or duplicate internal elastic lamina. Intuitively speaking, the presence of a sinutubular ridge, an inevitable fate in humans based on the results of this study, provides an irreversible atherosclerotic process as there is no evidence that the promoting ridge regresses. This is an alarming situation in those individuals who will eventually develop cardiovascular risk factors, whether through inevitable genetic manifestations or by means of exogenous environmental causes.

We analysed the morphological features of the human surgical specimens of supraspinatus tendon from patients with rotator cuff tears. Tendon samples were harvested from 31 subjects (21 men and 10 women; mean age 51 years, range 38-64) who underwent arthroscopic repair of a rotator cuff tear, and from five male patients who died of cardiovascular events (mean age, 69.6 years). Histological examination was performed using Haematoxylin and Eosin, Masson's Trichrome and Van Gieson's connective tissue stain. The specimens were examined twice by the same examiner under white light and polarized light microscopy. Particular effort was made to assess any evidence of the changes associated with tendinopathy. Within each specific category of tendon abnormalities, the chi-square test showed significant differences between the control and ruptured tendons (P < 0.05). Using the kappa statistics, the agreement between the two readings ranged from 0.57 to 0.84. We found thinning and disorientation of collagen fibres and chondroid metaplasia to be more pronounced on the articular side of the specimens from patients with rotator cuff tear (P < 0.05). The present study provides a description of the histological architecture of human surgical specimens of normal supraspinatus tendon from patients with rotator cuff tears and demonstrates more frequent tendon changes on the articular side of the rotator cuff.

Intestinal neuronal dysplasia (IND) is an intestinal motility disorder, which clinically resembles Hirschsprung's disease (HD). Adventitial fibromuscular dysplasia (AFMD) consists of proliferation of smooth muscle cells and collagen fibers in the adventitia of blood vessels. The purpose of this study was to investigate vascular abnormalities in large bowel biopsies from patients with isolated HD, IND associated with HD and isolated IND. Large bowel biopsies from patients presenting with isolated HD ( n=23), IND associated with HD ( n=11), isolated IND ( n=16) and normal bowel as controls ( n=6) were investigated using acetylcholinesterase (AChE) histochemistry, van Gieson staining and alpha-smooth muscle actin (alpha-SMA) immunohistochemistry. Increased AChE activity around submucosal vessels was found in 9/16 (56%) cases with isolated IND, 3/11 (27%) cases of IND associated with HD, 5/23 (21%) isolated HD cases and 0/6 controls. AFMD was found in 10/16 (62%) of the isolated IND cases, 4/11 (362) of the cases with IND associated with HD and 4/23 (17%) cases of HD without IND using van Gieson staining. None of the control specimens revealed AFMD. Increased alpha-SMA immunoreactivity filaments were demonstrated in the submucosal vessel wall in 9/16 (56%) of isolated IND and 2/11(18%) of IND associated with HD cases. Normal alpha-SMA immunoreactivity around submucosal vessels was seen in isolated HD and controls. Abnormal submucosal vasculature is a common histological finding in isolated IND and IND associated with HD and may be a useful additional diagnostic feature in these patients.

The aim of this study was to determine the relationship between changes in microporosity and the osseointegration and the osseoconductivity of hydroxyapatite (HAp). HAp cylinders were manufactured by a combination of uniaxial powder pressing and different sintering conditions, with different percentages of microporosity: HAp-I with 3.96+/-0.75% microporosity, HAp-II with 15.66+/-1.60% microporosity, and HAp-III with 29.72+/-0.69% microporosity. These HAp cylinders were surgically implanted in rabbit femurs. After 8 and 12 weeks, the femurs were removed, fixed, sectioned, ground, and stained by Stevenel's blue/Van Gieson for light microscopy and histomorphometry. Some ground sections were routinely processed for SEM. The osseointegration and the osseoconductivity were determined by means of image analysis and the data were submitted to ANOVA. In all cases the cortical bone was repaired and the HAp facing the medullary canal was lined with endosteum, which in some areas exhibited thin bone tissue formation. SEM observations showed no differences in the morphology of tissue-HAp interfaces for the three different porosities of HAp. There were no statistical differences between the groups related to either osseointegration or osseoconductivity. These results suggest that neither osseointegration nor osseoconductivity of HAp are influenced by changes in HAp microporosity.

Study design: Report of two cases of acute lumbar nerve root compression caused by myxomatous degeneration of the ligamentum flavum. Objective: To report a rare cause of acute lumbar nerve root compression. Setting: Orthopaedic department, Osaka, Japan. Summary of background data: Two patients, both 50-year-old men presenting with signs and symptoms suggestive of acute lumbar nerve root compression were found to have a ligamentum flavum mass. The masses were removed and the patients regained normal function postoperatively. Methods: To reveal the nature of the mass, histopathological studies were made. Continuous sections were prepared from the removed mass lesions. The sections were stained with hematoxylin and eosin, van Gieson's stain, azan stain, periodic acid Schiff reaction, Alcian blue stain and von Kossa's stain. Results: Histological examination revealed myxomatous degeneration of the ligamentum flavum. No elastic fibers were found at the degeneration site. Diffuse mucopolysaccharide deposition was found at the degeneration site, however, no cyst was found. Collagen fibers were not increased. Hypertrophy or ossification of the ligamentum flavum was not recognized in the sections. At a follow-up examination over 2 years later, the patients were free of symptoms and the findings of a neurological examination were normal. Conclusion: Two cases of myxomatous degeneration of the ligamentum flavum of the lumbar spine were reported, which have seldom been described as the cause of acute lumbar nerve root compression. Spinal Cord (2001) 39, 488–491.

Purpose: Disturbances of the ocular tear film layer and dry eye symptoms are common complications following retinal surgery and ocular tumour therapy. Examined were the histopathological changes of the conjunctiva following posterior segment surgery and plaque radiotherapy. Methods: Biopsy specimens of the superior bulbar conjunctiva were obtained during cataract surgery between 2 weeks and 7 years following vitrectomy (n=92) or plaque radiotherapy for uveal melanoma (n=20) and from control subjects without previous ocular surgery (n=29). These were examined using conventional histology (HE, PAS, Van Gieson) and immunochemistry [APAAP, using antibodies directed against MUC1, MUC5AC, syndecan-1 and tenascin-C (TN-C)]. The histopathological changes were graded and statistical analysis was performed using Wilcoxon and Kruskal-Wallis rank sum tests. Results: Conjunctival specimens of patients following vitrectomy or plaque radiotherapy for uveal melanoma demonstrated increased epithelial stratification, a significant decrease in the number of PAS- and MUC5AC-positive goblet cells, and distributional changes in expression of MUC1, syndecan-1 and TN-C within conjunctival epithelium or stroma. These alterations – in particular the goblet cell reduction and stromal fibrosis – were most prominent in those patients who had undergone radiotherapy. Conclusions: Posterior segment surgery can lead to morphological alterations of the conjunctiva and distributional changes in ocular mucins, which may cause dry eye symptoms.

The significance of serotonin in the pathogenesis of monocrotaline-induced pulmonary hypertension (MCT-PH) in rats, plasma serotonin concentrations, and the effect of a serotonin receptor antagonist administration in association with the number of proliferative cells were investigated. The thickness of the media of the small pulmonary arteries and the weight ratio of the RV to that of LV + S (RV/[LV + S] weight ratio) were used as indices of the severity of PH. Plasma serotonin concentrations were measured by high-performance liquid chromatography. Histopathologic analysis of the lung tissue was performed by hematoxylin-eosin and elastin van Gieson staining. Immunohistopathologic staining for proliferating cell nuclear antigen (PCNA) was performed to identify proliferative cells. The severity of PH as determined by the medial thickness of the small pulmonary arteries and RV/(LV + S) weight ratio in rats with MCT-PH was significantly reduced after treatment with MCI-9042 (p < 0.01 and p < 0.05, respectively). The serotonin concentration was significantly greater in MCT-PH rats than in normal control rats (p < 0.05). The scores for histopathologic changes, such as thickening of the alveolar walls and interstitial inflammatory cell infiltration in MCT-PH rats, were significantly reduced after treatment with MCI-9042 (p < 0.05 and p < 0.01, respectively). The number of PCNA-positive cells was significantly greater in MCT-PH rats than in normal control rats (p < 0.0001) and was reduced after treatment with MCI-9042 (p < 0.0001). Treatment with MCI-9042 significantly inhibited the development of MCT-PH along with a decrease in the number of PCNA-positive cells, suggesting a pivotal role of serotonin in the development of PH induced by MCT.

Myocardial pathology and cardiac functional abnormalities known to occur in association with chronic alcohol abuse have been attributed to a direct toxic effect of ethanol on the intra-myocardial vasculature: alcohol-induced occlusion of small arteries with consequent secondary ischaemia leads to individual myocyte loss, focal fibrosis and compensatory cardiac hypertrophy. To assess the intra-myocardial arterial vessels an interactive semi-automated, computerised, high-resolution, video image-analysing system was used and the findings from hearts of alcoholics were compared with normal hearts collected at autopsy. The elastic laminae, stained with Van Gieson's stain, acted as the reference points for measurement of the diameters, circumferences and thicknesses of the vessels assessed. It was not possible to demonstrate any statistically significant morphometric changes within intra-myocardial vasculature. The same technique used is readily adaptable for the assessment of the blood vessels in other organs and tissues.

Vaginal fibrosarcomas are unusual mesenchymal tumours in cows. This report describes the clinical investigation, gross and histopathological findings, surgical treatment and postoperative course of a vaginal fibrosarcoma in a cow. Fibropapillomas are the most commonly encountered type of tumours in the vagina and vulva of the cow. They are usually pedunculated and can be removed surgically. Although they do not cause infertility, they may be associated with dystocia [6]. Besides fibropapillomas, cases of squamous cell carcinoma, leiomyoma, fibroma, haemangioma, leiomyosarcoma and melanoma have also been reported in the vagina and vulva of cows [7]. Fibrosarcomas can be found in any location of the body. However, they are unusual mesenchymal tumours of the bovine vagina [2,1,5]. Fibromas, fibro-papillomas and fibrosarcomas have been reported as mushroom-shaped growths, and can be attached either by a broad base or by a long pedicle that allows part of the tumour to protrude from the vulva [7]. An eight-year-old Holstein Freisian cow was admitted for the evaluation of a pedunculated vaginal mass that was attached to the right lateral vaginal wall and partially protruded from the vagina (Figure ​(Figure1).1). The mass had a wet surface with a mucoid, sanguinous discharge. Appetite was normal and the general physical examination revealed no other abnormalities. Figure 1 Nodular appearance of the tumoral mass. Haematological examination prior to surgery revealed a leucocytosis. The total WBC count was elevated to 16.1 cells/μL (range 4-12), with a high neutrophil count of 8090 cells/μL (range 600-4000) and a lymphocyte count at the high end of physiological normal (7490 cells/μL; range 2500-7500). Other parameters, such as RBC, HGB and HCT were slightly below physiological range, which may be an indicator of mild anaemia due to the continuous haemorrhagic discharge from the mass. Serum biochemistry showed minor increases in ALT and creatinine but AST, total protein, triglycerides, phosphorus and AST were all within the normal range. The cow was restrained and the tail bandaged. Local epidural anaesthesia was performed with the administration of 8 ml of 2% lidocaine (Jetokain; Adeka, Turkey). Additional local infiltration anaesthesia, within the vaginal mucosa surrounding the pedicle of the tumoral mass, was performed with the same anaesthetic agent using a volume of approximately 15-20 ml. Following anaesthesia, the vulva was retracted from either side with uterine forceps and the mass was revealed. An oval incision was made on the mucoasl surface at a distance of approximately 2 cm from the margin of the mass. The total length of incision was 15 cm. Following this, blunt dissection with scissors was used to increase the depth of the incision without interfering with the edge of the mass. The major blood vessels were ligated where necessary. The defect created after the removal of the mass was closed in two steps. Firstly, interrupted cruciate sutures were applied and, secondly, these were supported with a superficial continuous suture pattern, both using a chromic gut of USP size 2. Postoperative parenteral antibiotics (Clemipen-Strep; Topkim, Turkey) for four days and local wound healing agents (Bepanthene plus; Roche, Turkey) were administered daily for two weeks. For structural differentiation, the tumoral mass was fixed in 10% formalin solution, routinely paraffin-embedded, sectioned at 4-5 μ and stained with haematoxylin-eosin (H&E) stain. Sections were stained with van Gieson's and Masson's trichrome stains for the detection of collagen fibers [3,4]. Macroscopically, the tumoral mass was located on the ventral vaginal wall. It was 12 cm × 6 cm × 4 cm in size, weighed 244 g, was greyish-yellow in colour, firm to the touch and had a nodular appearance (Figure ​(Figure1).1). The cut surface was homogeneously white in colour. Microscopically, the tumour was composed of spindle-shaped tumour cells forming interlacing bundles or arranged in herringbone patterns. The cells had marked cellular pleomorphism with oval or round and slightly hyperchromatic, nuclei with abundant eosinophilic cytoplasm. Mitotic figures were common and multinucleated giant cells were generally observed (Figure ​(Figure2).2). The stroma of the tumour was made up of collagen fibres which were detected by van Gieson's and Masson's trichrome stains. According to these histopathological findings, the tumour was diagnosed as a fibrosarcoma. In contrast to a fibropapilloma, the tumour did not consist of proliferating fibrous tissue with an epithelial covering of variable thickness. Figure 2 Fibrosarcoma with marked cellular pleomorphism and multinucleated giant cell. Haemotoxylin and Eosin. Original magnification × 480. The cow was examined two weeks after the operation, and excellent wound healing was observed with no evidence of postoperative infection. A further examination was performed following a six-month period, no evidence of regrowth of any tumoral tissue could be found and the general condition of the cow was normal. This suggests that a favourable prognosis may be expected following proper extirpation of pedunculated tumoral masses in cows.

Adenomyosis is a nonneoplastic hyperplastic lesion, characterized by invagination of proliferating endometrial glands into myometrium. In dogs, uterine adenomyosis is relatively rare and it is important in Toxicologic Pathology to differentiate other non-neoplastic and neoplastic lesions in uterus. In the present study, we report two cases of adenomyosis in the female beagle dogs used for a chemical toxicity test. Clinically, one out of the two female beagle dogs, 15 months of age, had vaginal bleeding for 2 weeks and the other one, 11 months of age, showed swelling of vulva for a week. At necropsy, the weight of uterus was markedly increased to 27.9 g and 15.8 g, compared with the mean value (4.01±2.37, n=6) of that of other normal dogs, respectively. The parameters of hematology and serum chemistry were ranged normal in both of the dogs with enlarged uterus. For differentiation of connective tissue with muscle fibers, Van Gieson stain was also performed in the serial tissue sections. Histopathologically, the lesions of the enlarged uteruses were characterized by proliferating endometrial glands into myometrium, surrounded by connective tissue. The endometrial glands were proliferating downward to myometrium or embedded in multiple clustered glands in deeper myometrium without compressing the adjacent muscle fibers. The gland epithelial cells are uniformly cuboidal shape with a dense and bottom-located nucleus. These gross and histological findings were consistent with adenomyosis.

The aim was to analyze the morphological differences of the intervertebral disc and endplates at different levels. Forty-five vertebral motion segments were obtained from the spine of nine 3 to 4-year-old cynomolgus monkeys (Macaca fascicularis). From every spine, five discs were sectioned (C5–C6, T3–T4, T9–T10, L2–L3, L4–L5). In all the groups, tissue samples were collected and sections were stained with Masson’s trichrome, Safranine-O and van Gieson’s connective tissue stain to analyze the intervertebral discs. Immunohistochemistry was performed, using specific antibodies to detect collagens I and II. The intervertebral disc height, the maximum nucleus pulposus height, the superior and inferior endplate heights were histomorphometrically measured and different indexes were calculated to compare the differences between specimens of the same animal and between discs of the same level, and finally the differences between groups of discs of different levels. There were no differences existing in annular fibers anchoring on the endplate between discs of different levels. A positive immune reaction for type I collagen was observed in the longitudinal ligaments and in the annular region adjacent to them. Collagen II immune reactivity was found in the annulus close to the nucleus pulposus, in the endplates and in the nucleus. There were no differences between discs of different levels in the collagen I and II localization. The height of the discs varied along the spine. The smallest value was measured in T3–T4, with a larger increase caudally than cranially. The highest value was measured in L2–L3. A cervical disc was 55% the height of a lumbar one. The endplate height increased along the length of the spine. The inferior EP was always higher than the superior. The study provides a detailed structural characterization of the intervertebral disc and may be useful for further investigations on the disc degeneration process.

Pulmonary interstitial fibrosis is an important component of the pathology of bronchopulmonary dysplasia (BPD). There is increasing evidence to support the role of infection, especially sepsis, in the pathogenesis of BPD. We have investigated the effects of combining chronic oxygen toxicity and lipopolysaccharide endotoxin in rats. Our hypothesis is that endotoxin activates pulmonary macrophages in an O2 damaged lung, which in turn activates pulmonary lipocytes to release stored retinoids and, with chronicity, initiates production of TGF-b by these cells. In the liver paradigm of fibrosis, this is followed by development of receptors for PDGF on lipocytes leading to autocrine proliferation, transformation into myofibroblasts and the production of cross-linked collagens. Rats treated with 100% O2 for 36 hours, 90% O2 for 36 hours, 80% O2 for 6 weeks and IP endotoxin for 3 days beginning at 72 hours, showed early immunostaining of lung epithelium, lung lipocytes and of liver lipocytes with TGF-β3, which disappeared with chronicity by 36 days. PDGF immunostaining also appeared early in lung and liver lipocytes and persisted for 36 days. Alpha smooth muscle actin, which appears with lipocyte transformation to myofibroblasts,showed immunoreactivity in cells around respiratory bronchioles and at septal tips, increasing greatly with chronicity. The presence of collagen in alveolar walls and septal tips also increased with time as shown by van Gieson staining and by electron microscopy. We suggest that activation of pulmonary lipocytes is an important component of the production of cross-linked collagen and its deposition in pulmonary interstitial fibrosis.

Fresh frozen tissue sections of human articular cartilage was treated without and with human testicular hyaluronidase (2 x 10(6) units/l) for 60 min at 37 degrees C and stained by the indirect immunoperoxidase technique with rabbit antihuman fibronectin. The rabbit antihuman fibronectin was purified by affinity chromatography on human fibronectin-Sepharose. Fibronectin was only found on the acellular surface of the articular cartilage in tissue sections not treated with hyaluronidase. In this surface layer, probably identical to "lamina splendens", the arrangement of fibronectin was as a membrane. No collagen was seen in this area by van Gieson staining. No staining for fibronectin was found in the cartilage matrix or in the chondrocytes. Treatment of the cartilage tissue with hyaluronidase resulted in visualization of high amount of fibronectin in the cartilage matrix, with the highest intensity around the chondrocytes. The staining of the acellular surface layer of the articular cartilage was identical with the results obtained without hyaluronidase treatment. These results indicate that articular cartilage is rich in fibronectin probably in complex with hyaluronic acid, and that the chondrocytes produce fibronectin in situ. It also demonstrates the steric hindrance of hyaluronic acid aggregates in diffusion of the antibody and the value of hyaluronidase treatment of tissue before demonstration of fibronectin.

The sequential changes in the presence of fibronectin in the synovial membrane during the development of antigen-induced arthritis in rabbits were studied using an indirect immunoperoxidase technique on the tissue specimens fixed in formaldehyde, embedded in paraffin and pre-treated with pepsin and testicular hyaluronidase. The relation to the distribution of fibronectin and connective tissue fibres, demonstrated as either argyrophilic or red by van Gieson method, was studied. Initial after the induction of the arthritis the synoviocytes became increased in size and number. The subsynoviocytial tissue was invaded by granulocytes and the number of vessels was increased. Fibronectin in increased amount was seen around the lining cells. After 2-4 weeks a markedly reduced amount of granulocytes were seen together with an increase in the number of macrophages. At this stage, fibronectin was also found together with argyrophilic fibres in the subsynoviocytial connective tissue. After 8-13 weeks the synovial membrane was found hypertrophic and folded. The lining layer was unchanged, but in the subsynoviocytial tissue lymphocytes and plasma cells were more focally arranged. At that time fine fibres, stained by the van Gieson method, were present together with fibronectin and argyrophilic fibres in the subsynoviocytial tissue. The morphological change and the distribution of fibronectin in experimentally induced arthritis correlated temporally to the morphological change and the presence of fibronectin found in experimentally induced granulation tissue.

Modern methods of reparative surgery of the blood vessels enable the patency of the main arteries of the lower limbs to be restored comparatively quickly after acute occlusion. However, operative revascularization is not always effective, ischemic changes in the skeletal muscles are progressive, and under these conditions amputation of the affected limb cannot be avoided [3-6, 8, 15]. Development and implementation of timely ahd effective correction of acute ischemia of the limbs would undoubtedly be aided by knowledge of the dynamics: of the structural and metabolic changes taking place in skeletal muscles during acute occlusion of the main arteries.* Data in the literature on this problem do not deal with the problem as a whole, but only with certain of its aspects, and as a rule, they do not give an objective and quantitative evaluation of the morphological changes discovered [1, 7, 10, 12, 14]. The aim of this investigation was a combined quantitative morphological analysis of changes in the skeletal muscles during acute arterial occlusion of the limbs. EXPERIMENTAL METHOD Skeletal muscles from the leg of 36 dogs weighing 13-18 kg, with experimental acute occlusion of the terminal part of the aorta [2], were studied. The duration of ischemia was 0 (control), 3, 6, 9, and 12 h. Sections through the leg muscles, after embedding in paraffin wax, were stained with hematoxylin and eosin, by Van Gieson's Regaud's, and Lie's methods, and by the trichrome method of Goldner [9, 11]. Microscopic preparations were studied in ordinary and polarized light. Tissue stereologic analysis of volumes of intact and injured muscle fibers and the edematous stroma was carried out on sections stained by Regaud's method in 10 fields of vision on the "Struktura" apparatus (Central Design Bureau, Academy of Medical Sciences of the USSR). The following histochemical methods were used: the diaminobenzidine method for myoglobin [13], staining with Sudan Black B and Oil Red for phospholipids and neutral fats, the PAS reaction with amylase control to detect glycogen and neutral glycosaminoglyca ne. The enzyme-histologi cal investigation of the soleus muscle was undertaken on frozen section 10 # thick. Activity of the following enzymes was determined by the usual methods [11]: succinate (SDH), isocitrate (ICDH), malate (MDH), ~-giycerophospha te (~-GPDH), lactate (LDH), glucose-6-phosph ate (G-6-PDH), glutamate (GDH), and fi-hydroxybutyrate (fi-HBDH) dehydrogenases; NADand NADP-diaphorases , alkaline and acid phosphatases (AlP and AcP respectively), myosin ATPase, and phosphorylase. Changes in SDH, LDH, fl-HBDH, NAD, NADP, and ATPase were estimated quantitative on an MIF-7 integrating photometric microscope. In one transverse section 66 areas of red and white muscle fibers (R~F and WMF respectively) were measured photometrically. All numerical data were subjected to statistical analysis. The significance of differences between the values for the parameters was determined by Student's test. EXPERIMENTAL RESULTS After ischemia for 3 h damage to the muscle fibers was found. Tissue analysis showed changes in the ratio of the relative frequency of areas of intact and injured fibers and stroma compared with the control. In the control group, for instance, the ratio was 67:6:22%, and after ischemia for 3 h it was 6~.13:24%. The focal and more widespread increase in cross-striation of the muscle fibers will be noted (Fig. la), and in polarized light this was revealed as more intensive fluorescence of the A-disks. The time course of contraetura[ injuries to the muscle fibers could be discerned: localized areas of approximation of the A-disks, which merged *Academician of the Academy of Medical Sciences of the USSR.

Twenty humeral heads were collected from 10 adult English Pointer dogs, fixed in 15% formalin containing cetylpyridinium chloride, decalcified, processed for paraffin sections, and cut serially. The articular cartilage was studied by staining principally with Alcian Blue in the presence of 0.4 or 0.9m MgCl2 with and without a van Gieson counterstain. The results of the differential staining procedures demonstrated the existence of two groups of chondrocytes with distinctly different staining affinities. One group reacted intensely for the presence of protein-polysaccharides within its cytoplasm while the other group completely lacked this property. An approximate proportionality of 3∶1 of the protein polysaccharide-positive and-negative chondrocytes was observed in the tangential layer and upper intermediate zone. In the lower intermediate zone, radiate zone, and zone of calcified cartilage, the chondrocyte types were present in equal proportions. Staining with Alcian Blue in the presence of 0.9m MgCl2 with and without a van Gieson counterstain indicated a further subdivision of the protein-polysaccharide positive group of chondrocytes. This blocking technique has been reported to distinguish between chondroitin sulphate and high mol. wt. keratosulphate. Thus, based upon a greatly decreased number of the blue-stained chondrocytes after staining with Alcian Blue in the presence of 0.9m MgCl2 the hypothesis is put forward that some chondrocytes produce primarily chondroitin suphate and others produce both chondroitin sulphate and keratosulphate.

The behavior of the connective tissue during the healing of transsected nerves was studied. Experiments were carried out with rahbits. The sciatic nerve was trans-sected and repaired using different techniques. Postoperatively the animals were all treated in exactly the same way. The clinical course was registrated carefully, with special attention to the return of the motor function and the occurence and extension of decubital ulcers. Finally the animals were sacrified, and the site of nerve repair was excised and histologically examined using Hamatoxilin, Eosin. Van Gieson, Kresylviolett, Kluver-Barriera, Sudan-Schwarz B, Bodian staining. Two different techniques were compared by operating on the two sciatique nerves of the same animal. Individual factors could be excluded as the result of each two operations was compared in the same individual. On the basis of the experimental experience the following operative technique of nerve grafting was developed: The epineurium of the two nerve stumps is resected. A dissection of the fasciculi of the nerve stumps is carried out. Major fasciculi are isolated individually. Minor fasciculi are isolated in groups of 3–5. The ends are resected at different levels according to the amount of trauma or fibrosis present. A sketch is made of the fascicular structure of the two nerve stumps. Using this sketch we try to define the corresponding fasciculi or groups of fasciculi. Between the corresponding fasciculi or groups of fasciculi nerve grafts (sural nerve) are introduced. The grafts are larger than the defect in position of function. Under microscopic control a careful adaptation is achieved. One suture (Nylon 10×0) at each end of each graft is used to secure the union. Under certain conditions we have relied only on the natural union and did not use any suture at all. As the fasciculi respectively groups of fasciculi had been resected at different levels, stumps and grafts are interdigitated with each other. For a median nerve 4–6, and for an ulnar nerve 3–5 individual nerve grafts were used. This technique was used in more than 200 cases with satisfactory results. The results even after bridging long defects (more than 5 cm) are good, and much better than the results published by Asworth, Boyes and Stark (1971) who used end-to-end suture to overcome large defects of peripheral nerves.

An inquiry was made into the action of weak solutions of acetic acid, alkali, and citrate buffer on precollagen and collagen fibers of regenerating connective tissue. Precollagen and collagen fibers were studied with the aid of Gomori's method, the method for acylamidcarboxyl groups, and van Gieson's method. Precollagen fibers in the regenerating connective tissue are hardly soluble in the citrate buffer solution and acetic acid but they are readily soluble in alkali. From the moment of its appearance in the regenerating connective tissue, procollagen is well soluble in the citrate buffer solution, alkali, and acetic acid. Precollagen solubility falls with the maturation of connective tissue.