Light Microscopic Evaluation Research Articles

Junctional Epidermolysis Bullosa in Sprague Dawley Rats Caused by a Frameshift Mutation of Col17a1 Gene

Junctional epidermolysis bullosa is an intractable cutaneous disorder in humans causing skin fragility and blistering due to mutations in genes encoding essential molecules adhering epidermis and dermis including collagen XVII. However, the pathogenesis still remains to be not fully understood perhaps because of a lack of appropriate animal models. In this study, we report novel mutant rats experiencing junctional epidermolysis bullosa, which was confirmed to be caused by a frameshift mutation of Col17a1 gene, as a rat model for investigating the underlying mechanism of pathogenesis. The mutant rats completely lacked the expression of collagen XVII and had blisters leading to infantile deaths as a homozygous condition, although their skin was apparently normal at birth by light microscopic evaluation except that immunohistochemical examination could not detect collagen XVII in any organs. These observations suggest that collagen XVII is not essential for the development of skin during the prenatal period but is indispensable for keeping epidermal-dermal connections stable after birth. Subsequent electron microscopic examinations further revealed an absence of hemidesmosomal inner plaques being composed of BP230, a binding partner of collagen XVII, and plectin in Col17a1-null newborns, albeit mRNA expressions of these molecules seemed to be unaffected at least during the fetal period. These results suggest that the lack of collagen XVII induces attenuation of hemidesmosomal inner plaques, which in turn destabilizes the epidermis-dermis connection and results in deterioration of epidermal physiology with formation of blisters after birth.

Detection of disease-specific signatures in B cell repertoires of lymphomas using machine learning.

The classification of B cell lymphomas-mainly based on light microscopy evaluation by a pathologist-requires many years of training. Since the B cell receptor (BCR) of the lymphoma clonotype and the microenvironmental immune architecture are important features discriminating different lymphoma subsets, we asked whether BCR repertoire next-generation sequencing (NGS) of lymphoma-infiltrated tissues in conjunction with machine learning algorithms could have diagnostic utility in the subclassification of these cancers. We trained a random forest and a linear classifier via logistic regression based on patterns of clonal distribution, VDJ gene usage and physico-chemical properties of the top-n most frequently represented clonotypes in the BCR repertoires of 620 paradigmatic lymphoma samples-nodular lymphocyte predominant B cell lymphoma (NLPBL), diffuse large B cell lymphoma (DLBCL) and chronic lymphocytic leukemia (CLL)-alongside with 291 control samples. With regard to DLBCL and CLL, the models demonstrated optimal performance when utilizing only the most prevalent clonotype for classification, while in NLPBL-that has a dominant background of non-malignant bystander cells-a broader array of clonotypes enhanced model accuracy. Surprisingly, the straightforward logistic regression model performed best in this seemingly complex classification problem, suggesting linear separability in our chosen dimensions. It achieved a weighted F1-score of 0.84 on a test cohort including 125 samples from all three lymphoma entities and 58 samples from healthy individuals. Together, we provide proof-of-concept that at least the 3 studied lymphoma entities can be differentiated from each other using BCR repertoire NGS on lymphoma-infiltrated tissues by a trained machine learning model.

Investigation of the Effects of Allicin on the Nasal Mucosa.

Objectives: We investigated the effects of allicin on nasal mucosa via an experimental study. Methods: In the study, 16 male New Zealand Albino (2.5-4.5 kg) rabbits were used. The right nasal passages of the 8 rabbits were included in the control group (Group 1, n = 8), and the right nasal passages of the 8 different rabbits were included in the study group (Group 2, n = 8). In the study group (Group 2), a Merocel tampon soaked in Allicin (Alli Tech; Dulwich Health) (0.5 mg/kg). In the control group (Group 1), a Merocel tampon soaked in serum physiologic was placed in the right nasal passage for 3 days (first to third days of the study). On the fourth day, nasal mucosa was excised and histopathological examinations were performed. Results: Our results showed that there were no significant differences in terms of bleeding, congestion, inflammation, calcification, and seromucous gland density between the study and control groups (P > .05). In light microscopic evaluation, moderate density of lymphocytic cells beneath the surface epithelium and, further down, seromucous gland structures, dense seromucous glands, and occasional ductal structures were observed in the study group. Congested vascular structures beneath the respiratory epithelium and adjacent to a thick-walled vascular structure, coarse calcification is observed in the control group. Conclusion: It has been demonstrated that the Allicin-soaked Merocel pack does not have adverse effects on rabbit nasal mucosa, and it does not lead to mucosal bleeding, congestion, inflammation, and calcification, and changes in the seromucous gland density. Considering the antibacterial and antiviral effects of allicin, it is appropriate to plan research in humans to evaluate its use in nasal packs applied during epistaxis, septoplasty, rhinoplasty, and endoscopic sinus surgery.

Primeiro registro de infestação por Leporacarus gibbus em coelhos domésticos (Oryctolagus cuniculus) no sul do Rio Grande do Sul, Brasil

Leporacarus gibbus is an ectoparasite of rabbits, in which infestation is usually subclinical and can, sometimes, be underdiagnosed. This case report presents a symptomatic infestation by L. gibbus in rabbits, for the first time, at southern Rio Grande do Sul, Brazil. Forty-four rabbits from a vivarium in the city of Capão do Leão, Rio Grande do Sul State, Brazil, were evaluated. Dermatitis and alopecia were observed on the hind legs, neck, and back of the animals. External inspection revealed the presence of ectoparasites. Skin scraping, adhesive tape impression, and hair plucking were performed for diagnosis. However, only after stereomicroscopic examination of the coat, the presence of mites was revealed. The specimens were clarified in phenol/xylene and mounted in a non-permanent preparation. After light microscopic evaluation, the specimens were taxonomically classified as L. gibbus. Ivermectin treatment was applied, with 100% efficacy after 7 days. This report describes, for the first time, a case of L. gibbus infestation in domestic rabbits in the region, with dermatological manifestation.

Comparative analysis of effects of conditioned mediums obtained from 2D or 3D cultured mesenchymal stem cells on kidney functions of diabetic rats: Early intervention could potentiate transdifferentiation of parietal epithelial cell into podocyte precursors

AimThe secretome of mesenchymal stem cells (MSCs) could be a potential therapeutic intervention for diabetes and associated complications like nephropathy. This study aims to evaluate the effects of conditioned mediums (CMs) collected from umbilical cord-derived MSCs incubated under 2-dimensional (2D) or 3D culture conditions on kidney functions of rats with type-I diabetes (T1D). Main methodsSprague-Dawley rats were treated with 20 mg/kg streptozocin for 5 consecutive days to induce T1D, and 12 doses of CMs were applied intraperitoneally for 4 weeks. The therapeutic effects of CMs were comparatively investigated by biochemical, physical, histopathological, and immunohistochemical analysis. Key findings3D-CM had significantly higher total protein concentration than the 2D-CM Albumin/creatinine ratios of both treatment groups were significantly improved in comparison to diabetes. Light microscopic evaluations showed that glomerular and cortical tubular damages were significantly ameliorated in only the 3D-CM applied group compared to the diabetes group, which were correlated with transmission electron microscopic observations. The nephrin and synaptopodin expressions increased in both treatment groups compared to diabetes. The WT1, Ki-67, and active caspase-3 expressions in glomeruli and parietal layers of the treatment groups suggest that both types of CMs suppress apoptosis and promote possible parietal epithelial cells' (PECs') transdifferentiation towards podocyte precursor cells by switching on WT1 expression in parietal layer rather than inducing new cell proliferation. Significance3D-CM was found to be more effective in improving kidney functions than 2D-CM by ameliorating glomerular damage through the possible mechanism of transdifferentiation of PECs into podocyte precursors and suppressing glomerular apoptosis.

Use of Circularly Polarized Light on the Evaluation of Injectable Platelet Rich Fibrin in the Early Formation of Woven and Lamellar Bone: A Specific Method in the Study of Osseous Tissue

Purpose: In bone grafting, the goal is the formation of 100% vital bone. The ideal graft material is osteogenic, osteoconductive and osteoinductive and can be evaluated by histology and histomorphometric methods to calculate percentages of vital bone formation, residual graft material and connective tissue. Bone graft substitute materials currently used include allogeneic, xenogeneic and alloplastic materials. The goal of this study is a histological evaluation of the early effects of the liquid (injectable) form of Platelet Rich Fibrin (i-PRF) on the formation of woven and lamellar bone using Circularly Polarized Light (CPL). Patients and Methods: This is a retrospective cohort study of forty-two patients from 2021 to 2022 who completed bone grafting after a non-restorable endodontically treated molar tooth was extracted in preparation for future implant surgery. Each bone graft was augmented with i-PRF. To process i-PRF, Relative Centrifugation Force (RCF) was reduced from 2,700 RPM to 700 RPM (60 g). Centrifugation time was reduced from 12 minutes to 3 minutes. After an average bone graft healing time of 8 to 12 weeks, bone core samples were obtained at the time of implant surgery for histological and histomorphometric analysis. Bone cores were stained with Stevenel’s blue and van Gieson’s picrofuchsin for histologic analysis by means of brightfield and circular polarized light microscopic evaluation to evaluate the early effects of the liquid form of PRF on formation of woven and lamellar bone using allogeneic bone. Results: Forty-two bone cores were harvested at the time of implant surgery for histological and histomorphometric analysis. The liquid form of PRF resulted in greater percentages of vital bone formation (average 87.4 %) compared to the existing published literature. CPL analysis of forty-two bone core specimens showed the positive effects of i-PRF on de novo bone formation. Conclusion: The results of this study using circularly polarized light demonstrate that the liquid form of PRF (i-PRF) resulted in greater percentages of early, new vital bone formation compared to the published literature. Circularly polarized light should be considered as an adjunctive method to determine bone maturity and structure.

Gross and histological studies of the female reproductive tract of the spur-winged goose (<i>Plectropterus gambensis</i>)

The extinction of most wild avian species is becoming apprehensive and some water fowls like the spur-winged goose (Plectropterus gambensis) may represent a good model for research, to advance wildlife conservation of related avian species. Currently, there are no baseline information regarding the anatomic characteristics of the reproductive tract of this species, hence; the present study aimed at investigating the gross and histological features of the ovary and oviduct of the wild spur-winged goose. A total of five female adults spur-winged geese with ages between eight to ten months old were used for this study. The bird were purchased from a farm and transported to the Laboratory for quick decapitation and tissue processing. The birds were allowed to acclimatized for seven days, before humanely sacrificed by quick cervical dislocation and reproductive organs (ovary and oviduct) were harvested for both gross and histological evaluations. Thereafter, the ovary and oviduct were fixed, and stained with H & E for light microscopic evaluation using DB2 -180M Digital Biological Microscope at different magnifications (×40, ×100, and ×400). The gross finding on the ovary revealed that it is ovoid in shape and appeared as a cluster of sacs showing yellowish to whitish follicles of different sizes and have a poorly defined oviductal segment. The oviduct appeared to be the longest part of the female reproductive tract (12.5-14.5cm). Histologically, the ovary is characterized by a membrana granulosa surrounding the oocytes at various stages of development. Also, the oviduct is by simple cuboidal to columnar ciliated secretory epithelium, and connects the ovary to the cloaca as a thin and un-convoluted tube. In conclusion, this study provided valuable information to serve as a baseline model to further research and understand the reproductive anatomy of other related domestic and wild waterfowl species.

Protective role of hesperidin in finasteride-induced testicular toxicity in adult male Wistar rats: Insights into oxidative stress, apoptosis, and ultrastructure of seminiferous tubules

A negative impact of finasteride on fertility has been reported, in which over production of reactive oxygen species and apoptosis were implicated. Hesperidin, a plant-derived bioflavonoid with antioxidant and anti-apoptotic effects, may mitigate these adverse effects. In order to investigate the possible protective role of hesperidin against finasteride-induced seminiferous tubules toxicity in adult male Wistar rats, 60 rats were randomized into five groups (I-V) receiving distilled water, 0.5% sodium carboxymethylcellulose solution, hesperidin, finasteride, and combined hesperidin and finasteride respectively. Testicular weight, sperm count and motility were determined. Testicular tissue homogenates were prepared to measure the level of malondialdehyde (MDA), total antioxidant capacity (TAC), reduced glutathione (GSH) and the gene expression of caspase-3 and B-cell lymphoma 2 (Bcl2). Testes were processed for light and electron microscopic evaluation. Johnsen score was calculated. Administration of finasteride resulted in significantly decreased testicular weights, sperm count and motility, Johnsen score, tissue levels of TAC and GSH together with significant increase in tissue MDA. Gene expression revealed significantly increased caspase-3 and decreased Bcl2. Furthermore, finasteride disrupted the seminiferous tubules, causing degenerative changes affecting Sertoli cells and spermatogenic cells. Co-administration of hesperidin with finasteride resulted in improvement in testicular weights, TAC, GSH, Bcl2, Johnsen score, sperm count and motility as well as preservation of the structure of the seminiferous tubules. To conclude, hesperidin was found to have a protective potential on finasteride-induced oxidative stress, apoptosis and testicular structural damage.

FRI390 The Effects Of Chronic Asprosin Administration On Ovarian Tissue

Abstract Disclosure: H. Kelestimur: None. Z. Oz: None. M. Ozdede: None. I. Serhatlioglu: None. N. Kaya Tektemur: None. E. Kacar: None. Purpose: Asprosin is a novel glucogenic and orexigenic hormone produced by the fibrillin 1 (FBN1) gene that is secreted by white adipose tissue during fasting. Fibrillins (FBNs) are structural and signalling proteins found mainly in the ovarian stroma, tunica albuginea and theca layers. The aim of this study was to determine the effects of asprosin on ovarian tissue in the female rats. Materials and methods: Twenty-four female Sprague-Dawley rats were randomly divided into 2 groups (n=12) as control and asprosin. Asprosin and control groups were given intraperitoneal saline at doses of 500 ng/kg and 1 ml/kg, respectively, at 14.00 every day for eight weeks. After the application, decapitation was performed, and the rapidly removed ovarian tissues were placed in a 10% formaldehyde solution for fixation. Formalin-fixed ovaries were dehydrated in a graded alcohol series and graded ethanol, respectively After that, all tissues were embedded in paraffin wax. The ovaries were cut into 5-µm sections and stained with hematoxylin and eosin (H&E) and also Masson trichrome staining. Ovarian follicular reserve and fibrosis were evaluated by light microscopy. The follicles were histologically classified as primordial, primary, secondary, and Graaf and all follicular structures were counted in each section. The fibrosis was evaluated with Masson’s trichrome staining and scored from 0 to 3 semi-quantitatively (0 = no fibrosis, 1 = low fibrosis, 2 = intermediate fibrosis, 3 = severe fibrosis). Results: In the asprosin group, unlike the control group, occasional congestion was observed in the vessels located in the ovarian medulla. Apart from this, no histopathological changes were found. An increase in fibrosis was detected in the ovarian sections of the asprosin group when compared to the control group (p = 0,035). In the light microscopic evaluation of the ovarian sections of the rats in the asprosin group, the primary and secondary follicle numbers were significantly higher than those in the control group (p = 0,008 and p = 0,047 respectively). However, the difference between the control and asprosin groups in terms of the primordial follicle, Graafian follicle and corpus luteum numbers were not statistically significant. Conclusion: Our results have shown that chronic asprosin administration causes an increase in the number of primary and secondary follicles, suggesting that changes in ovarian steroidogenesis and folliculogenesis depending on the application may lead to positive effects on female fertility. Keywords: Asprosin, adipokine, ovarian follicle. Acknowledgement: This study was supported by TUBITAK (Project: 220S744). Presentation: Friday, June 16, 2023

Basic Three-Dimensional (3D) Intestinal Model System with an Immune Component.

There has been an increase in the use of in vivo and in vitro intestinal models to study the pathophysiology of inflammatory intestinal diseases, for the pharmacological screening of potentially beneficial substances, and for toxicity studies on potentially harmful food components. Of relevance, there is a current demand for the development of cell-based in vitro models to substitute animal models. Here, a protocol for a basic, "healthy tissue" three-dimensional (3D) intestinal equivalent model using cell lines is presented with the dual benefit of providing both experimental simplicity (standardized and easily repeatable system) and physiological complexity (Caco-2 enterocytes with a supporting immune component of U937 monocytes and L929 fibroblasts). The protocol also includes paraffin embedding for light microscopic evaluation of fixed intestinal equivalents, thereby providing the advantage of analyzing multiple visual parameters from a single experiment. Hematoxylin and eosin (H&E) stained sections showing the Caco-2 columnar cells forming a tight and regular monolayer in control treatments are used to verify the efficacy of the model as an experimental system. Using gluten as a pro-inflammatory food component, parameters analyzed from sections include reduced monolayer thickness, as well as disruption and detachment from the underlying matrix (H&E), decreased tight junction protein expression as shown from occludin staining (quantifiable statistically), and immune-activation of migrating U937 cells as evidenced from the cluster of differentiation 14 (CD14) staining and CD11b-related differentiation into macrophages. As shown by using lipopolysaccharide to simulate intestinal inflammation, additional parameters that can be measured are increased mucus staining and cytokine expression (such as midkine) that can be extracted from the medium prior to fixation. The basic three-dimensional (3D) intestinal mucosa model and fixed sections can be recommended for inflammatory status and barrier integrity studies with the possibility of analyzing multiple visual quantifiable parameters.

Protocol for the evaluation of cyclin D1 expression in tumor tissue and surgical margins in oral squamous cell carcinoma – a retrospective study

Oral squamous cell carcinoma (OSCC) is a malignant epithelial tumor with poor prognosis. There is an increase in the proliferation of epithelial cells in OSCC which is control by cell cycle regulators like Cyclin D1. Cyclin D1 is linked to a poor prognosis for patients, including aggressive malignancies, recurrence, protracted remission, etc. To evaluate Cyclin D1 immunoexpression in different grades and surgical margins in OSCC. All tissue sections will be processed for Cyclin D1 IHC. After that light microscopic evaluation of Cyclin D1 expression was classified as weak (scoring 1-4), moderate (score 5-8), or strong based on a final expression score that was obtained by multiplying the labelling index score by the intensity score (score 9–12). The present study will find the immunohistochemical expression of Cyclin D1 in OSCC. There will be variation in the immunoexpression of Cyclin D1 among different grades of OSCC its surgical margin and normal oral mucosa. We hypothesize that: As the disease condition goes towards poor grade, intensity of cyclin D1 expression immunohistochemically may increase. Hence, the intensity can be used as a prognostic marker.

A Method for Eliminating Fibroblast Contamination in Mouse and Human Primary Corneal Epithelial Cell Cultures

Purpose This study was designed to determine if previous approaches to eliminate fibroblast contamination in different cells types would be successful in eliminating fibroblast contamination from human and mouse primary corneal epithelial cell cultures, with the primary goal being to describe a simple, easy, and effective method to culture fibroblast-free primary mouse and human corneal epithelial cell cultures. Methods Primary human and mouse corneal stromal cells and epithelial cells were isolated and cultured from human corneal rims and mouse corneas, respectively. Several approaches previously used in other tissue types were evaluated using corneal epithelial cells and mixtures of fibroblasts and epithelial cells to determine the most effective purification method. Methods evaluated included 0.25% trypsin-EDTA, low temperature, mitomycin-C, and dispase. Degree of fibroblast contamination was examined using light microscopy evaluation of cell phenotype, immunofluorescence and western blotting using cell type-specific markers. Anti-pancytokeratin (PanCK) was used as the epithelial immunofluorescence label, and anti-α smooth muscle actin (αSMA) as the fibroblast immunofluorescence label. Epithelial western blot antibodies included PanCK, keratin 12, and E-cadherin, while αSMA, collagen 1A1 and collagen 3A1 were used to identify fibroblasts. Results Fibroblast contamination of human and mouse primary cornea epithelial cell cultures was best controlled using the 0.25% trypsin-EDTA method. The other methods examined were not effective at eliminating cornea fibroblast contamination. Conclusions Trypsin-EDTA digestion is a simple and effective method for controlling fibroblast contamination of cultured primary human and mouse corneal epithelial cells.

Improvements in morphometric and structural traits of Vitex trifolia L. to exogenous application of nano-silicon in vitro

The present study aimed to analyze the effect of silicon nanoparticles (SiNPs) on the in vitro proliferation of shoots, biomass production, foliar micro-structural features, and root-inducing ability in Vitex trifolia L. Bud breaking was achieved from axillary meristems on 2.5 mg L−1 of 6-benzylaminopurine (BAP) in Murashige and Skoog's (MS) medium. The shoots were proliferated (18.0 ± 0.39 shoots) on 0.75 mg L−1 BAP with 0.2 mg L−1 1-Naphthaleneacetic acid (NAA). Among the varied concentrations of Aerosil300 (hydrophilic silica) used, 6.0 mg L−1 SiNPs with optimal growth regulators into the medium had a positive impact on shoot proliferation (24.0 ± 0.30 shoots) and fresh biomass production. The light microscopic evaluation showed that the leaves developed without SiNPs possessed underdeveloped cuticle, trichomes, stomata, epidermal cells, and photosynthetic and vascular tissues. The SiNPs treatment resulted in well-differentiated tissue systems and favored an increase in width of the lamina, differentiation of palisade and spongy tissues, thickening of cell walls, increased density of trichomes, organized guard cells in stomata, and improved xylem and phloem tissues. The shoots were effectively rooted on half-strength MS medium containing 3.0 mg L−1 NAA, and the shoots derived from SiNPs treatment had the highest rooting efficiency (8.0 roots per shoot). The findings highlight that the use of SiNPs significantly promoted tissue systems and morphology of V. trifolia, which favored the survival of plantlets under in vivo conditions.

Protocol for evaluation and correlation of CD44 immunoexpression in tumor tissue and surgical margins of oral squamous cell carcinoma with three-year survival: A retrospective study

Introduction: The commonest type of cancer in the head and neck region is oral squamous cell carcinoma (OSCC) due to its high rates of occurrence and mortality. Cluster of differentiation 44 (CD44) is a surface glycoprotein present on tumor cells. It regulates cell proliferation, adhesion, migration an invasion of cancer stem cell. Immunoexpression of CD44 in surgical margins indicates poor prognosis of disease and increased risk of local recurrence.[4] Objectives: To evaluate and correlate CD44 immunoexpression in tumor tissue and surgical margins OSCC with three-year survival. Methodology: All tissue sections will be processed for CD44 immunohistochemistry. After that, light microscopic evaluation of CD44 expression will be done. A dark brown color of membranous staining of neoplastic cells will be considered for CD44 positivity; this must be observed in the tumor core, in the basal layer because most stem cells are present in the basal layer of oral mucosa and in the invasive front of tumor. Expected results: The present study will find the immunohistochemical expression of CD44 tumor tissue and surgical margins of OSCC, in order to evaluate the prognosis of OSCC. Immunoexpression may vary in according to their different grades. Conclusions: We hypothesize that, as the disease stage advances, intensity of CD44 expression immunohistochemically increases and it can adversely affect overall survival; hence it is considered as an independent predictor of prognosis of OSCC.

Poster 105: Rotator cuff healing using demineralised bone fibers in ovine infraspinatus tendon models

Objectives: Rotator cuff tears are a common cause of shoulder pain and functional deficits. Although surgical repair is often able to restore function and resolve pain, high rates of postoperative structural failures have been reported. To improve the outcomes and reduce re-tear rates biologic tissue scaffolds have been used to augment and reinforce the primary tendon repair by providing mechanical support of the initial repair and ideally improving the rate and/or quality of biological healing at the tendon-bone interface. Demineralised bone fibers (DBF) are bone collagen and can be formed into a patch providing a scaffold and inductive bone morphogenetic proteins known to facilitate healing. This study evaluated the in vivo response of human DBF Patches (hDBF-P) as an onlay over the infraspinatus tendon (IST) and at the tendon-bone interface in an ovine model. Methods: The hDBF-P was characterised using stereozoom (Figure 1A) and environment electron microscopy (Figure 1B). The osteoinductivity (OI) was evaluated in the standard nude rat intramuscular implantation (IM) model at 28 days using mCT and paraffin histology. A bilateral model of an onlay or an enthesis repair was performed in 12 adult wethers following ethical approval. Four groups were evaluated at 6 and 12 weeks (n=3 per time point): Onlay control and hDBF treated; Enthesis repair control and hDBF treated. The onlay model simulated a tendon injury by 4 longitudinal slits along the IST and lateral drill holes in the proximal humerus in the onlay control group [1]. In the onlay treated group the hDBF-P was hydrated in saline and secured to the IST with interrupted 2-0 non-resorbable sutures (Figure 2A). On contralateral side, the central third of the IST was sharply dissected, the footprint burred, and 2 medial row anchors inserted. The sutures were passed through the IST in the enthesis repair control group. In the enthesis repair treatment group, the sutures were passed through the hDBF-P which was placed at the tendon bone interface followed by sutures through the IST. Two lateral push lock anchors were used for a suture bridge (Figure 2B) in the control and treated groups. Endpoints included clinical recovery, macroscopic dissection, 3T MRI, radiographs, mCT and paraffin histology processing, H&E staining, and light microscopic evaluations to assess local cell and tissue responses and healing. Results: OI was confirmed on IM implantation in nude rats. The sheep recovered uneventfully with no adverse events macroscopically or radiographically. MRI in the onlay control group revealed little response and no increase in tendon thickness which was confirmed histologically. In contrast the onlay hDBF-P treated demonstrated increased tendon thickness and hDBF resorption with time. Histology showed some initial inflammatory cells that decrease with time in the onlay group. MRI in the hDBF-P treated enthesis group supported increased activity at the bone interface at 6 and 12 weeks compared to controls. Histology at the interface with hDBF-P showed evidence of new bone formation at 6 weeks that matured with time support enthesis reformation. Conclusions: hDBF-P is a collagen graft composed of demineralized bone fibers that have osteoconductive and osteoinductive potential. When formed into a patch, this implant provides a scaffold that can participate with the local biology. hDBF-P used as on-lay in a tendon injury model increased tendon thickness. hDBF-P when used at the interface between the tendon and bone facilitated enthesis reformation through its conductive and inductive potential as well as scaffold architecture. DBF patches represent an exciting allograft implant with the potential to address the clinical problems in rotator cuff surgery. Literature cited Van Kampen, C., et al. (2013). Tissue-engineered augmentation of a rotator cuff tendon using a reconstituted collagen scaffold: a histological evaluation in sheep. Muscles, ligaments and tendons journal, 3(3), p.229.

#4694 AUTOMATIC SEGMENTATION OF GLOMERULAR SUBSTRUCTURES BY DEEP LEARNING

Abstract Background and Aims Electron microscopy (EM) complements light microscopy (LM) evaluation of the kidney biopsy. Foot process effacement, as assessed by EM, helps in diagnosing podocytopathies. However, human interpretation of EM images is time-intensive and often subjective. In this pilot, we investigate how deep learning techniques can help in adequate segmentation of the glomerular basement membranes (GBM) and podocytes in EM images. The ultimate goal would be to design an automatic tool for reliable and fast assessment of foot process effacement. Method Podocytes and glomerular basement membranes (GBM) of 10 patients, 5 with podocyte disease and 5 without glomerular changes (where biopsy showed tubulointerstitial pathology on LM) were annotated with a combination of manual annotation and thresholding for providing ground truth. After data preprocessing including splitting, flipping, rotating and tiling, a modified U-net architecture was applied (‘baseline model’). This baseline model was compared to a combination of U-net and a self-supervised contrastive learning approach with pretraining on 100 additional images (SimCLR framework, ‘fine-tuned model’). Segmentation performance was measured by IoU score. Results Segmentation of the glomerular basement membrane was best achieved by the baseline model and resulted in an IoU score of 0.711±0.089 compared to a IoU score of 0.675 ±0.093 in the fine-tuned model Segmentation of the podocytes was most successful in the fine-tuned model, with a IoU score of 0.609±0.118 compared to a IoU score of 0.591 ±0.118 in the baseline model Conclusion This study pioneers in segmenting glomerular substructures on EM images by means of a modified U-net architecture. The next step is training and validation in larger datasets. Data annotation remains a challenge. Inclusion of more images is expected to greatly improve the performance of the model. Guideline 1500-3600 characters

Endometrial Staining of CD56 (Uterine Natural Killer), BCL-6, and CD138 (Plasma Cells) Improve Diagnosis and Clinical Pregnancy Outcomes in Unexplained Infertility and Recurrent IVF Failures: Standardization of Diagnosis with Digital Pathology.

In women with unexplained infertility (UI) and recurrent in vitro fertilization (IVF) failures, the etiology is often unclear. Endometrial immune perturbations and the use of immune markers associated with these dysregulations are of great interest in the diagnosis and treatment of UI. However, reliable biomarkers and standardized quantification methods are lacking. Here, to address endometrial immune dysregulation in UI patients with recurrent IVF failures, we performed endometrial tissue sampling and immunostaining of CD56 (uNK), CD138, and BCL-6. Of these cases, 57.9% had positive CD56 in the endometrial stroma, while 46.1% had positive BCL-6 in the glandular epithelium, and 14.5% of the cases were found to be positive for CD138. Combined staining rates were 60.5%, 68.4%, and 71.05% for (CD56 or BCL-6), (CD56 or CD138), and (CD56, BCL-6, or CD138), respectively. There was a significant correlation between CD56 and BCL-6 positivity, while CD138 positivity was an independent parameter. After the recommended targeted therapy, pregnancy rates were found to increase from 58.5% to 61.6% and 73.8% in CD56-positive, (CD56- or BCL-6-positive), and (CD56-, BCL-6-, or CD138-positive) cases, respectively. Notably, a retrospective evaluation of digital pathology and light microscopy results showed a significant correlation. This study suggests that the examination of CD56, BCL-6, and CD138 in the same endometrial sample may be an effective method in determining the etiology of UI and reaching an early diagnosis and treatment options. Moreover, digital pathology can be used in the evaluation of CD56 and BCL-6 to provide objective, rapid, and reliable results.

Morphological characteristics of the placenta of Balami and Yankasa ewes at different stages of gestation in Maiduguri, Nigeria

The morphological characteristics of the placenta of Yankasa and Balami ewes were evaluated. Uteri of 20 pregnant Yankasa and Balami ewes were collected as abattoir waste from Maiduguri Municipal abattoir and were placed in containers containing 10% neutral buffered formalin. Using the crown-rump length (CRL) method of estimating the ages of the foetuses, they were grouped into three gestational stages. In both breeds, 50% of pregnancy occurred in the left uterine horn; in the Yankasa, 20% and 30% occur in the right uterine horn, and both (twining), respectively. While in the Balami, 40% and 10% occurred in the right and both (twinning) uterine horns, respectively. The results showed that the placenta-maternal contact in both breeds was fragile in the first stage, firm in the second stage, and loose in the third stage. The results showed that there were significant changes (P<0.001) in the mean weight, length, and width of the placentomes in all three stages. Three types of placenta appearances were identified in the Yankasa ewe: concave, convex, and flat, while two types of appearances in the Balami ewe were concave and convex. In the light microscopy evaluation, the trophoblastic epithelia of both ewes were composed of mononucleated, binucleated cells and syncytia. Endometrial glands were also observed in the inter-placental space. In conclusion, this study has shown that in both breeds, the cotyledonary surfaces of the placentae were mostly concave, followed by convex, and a mixture of the two (concave/convex). The trophoblast epithelia are positioned to control the movement of substances across foetal and maternal tissues. Even though little is still known about the regulation of these cells in the trophoblast epithelia, they are believed to be responsible for the synthesis of hormones such as lactogen for the maintenance of the pregnancy before parturition.

Corneal Guttae After Descemet Membrane Endothelial Keratoplasty.

The aim of this study was to report on the occurrence of corneal guttae after Descemet membrane endothelial keratoplasty (DMEK). In this retrospective case series, 13 eyes of 13 patients who underwent DMEK at 2 tertiary referral centers between 2007 and 2021 (average available follow-up 73 ± 52 months, range 18-174 months) and showed corneal guttae during postoperative examinations were included. Eye bank images were retrospectively reviewed. Occurrence of guttae was observed by specular microscopy in 13 eyes. In 11 cases, presence of guttae was confirmed by confocal microscopy and in 1 case by histology. Five eyes showed an increase in guttae density during the postoperative course. Surgery indications were Fuchs endothelial corneal dystrophy (n = 11), pseudophakic bullous keratopathy (n = 1), and DMEK graft failure after allograft rejection (n = 1); the latter eye had shown no signs of guttae after primary DMEK. Two eyes with guttae required a repeat DMEK due to graft failure. At the last available follow-up, all 11 remaining eyes had clear corneas and 10 eyes had a best-corrected visual acuity of ≥0.9 (decimal). During donor cornea processing in the eye bank, no guttae were observed on the donor tissue. Corneal guttae can occur after DMEK including in eyes operated for indications other than Fuchs endothelial corneal dystrophy and most likely guttae were present on the donor graft but were not detectable by routine slit-lamp and light microscopy evaluation in the eye bank. Postoperative guttae density varies among patients and especially small isolated guttae do not seem to affect clinical outcomes.

Diagnosis of Anaplasma marginale in cattle at the Iowa State University veterinary diagnostic laboratory 2003–2021

Anaplasma marginale is a blood-borne rickettsia-like organism that infects cattle erythrocytes and causes anaplasmosis. This study reviews diagnostic data of all A. marginale diagnostics performed from 2003 to August 2021 in the Iowa State Veterinary Diagnostic Laboratory. Typically, the referring veterinarian's initial tentative diagnosis was based on presenting clinical signs or necropsy findings. Confirmatory testing at the ISU-VDL consisted of light microscopy evaluation of stained blood smears or molecular diagnostic procedures. A total of 94 cases were submitted with tissue samples from deceased animals, of which 79 were from Iowa and 15 were from other states. The most typical gross lesions were widespread yellow adipose tissue and splenomegaly. Typical histopathological lesions included marked bile stasis and hemosiderin-laden macrophages in the liver and spleen, respectively. Starting in 2013, when PCR was implemented to confirm cases of anaplasmosis, 315/1125 (28%) were positive to A. marginale, and 810 were negative, using a cut-off of 35.0 Ct. The average (±SD) of the positive PCR Ct was 19.5 (±6.0), and the first and third quartiles were 14.9 and 23.4. Most cases occurred between August and November, peaking in September, whether from necropsies or positive blood samples by PCR. The most common tick observed in Iowa, Dermacentor variabilis, is likely the main vector for transmission. Further surveys should be conducted to estimate seroprevalence by geographical location, the density of cattle populations, distribution of known vectors according to season, and strains of A. marginale.