Current applications of ex-vivo fluorescent confocal microscope in urological practice: a systematic review of literature.

BACKGROUND: Histopathological examination, a cornerstone in diagnosing cancer, faces challenges due to its time-consuming nature. This review explores the potential of ex-vivo fluorescent confocal microscopy (FCM) in urology, addressing the need for real-time pathological assessment, particularly in prostate cancer. This systematic review aims to assess the applications of FCM in urology, including its role in prostate cancer diagnosis, surgical margin assessment, and other urological fields. METHODS: Following Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines, a systematic search of PubMed and SCOPUS was conducted, focusing on English written original articles published after January 1, 2018, discussing the use of FCM in urological practice. The search included keywords related to FCM and urological terms. The risk of bias assessment was performed using Quality Assessment of Diagnostic Accuracy Studies-2 (QUADAS-2) tool. RESULTS: A total of 17 relevant studies were included in the review that focuses on three main urological issues: prostate cancer (15 articles), bladder cancer (1 article), and renal biopsy (1 article). FCM exhibited significant promise in diagnosing prostate cancer. These studies reported an accuracy range of 85.33% to 95.1% in distinguishing between cancerous and non-cancerous prostate tissues. Moreover, FCM proved valuable for assessing surgical margins in real-time during radical prostatectomy, reducing the need for frozen section analysis. In some investigations, researchers explored the integration of artificial intelligence (AI) with FCM to automate diagnostic processes. Concerning bladder cancer, FCM played a beneficial role in evaluating urethral and ureteral margins during radical cystectomy. Notably, it showed substantial agreement with conventional histopathology and frozen section examination. In the context of renal biopsy, FCM demonstrated the potential to differentiate normal renal parenchyma from cancerous tissue, although the available evidence is limited in this area. The main limitation of the current study is the scarcity of data regarding the topic of interest. CONCLUSIONS: Ex-vivo FCM holds promise in urology, particularly in prostate cancer diagnosis and surgical margin assessment. Its real-time capabilities may reduce diagnostic delays and patient stress. However, most studies remain experimental, requiring further research to validate clinical utility.

Zirconia Hybrid Dental Implants Influence the Biological Properties of Neural Crest-Derived Mesenchymal Stromal Cells.

Dental implants are regularly employed in tooth replacement, the good clinical outcome of which is strictly correlated to the choice of an appropriate implant biomaterial. Titanium-based implants are considered the gold standard for rehabilitation of edentulous spaces. However, the insurgence of allergic reactions, cellular sensitization and low integration with dental and gingival tissues lead to poor osseointegration, affecting the implant stability in the bone and favoring infections and inflammatory processes in the peri-implant space. These failures pave the way to develop and improve new biocompatible implant materials. CERID dental implants are made of a titanium core embedded in a zirconium dioxide ceramic layer, ensuring absence of corrosion, a higher biological compatibility and a better bone deposition compared to titanium ones. We investigated hDPSCs' biological behavior, i.e., cell adhesion, proliferation, morphology and osteogenic potential, when seeded on both CERID and titanium implants, before and after cleansing with two different procedures. SEM and AFM analysis of the surfaces showed that while CERID disks were not significantly affected by the cleansing system, titanium ones exhibited well-visible modifications after brush treatment, altering cell morphology. The proliferation rate of DPSCs was increased for titanium, while it remained unaltered for CERID. Both materials hold an intrinsic potential to promote osteogenic commitment of neuro-ectomesenchymal stromal cells. Interestingly, the CERID surface mitigated the immune response by inducing an upregulation of anti-inflammatory cytokine IL-10 on activated PBMCs when a pro-inflammatory microenvironment was established. Our in vitro results pave the way to further investigations aiming to corroborate the potential of CERID implants as suitable biomaterials for dental implant applications.

Human dental pulp stem cells (hDPSCs) promote the lipofibroblast transition in the early stage of a fibro-inflammatory process.

Introduction: In autoimmune diseases, particularly in systemic sclerosis and chronic periaortitis, a strict correlation between chronic inflammation and fibrosis exists. Since the currently used drugs prove mostly effective in suppressing inflammation, a better comprehension of the molecular mechanisms exerted by cell types implicated in fibro-inflammation is needed to develop novel therapeutic strategies. Mesenchymal stromal/stem cells (MSCs) are being matter of deep investigation to unveil their role in the evolution of fibrogenetic process. Several findings pointed out the controversial implication of MSCs in these events, with reports lining at a beneficial effect exerted by external MSCs and others highlighting a direct contribution of resident MSCs in fibrosis progression. Human dental pulp stem cells (hDPSCs) have demonstrated to hold promise as potential therapeutic tools due to their immunomodulatory properties, which strongly support their contribution to tissue regeneration. Methods: Our present study evaluated hDPSCs response to a fibro-inflammatory microenvironment, mimicked in vitro by a transwell co-culture system with human dermal fibroblasts, at early and late culture passages, in presence of TGF-ß1, a master promoter of fibrogenesis. Results and Discussion: We observed that hDPSCs, exposed to acute fibro-inflammatory stimuli, promote a myofibroblast-to-lipofibroblast transition, likely based on BMP2 dependent pathways. Conversely, when a chronic fibro-inflammatory microenvironment is generated, hDPSCs reduce their anti-fibrotic effect and acquire a pro-fibrotic phenotype. These data provide the basis for further investigations on the response of hDPSCs to varying fibro-inflammatory conditions.

Wound Healing after Acellular Dermal Substitute Positioning in Dermato-Oncological Surgery: A Prospective Comparative Study.

BACKGROUND: MatriDerm and Integra are both widely used collagenic acellular dermal matrices (ADMs) in the surgical setting, with similar characteristics in terms of healing time and clinical indication. The aim of the present study is to compare the two ADMs in terms of clinical and histological results in the setting of dermato-oncological surgery. METHODS: Ten consecutive patients with medical indications to undergo surgical excision of skin cancers were treated with a 2-step procedure at our Dermatologic Surgery Unit. Immediately after tumor removal, both ADMs were positioned on the wound bed, one adjacent to the other. Closure through split-thickness skin grafting was performed after approximately 3 weeks. Conventional histology, immunostaining and ELISA assay were performed on cutaneous samples at different timepoints. RESULTS: No significant differences were detected in terms of either final clinical outcomes or in extracellular matrix content of the neoformed dermis. However, Matriderm was observed to induce scar retraction more frequently. In contrast, Integra was shown to carry higher infectious risk and to be more slowly reabsorbed into the wound bed. Sometimes foreign body-like granulomatous reactions were also observed, especially in Integra samples. CONCLUSIONS: Even in the presence of subtle differences between the ADMs, comparable global outcomes were demonstrated after dermato-oncological surgery.

Flow-dependent shear stress affects the biological properties of pericyte-like cells isolated from human dental pulp.

BACKGROUND: Human dental pulp stem cells represent a mesenchymal stem cell niche localized in the perivascular area of dental pulp and are characterized by low immunogenicity and immunomodulatory/anti-inflammatory properties. Pericytes, mural cells surrounding the endothelium of small vessels, regulate numerous functions including vessel growth, stabilization and permeability. It is well established that pericytes have a tight cross talk with endothelial cells in neoangiogenesis and vessel stabilization, which are regulated by different factors, i.e., microenvironment and flow-dependent shear stress. The aim of this study was to evaluate the effects of a pulsatile unidirectional flow in the presence or not of an inflammatory microenvironment on the biological properties of pericyte-like cells isolated from human dental pulp (hDPSCs). METHODS: Human DPSCs were cultured under both static and dynamic conditions with or without pre-activated peripheral blood mononuclear cells (PBMCs). Pulsatile unidirectional flow shear stress was generated by using a specific peristaltic pump. The angiogenic potential and inflammatory properties of hDPSCs were evaluated through reverse phase protein microarrays (RPPA), confocal immunofluorescence and western blot analyses. RESULTS: Our data showed that hDPSCs expressed the typical endothelial markers, which were up-regulated after endothelial induction, and were able to form tube-like structures. RPPA analyses revealed that these properties were modulated when a pulsatile unidirectional flow shear stress was applied to hDPSCs. Stem cells also revealed a downregulation of the immune-modulatory molecule PD-L1, in parallel with an up-regulation of the pro-inflammatory molecule NF-kB. Immune-modulatory properties of hDPSCs were also reduced after culture under flow-dependent shear stress and exposure to an inflammatory microenvironment. This evidence was strengthened by the detection of up-regulated levels of expression of pro-inflammatory cytokines in PBMCs. CONCLUSIONS: In conclusion, the application of a pulsatile unidirectional flow shear stress induced a modulation of immunomodulatory/inflammatory properties of dental pulp pericyte-like cells.

PEDOT: PSS promotes neurogenic commitment of neural crest-derived stem cells.

Poly (3,4-ethylendioxythiophene) polystyrene sulphonate (PEDOT:PSS) is the workhorse of organic bioelectronics and is steadily gaining interest also in tissue engineering due to the opportunity to endow traditional biomaterials for scaffolds with conductive properties. Biomaterials capable of promoting neural stem cell differentiation by application of suitable electrical stimulation protocols are highly desirable in neural tissue engineering. In this study, we evaluated the adhesion, proliferation, maintenance of neural crest stemness markers and neurogenic commitment of neural crest-derived human dental pulp stem cells (hDPSCs) cultured on PEDOT:PSS nanostructured thin films deposited either by spin coating (SC-PEDOT) or by electropolymerization (ED-PEDOT). In addition, we evaluated the immunomodulatory properties of hDPSCs on PEDOT:PSS by investigating the expression and maintenance of the Fas ligand (FasL). We found that both SC-PEDOT and ED-PEDOT thin films supported hDPSCs adhesion and proliferation; however, the number of cells on the ED-PEDOT after 1 week of culture was significantly higher than that on SC-PEDOT. To be noted, both PEDOT:PSS films did not affect the stemness phenotype of hDPSCs, as indicated by the maintenance of the neural crest markers Nestin and SOX10. Interestingly, neurogenic induction was clearly promoted on ED-PEDOT, as indicated by the strong expression of MAP-2 and ß -Tubulin-III as well as evident cytoskeletal reorganisation and appreciable morphology shift towards a neuronal-like shape. In addition, strong FasL expression was detected on both undifferentiated or undergoing neurogenic commitment hDPSCs, suggesting that ED-PEDOT supports the expression and maintenance of FasL under both expansion and differentiation conditions.

Effects of Energy Drink Acute Assumption in Gastrointestinal Tract of Rats.

Energy drinks (EDs) are non-alcoholic beverages containing high amounts of caffeine and other psychoactive substances. EDs also contain herbal extract whose concentration is usually unknown. EDs can have several adverse effects on different organs and systems, but their effects on the gastrointestinal (GI) tract have been poorly investigated. To determine the acute effects of EDs on the GI tract, we administered EDs, coffee, soda cola, or water to Sprague-Dawley rats (n = 7 per group, randomly assigned) for up to five days, and analyzed the histopathological changes in the GI tract. Data were compared among groups by Kruskal-Wallis or Mann-Whitney tests. We found that, while EDs did not cause any evident acute lesion to the GI tract, they triggered eosinophilic infiltration in the intestinal mucosa; treatment with caffeine alone at the same doses found in EDs leads to the same effects, suggesting that it is caffeine and not other substances present in the EDs that causes this infiltration. The interruption of caffeine administration leads to the complete resolution of eosinophilic infiltration. As no systemic changes in pro-inflammatory or immunomodulating molecules were observed, our data suggest that caffeine present in ED can cause a local, transient inflammatory status that recruits eosinophils.

Role of PD-L1 in licensing immunoregulatory function of dental pulp mesenchymal stem cells.

BACKGROUND: Dental pulp stem cells (DPSCs) are low immunogenic and hold immunomodulatory properties that, along with their well-established multi-potency, might enhance their potential application in autoimmune and inflammatory diseases. The present study focused on the ability of DPSCs to modulate the inflammatory microenvironment through PD1/PD-L1 pathway. METHODS: Inflammatory microenvironment was created in vitro by the activation of T cells isolated from healthy donors and rheumatoid arthritis (RA) patients with anti-CD3 and anti-CD28 antibodies. Direct and indirect co-cultures between DPSCs and PBMCs were carried out to evaluate the activation of immunomodulatory checkpoints in DPSCs and the inflammatory pattern in PBMCs. RESULTS: Our data suggest that the inflammatory stimuli trigger DPSCs immunoregulatory functions that can be exerted by both direct and indirect contact. As demonstrated by using a selective PD-L1 inhibitor, DPSCs were able to activate compensatory pathways targeting to orchestrate the inflammatory process by modulating pro-inflammatory cytokines in pre-activated T lymphocytes. The involvement of PD-L1 mechanism was also observed in autologous inflammatory status (pulpitis) and after direct exposure to pre-activated T cells from RA patients suggesting that immunomodulatory/anti-inflammatory properties are strictly related to their stemness status. CONCLUSIONS: Our findings point out that the communication with the inflammatory microenvironment is essential in licensing their immunomodulatory properties.

GD2 CAR T cells against human glioblastoma.

Glioblastoma is the most malignant primary brain tumor and is still in need of effective medical treatment. We isolated patient-derived glioblastoma cells showing high GD2 antigen expression representing a potential target for CAR T strategy. Data highlighted a robust GD2 CAR antitumor potential in 2D and 3D glioblastoma models associated with a significant and CAR T-restricted increase of selected cytokines. Interestingly, immunosuppressant TGF ß1, expressed in all co-cultures, did not influence antitumor activity. The orthotopic NOD/SCID models using primary glioblastoma cells reproduced human histopathological features. Considering still-conflicting data on the delivery route for targeting brain tumors, we compared intracerebral versus intravenous CAR T injections. We report that the intracerebral route significantly increased the length of survival time in a dose-dependent manner, without any side effects. Collectively, the proposed anti-GD2 CAR can counteract human glioblastoma potentially opening a new therapeutic option for a still incurable cancer.

Current and future perspectives of digital microscopy with fluorescence confocal microscope for prostate tissue interpretation: a narrative review.

Fluorescence confocal microscopy (FCM) is an optical imaging technique providing digital microscopical images of fresh tissue in a real time fashion, without conventional processing. FCM has been widely applied in several fields of dermatology, including the detection of basal cell carcinoma and of cutaneous inflammatory diseases. The aim of the paper is to provide an overview of FCM applications in the field of prostate tissue interpretation and prostate cancer (PCa) detection. A Literature search (PubMed & Web of Science) was performed to identify articles concerned with the clinical and surgical applications of FCM in prostatic and periprostatic tissues interpretation. Overall, six articles were identified. All articles investigated the level of agreement between FCM and conventional histopathological analysis (hematoxylin-eosin, HE) for the discrimination between normal and PCa tissues. An investigative article on prostate samples retrieved from radical prostatectomy (RP) specimens and an atlas of FCM digital images from the same series were found. Two prospective clinical trials, comparing FCM and HE, pointed out a "substantial" to "almost perfect" discriminative performance of FCM for the diagnosis of PCa on prostate biopsy core. Finally, two studies investigated the intra-operative role of FCM during RP for the control of surgical dissection. In this setting, FCM could be used to analyse samples retrieved from suspicious peri-prostatic areas; FCM has also been tested for an en-face evaluation of flat slices obtained from the systematic sampling of the posterolateral aspects of the prostate, in a NeuroSAFE-like approach. Generally, FCM provides digital microscopical images of fresh tissue in a real time fashion, without requiring conventional processing. Currently, available studies confirmed a high concordance with conventional pathology for the detection of PCa. Further studies are required to validate the technology, to evaluate ISUP score attribution and to implement the fields of application of FCM for the treatment of prostate diseases.

Evaluation of Antimicrobial Effect of Air-Polishing Treatments and Their Influence on Human Dental Pulp Stem Cells Seeded on Titanium Disks.

Dental implants are one of the most frequently used treatment options for tooth replacement, and titanium is the metal of choice due to its demonstrated superiority in resisting corrosion, lack of allergic reactions and mechanical strength. Surface roughness of titanium implants favors the osseointegration process; nevertheless, its topography may provide a suitable substrate for bacterial biofilm deposition, causing peri-implantitis and leading to implant failure. Subgingival prophylaxis treatments with cleansing powders aimed to remove the bacterial accumulation are under investigation. Two different air-polishing powders-glycine and tagatose-were assayed for their cleaning and antimicrobial potential against a Pseudomonas biofilm and for their effects on human dental pulp stem cells (hDPSCs), seeded on sandblasted titanium disks. Immunofluorescence analyses were carried out to evaluate cell adhesion, proliferation, stemness and osteogenic differentiation. The results demonstrate that both the powders have a great in vitro cleaning potential in the early period and do not show any negative effects during hDPSCs osteogenic differentiation process, suggesting their suitability for enhancing the biocompatibility of titanium implants. Our data suggest that the evaluated cleansing systems reduce microbial contamination and allow us to propose tagatose as an adequate alternative to the gold standard glycine for the air-polishing prophylaxis treatment.

Digital Biopsy with Fluorescence Confocal Microscope for Effective Real-time Diagnosis of Prostate Cancer: A Prospective, Comparative Study.

BACKGROUND: A microscopic analysis of tissue is the gold standard for cancer detection. Hematoxylin-eosin (HE) for the reporting of prostate biopsy (PB) is conventionally based on fixation, processing, acquisition of glass slides, and analysis with an analog microscope by a local pathologist. Digitalization and real-time remote access to images could enhance the reporting process, and form the basis of artificial intelligence and machine learning. Fluorescence confocal microscopy (FCM), a novel optical technology, enables immediate digital image acquisition in an almost HE-like resolution without requiring conventional processing. OBJECTIVE: The aim of this study is to assess the diagnostic ability of FCM for prostate cancer (PCa) identification and grading from PB. DESIGN, SETTING, AND PARTICIPANTS: This is a prospective, comparative study evaluating FCM and HE for prostate tissue interpretation. PBs were performed (March to June 2019) at a single coordinating unit on consecutive patients with clinical and laboratory indications for assessment. FCM digital images (n = 427) were acquired immediately from PBs (from 54 patients) and stored; corresponding glass slides (n = 427) undergoing the conventional HE processing were digitalized and stored as well. A panel of four international pathologists with diverse background participated in the study and was asked to evaluate all images. The pathologists had no FCM expertise and were blinded to clinical data, HE interpretation, and each other's evaluation. All images, FCM and corresponding HE, were assessed for the presence or absence of cancer tissue and cancer grading, when appropriate. Reporting was gathered via a dedicated web platform. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The primary endpoint is to evaluate the ability of FCM to identify cancer tissue in PB cores (per-slice analysis). FCM outcomes are interpreted by agreement level with HE (K value). Additionally, either FCM or HE outcomes are assessed with interobserver agreement for cancer detection (presence vs absence of cancer) and for the discrimination between International Society of Urologic Pathologists (ISUP) grade = 1 and ISUP grade > 1 (secondary endpoint). RESULTS AND LIMITATIONS: Overall, 854 images were evaluated from each pathologist. PCa detection of FCM was almost perfectly aligned with HE final reports (95.1% of correct diagnosis with FCM, κ = 0.84). Inter-rater agreement between pathologists was almost perfect for both HE and FCM for PCa detection (0.98 for HE, κ = 0.95; 0.95 for FCM, κ = 0.86); for cancer grade attribution, only a moderate agreement was reached for both HE and FCM (HE, κ = 0.47; FCM, κ = 0.49). CONCLUSIONS: FCM provides a microscopic, immediate, and seemingly reliable diagnosis for PCa. The real-time acquisition of digital images-without requiring conventional processing-offers opportunities for immediate sharing and reporting. FCM is a promising tool for improvements in cancer diagnostic pathways. PATIENT SUMMARY: Fluorescence confocal microscopy may provide an immediate, microscopic, and apparently reliable diagnosis of prostate cancer on prostate biopsy, overcoming the standard turnaround time of conventional processing and interpretation.

Effects of a Novel Bioactive Glass Composition on Biological Properties of Human Dental Pulp Stem Cells.

Functional reconstruction of bone defects represents a clinical challenge in the regenerative medicine field, which targets tissue repair following traumatic injuries and disease-related bone deficiencies. In this regard, the optimal biomaterial should be safe, biocompatible and tailored in order to promote the activation of host progenitor cells towards bone repair. Bioactive glasses might be suitable biomaterials due to their composition being able to induce the host healing response and, eventually, anti-bacterial properties. In this study we investigated whether and how an innovative bioactive glass composition, called BGMS10, may affect cell adhesion, morphology, proliferation, immunomodulation and osteogenic differentiation of human dental pulp stem cells (hDPSCs). When cultured on BGMS10, hDPSCs maintained their proliferation rate and typical fibroblast-like morphology, showing the expression of stemness markers STRO-1 and c-Kit. Moreover, the expression of FasL, a key molecule in mediating immunomodulation effects of hDPSCs, was maintained. BGMS10 also proved to trigger osteogenic commitment of hDPSCs, as confirmed by the activation of bone-related transcription factors RUNX2 and Osx and the ongoing deposition of extracellular matrix supported by the expression of OPN and OCN. Our findings suggest that BGMS10 not only maintains the typical biological and immunomodulatory properties of hDPSCs but also favors the osteogenic commitment.

Modulation of Cell Death and Promotion of Chondrogenic Differentiation by Fas/FasL in Human Dental Pulp Stem Cells (hDPSCs).

Human dental pulp stem cells (hDPSCs) are characterized by high proliferation rate, the multi-differentiation ability and, notably, low immunogenicity and immunomodulatory properties exerted through different mechanisms including Fas/FasL pathway. Despite their multipotency, hDPSCs require particular conditions to achieve chondrogenic differentiation. This might be due to the perivascular localization and the expression of angiogenic marker under standard culture conditions. FasL stimulation was able to promote the early induction of chondrogenic commitment and to lead the differentiation at later times. Interestingly, the expression of angiogenic marker was reduced by FasL stimulation without activating the extrinsic apoptotic pathway in standard culture conditions. In conclusion, these findings highlight the peculiar embryological origin of hDPSCs and provide further insights on their biological properties. Therefore, Fas/FasL pathway not only is involved in determining the immunomodulatory properties, but also is implicated in supporting the chondrogenic commitment of hDPSCs.

Assessment of the In Vivo Antioxidant Activity of an Anthocyanin-Rich Bilberry Extract Using the Caenorhabditis elegans Model.

Anthocyanins have been associated with several health benefits, although the responsible mechanisms are not well established yet. In the present study, an anthocyanin-rich extract from bilberry (Vaccinium myrtillus L.) was tested in order to evaluate its capacity to modulate reactive oxygen species (ROS) production and resistance to thermally induced oxidative stress, using the nematode Caenorhabditis elegans as an in vivo model. The assays were carried out with the wild-type N2 strain and the mutant strains daf-16(mu86) I and hsf-1(sy441), which were grown in the presence of two anthocyanin extract concentrations (5 and 10 µg/mL in the culture medium) and further subjected to thermal stress. The treatment with the anthocyanin extract at 5 µg/mL showed protective effects on the accumulation of ROS and increased thermal resistance in C. elegans, both in stressed and non-stressed young and aged worms. However, detrimental effects were observed in nematodes treated with 10 µg/mL, leading to a higher worm mortality rate compared to controls, which was interpreted as a hormetic response. These findings suggested that the effects of the bilberry extract on C. elegans might not rely on its direct antioxidant capacity, but other mechanisms could also be involved. Additional assays were performed in two mutant strains with loss-of-function for DAF-16 (abnormal DAuer Formation factor 16) and HSF-1 (Heat Shock Factor 1) transcription factors, which act downstream of the insulin/insulin like growth factor-1 (IGF-1) signaling pathway. The results indicated that the modulation of these factors could be behind the improvement in the resistance against thermal stress produced by bilberry anthocyanins in young individuals, whereas they do not totally explain the effects produced in worms in the post-reproductive development stage. Further experiments are needed to continue uncovering the mechanisms behind the biological effects of anthocyanins in living organisms, as well as to establish whether they fall within the hormesis concept.

Positive surgical margin during radical prostatectomy: overview of sampling methods for frozen sections and techniques for the secondary resection of the neurovascular bundles.

OBJECTIVE: The aim of the paper is to provide an overview of intraoperative sampling methods for frozen section (FS) analysis and of surgical techniques for a secondary neurovascular bundle (NVB) resection, as the method of surgical margin (SM) sampling and the management of a positive SM (PSM) at the nerve-sparing (NS) area are under evaluated issues. FS analysis during radical prostatectomy (RP) can help to tailor the plane of dissection based on cancer extension and thus extend the indications for NS surgery. EVIDENCE ACQUISITION: We performed a PubMed/Medical Literature Analysis and Retrieval System Online (MEDLINE), Web of Science, Cochrane Library, and Elton B. Stephens Co. (EBSCO)host search to include articles published in the last decade, evaluating FS analysis in the NS area and surgical attempts to convert a PSM to a negative status. EVIDENCE SYNTHESIS: Overall, 19 papers met our inclusion criteria. The ways to collect samples for FS analysis included: systematic (analysing the whole posterolateral aspect of the prostate specimen, i.e., neurovascular structure-adjacent frozen-section examination [NeuroSAFE]); magnetic resonance imaging (MRI)-guided (biopsies from MRI-suspicious areas, retrieved by the surgeon in a cognitive way); and random biopsies from the soft periprostatic tissues. Techniques to address a PSM in the NS area included: full resection of the spared NVB, from its caudal to cranial aspect, often including the rectolateral part of the Denonvilliers' fascia; partial resection of the NVB, in cases where sampling attempts to localise a PSM; incremental approach, meaning a partial or full resection that extends until no prostate tissue is found in the soft periprostatic environment. CONCLUSIONS: There is no homogeneity in prostate sampling for FS analysis, although most recent evidence is moving toward a systematic sampling of the entire NS area. The management of a PSM is variable and can be affected by the sampling strategy (difficult localisation of the persisting tumour at the NVB). The difficult identification of the exact soft tissue location contiguous to a PSM could be considered as the critical point of FS analysis and of spared-NVB management.

Ex vivo fluorescence confocal microscopy: prostatic and periprostatic tissues atlas and evaluation of the learning curve.

Ex vivo fluorescence confocal microscopy (FCM) is an optical technology that provides fast H&E-like images of freshly excised tissues, and it has been mainly used for "real-time" pathological examination of dermatological malignancies. It has also shown to be a promising tool for fast pathological examination of prostatic tissues. We aim to create an atlas for FCM images of prostatic and periprostatic tissues to facilitate the interpretation of these images. Furthermore, we aimed to evaluate the learning curve of images interpretation of this new technology. Eighty fresh and unprepared biopsies obtained from radical prostatectomy specimens were evaluated using the FCM VivaScope® 2500 M-G4 (Mavig GmbH, Munich, Germany; Caliber I.D.; Rochester NY, USA) by two pathologists. Images of FCM with the corresponding H&E are illustrated to create the atlas. Furthermore, the two pathologists were asked to re-evaluate the 80 specimens after 90 days interval in order to assess the learning curve of images' interpretation of FCM. FCM was able to differentiate between different types of prostatic and periprostatic tissues including benign prostatic glands, benign prostatic hyperplasia, high-grade intraepithelial neoplasm, and prostatic adenocarcinoma. As regards the learning curve, FCM demonstrated a short learning curve. We created an atlas that can serve as the base for urologists and pathologists for learning and interpreting FCM images of prostatic and periprostatic tissues. Furthermore, FCM images is easily interpretable; however, further studies are required to explore the potential applications of this new technology in prostate cancer diagnosis and management.