Preservation Of Morphology Research Articles

Optimization of Formic Acid-Formalin-Based Decalcification Protocol for Rat Calvarial Bone Histology

Decalcification is crucial in histological processing, particularly for studying mineralized tissues like bone. The choice of decalcification method can significantly impact the quality of histological sections and the preservation of tissue morphology. This study aims to establish a standardized protocol for decalcifying rat calvarial bone using a formic acid-formalin-based decalcification solution. The protocol was systematically optimized and evaluated based on various parameters, including decalcification time, formic acid concentration, and tissue integrity preservation. The decalcification process was evaluated through comprehensive assessments, including gross physical examination, chemical analysis, and radiographic imaging techniques. Our result demonstrated that the 10% formic acid concentration proved most effective for decalcifying rat calvarial bone samples within eight days, excelling in mineral content removal while preserving specimen structural integrity. In contrast, the 5% concentration failed to complete decalcification within ten days, and the 15% compromised sample quality within eight days. Histological analyses confirmed the efficacy of the 10% formic acid concentration in maintaining tissue integrity and achieving optimal staining quality. The standardized protocol presented in this study provides an effective and reliable approach for achieving consistent and high-quality histological sections of rat calvarial bone. An ideal decalcification agent should effectively remove calcium salts, preserve structural integrity and molecular components, facilitate rapid yet minimally damaging decalcification, and ensure ease of handling for laboratory personnel. Further exploration of its applicability to different bone types or species is recommended to broaden its research utility.

Self-supervised anatomical continuity enhancement network for 7T SWI synthesis from 3T SWI

Synthesizing 7T Susceptibility Weighted Imaging (SWI) from 3T SWI could offer significant clinical benefits by combining the high sensitivity of 7T SWI for neurological disorders with the widespread availability of 3T SWI in diagnostic routines. Although methods exist for synthesizing 7T Magnetic Resonance Imaging (MRI), they primarily focus on traditional MRI modalities like T1-weighted imaging, rather than SWI. SWI poses unique challenges, including limited data availability and the invisibility of certain tissues in individual 3T SWI slices. To address these challenges, we propose a Self-supervised Anatomical Continuity Enhancement (SACE) network to synthesize 7T SWI from 3T SWI using plentiful 3T SWI data and limited 3T–7T paired data. The SACE employs two specifically designed pretext tasks to utilize low-level representations from abundant 3T SWI data for assisting 7T SWI synthesis in a downstream task with limited paired data. One pretext task emphasizes input-specific morphology by balancing the elimination of redundant patterns with the preservation of essential morphology, preventing the blurring of synthetic 7T SWI images. The other task improves the synthesis of tissues that are invisible in a single 3T SWI slice by aligning adjacent slices with the current slice and predicting their difference fields. The downstream task innovatively combines clinical knowledge with brain substructure diagrams to selectively enhance clinically relevant features. When evaluated on a dataset comprising 97 cases (5495 slices), the proposed method achieved a Peak Signal-to-Noise Ratio (PSNR) of 23.05 dB and a Structural Similarity Index (SSIM) of 0.688. Due to the absence of specific methods for 7T SWI, our method was compared with existing enhancement techniques for general 7T MRI synthesis, outperforming these techniques in the context of 7T SWI synthesis. Clinical evaluations have shown that our synthetic 7T SWI is clinically effective, demonstrating its potential as a clinical tool.

Microwave properties of chlorinated viscose-based carbon fibers for X-band radars

Viscose-based carbon fibers (VBCFs) were chlorinated with CCl4 vapor to investigate the effect of chlorination temperature on their microwave properties. The maximum chlorine content of 2.0 mmol g−1 was obtained at 450 °C. TEM and SEM showed carbon nanostructures and surface morphology preservation, respectively. TGA and TPD MS confirmed the formation of carbon-oxygen and chlorine-carbon groups, including CCl3 groups. The prepared chlorinated VBCFs can effectively reflect X-band microwaves, with nearly constant reflection losses over a wide frequency range, and are characterized by minimum transmission losses of −22 dB.

Intranasal administration of Escherichia coli Nissle expressing the spike protein of SARS-CoV-2 induces long-term immunization and prevents spike protein-mediated lung injury in mice

While current anti-Spike protein (SP) vaccines have been pivotal in managing the pandemic, their limitations in delivery, storage, and the inability to provide mucosal immunization (preventing infections) highlight the ongoing necessity for research and innovation. To tackle these constraints, our research group developed a bacterial-based vaccine using a non-pathogenic E. coli Nissle 1917 (EcN) strain genetically modified to express the SARS-CoV-2 spike protein on its surface (EcN-pAIDA1-SP). We intranasally delivered the EcN-pAIDA1-SP in two doses and checked specific IgG/IgA production as well as the key immune mediators involved in the process. Moreover, following the initial and booster vaccine doses, we exposed both immunized and non-immunized mice to intranasal delivery of SARS-CoV-2 SP to assess the effectiveness of EcN-pAIDA1-SP in protecting lung tissue from the inflammation damage. We observed detectable levels of anti-SARS-CoV-2 spike IgG in serum samples and IgA in bronchoalveolar lavage fluid two weeks after the initial treatment, with peak concentrations in the respective samples on the 35th day. Moreover, immunoglobulins displayed a progressively enhanced avidity index, suggesting a selective binding to the spike protein. Finally, the pre-immunized group displayed a decrease in proinflammatory markers (TLR4, NLRP3, ILs) following SP challenge, compared to the non-immunized groups, along with better preservation of tissue morphology. Our probiotic-based technology provides an effective immunobiotic tool to protect individuals against disease and control infection spread.

Evaluation of the Effects of Alpha-Tocopherol, Quercetin and their Combination on Ethanol-Induced Pancreatic and Duodenal Mucosal injuries: An Experimental Study

Aim: In this study, the effects of alpha-tocopherol (AT), quercetin (QT) or their combination on ethanol-induced pancreatic and duodenal mucosal damage were investigated in rats using morphological and biochemical evaluations.
 Study Design: Experimental study.
 Place and Duration of Study: University of Ibadan, Ibadan, Nigeria.
 Methodology: Ethanol-induced injuries were produced by oral administration of 40% ethanol (0.2 ml/day) for 40 consecutive days, while a control group of rats was served distilled water. Other groups received AT (2.5 mg/kg), QT (50 mg/kg) or their combination with 40% ethanol during the experimental period.
 Results: Blood glucose level was significantly (p<0.05) increased in ethanol-treated rats relative to controls. Ethanol administration caused shrinkage of insulin-secreting islets tissues in the pancreas, while lesions such as erosions, loss of villi and severe inflammatory cell infiltrations of the mucosa and sub-mucosa were observed in the duodenum. These changes were accompanied by significant elevation in the levels of hydrogen peroxide (H2O2), malondialdehyde (MDA) and advanced oxidation protein products (AOPP) in the pancreas and duodenum, along with reduced activities of glutathione peroxidase (GPx) and glutathione S-transferase (GST). Treatment of rats with AT, QT, and especially their combination, yielded profound reversal of ethanol-induced effects indicated by restoration of blood glucose to control levels, preservation of pancreatic and duodenal morphology and the inhibition of ethanol-induced oxidative stress.
 Conclusion: Overall, dietary supplementation with AT and/or QT could potentially counteract the adverse effects associated with chronic alcohol consumption.

Germline knockout of Nr2e3 protects photoreceptors in three distinct mouse models of retinal degeneration

Retinitis pigmentosa (RP) is a common form of retinal dystrophy that can be caused by mutations in any one of dozens of rod photoreceptor genes. The genetic heterogeneity of RP represents a significant challenge for the development of effective therapies. Here, we present evidence for a potential gene-independent therapeutic strategy based on targeting Nr2e3, a transcription factor required for the normal differentiation of rod photoreceptors. Nr2e3 knockout results in hybrid rod photoreceptors that express the full complement of rod genes, but also a subset of cone genes. We show that germline deletion of Nr2e3 potently protects rods in three mechanistically diverse mouse models of retinal degeneration caused by bright-light exposure (light damage), structural deficiency (rhodopsin-deficient Rho-/- mice), or abnormal phototransduction (phosphodiesterase-deficient rd10 mice). Nr2e3 knockout confers strong neuroprotective effects on rods without adverse effects on their gene expression, structure, or function. Furthermore, in all three degeneration models, prolongation of rod survival by Nr2e3 knockout leads to lasting preservation of cone morphology and function. These findings raise the possibility that upregulation of one or more cone genes in Nr2e3-deficient rods may be responsible for the neuroprotective effects we observe.

Short- and Long-Term Storage of Non-Domesticated European Mouflon (Ovis aries musimon) Cumulus-Oocyte Complexes Recovered in Field Conditions.

Reproductive biotechnologies can be used as a supporting tool, through gamete conservation and in vitro embryo production, in the preservation of invaluable and irreplaceable animal genetic resources. In the present study, immature mouflon cumulus-oocyte complexes (COCs) collected from ovariectomized female ovaries underwent short- or long-term conservation (24 h maintained in Earle's/Hank's (EH) medium or vitrification) under field conditions and afterwards transported to the laboratory where they were cultured for in vitro maturation (IVM) and assessed for oocyte meiotic competence and bioenergetic-oxidative status. Utilization of both storage techniques led to COC morphology preservation, as well as cumulus expansion and oocyte meiotic resumption after the IVM procedure. Quantitative bioenergetic-oxidative parameters were reduced in vitrified oocytes compared with EH ones. Immature COC storage needs to be optimized in both domesticated and non-domesticated sheep as a part of the strategy to avoid the loss of valuable genotypes of these animal species.

Long-Term Porcine Retina Explants as an Alternative to InVivo Experimentation.

The porcine retina represents an optimal model system to study treatment approaches for inherited retinal dystrophies owing to close anatomical similarities to the human retina, including a cone enriched visual streak. The aim of this work was to establish a protocol to keep explants in culture for up to 28days with good morphological preservation. Two to four retina explants per eye were obtained from the central part of the retina and transferred onto a membrane insert with the photoreceptors facing down. Different medium compositions using Neurobasal-A medium containing 100 or 450mg/dL glucose and combinations of fetal calf serum, B-27 with or without insulin and N-2 were tested. We developed a tissue quality score with robust markers for different retinal cell types (protein kinase C alpha, peanut agglutinin and 4',6-diamidino-2-phenylindol). Retinae were kept until 28days with only little degradation. The best results were attained using Neurobasal-A medium containing 100mg/dL glucose supplemented with B-27 containing insulin and N-2. For an easy preparation process, it is necessary to minimize transport time and keep the eyes on ice until dissected. Heat-mediated decontamination by the butcher has to be avoided. Using a standardized protocol, porcine retina explants represent an easy to handle intermediate model between in vitro and in vivo experimentation. This model system is robustly reproducible and contributes to the implementation of the 3R principle to minimize animal experimentation. This model can be used to test future therapeutic approaches for inherited retinal dystrophies.

Assessing morphological preservation of gastrointestinal parasites from fecal samples of wild capuchin monkeys (Cebus imitator) stored in ethanol versus formalin

The copromicroscopic identification of gastrointestinal parasites is a common, cost-effective method vital to understanding host-parasite interactions. However, its efficacy depends on effective preservation of the samples. In this study, we compare the preservation of ethanol and formalin preserved gastrointestinal parasites collected from a wild population of Costa Rican capuchin monkeys (Cebus imitator). Fecal samples were collected, halved, and stored in either 10% formalin or 96% ethanol at ambient temperature, then microscopically screened for the presence of parasites. Parasites were morphologically identified and rated based on their preservation using a newly developed rubric. We identified more parasitic morphotypes in formalin-preserved samples but found no difference in the number of parasites per fecal gram (PFG) between mediums. There was no difference in the PFG of two most prevalent parasite morphotypes, Filariopsis barretoi larvae and Strongyle-type eggs, and while Filariopsis larvae were better preserved in formalin, strongyle eggs showed no preservation difference between mediums. Our results support the suitability of both ethanol and formalin for morphological parasite identification in samples stored over 1 year, describe the morphological changes and challenges associated with parasite degradation, and highlight the potential for future studies to use both morphological and molecular methods in non-invasively collected samples.

Preservation of Intrinsically Photosensitive Retinal Ganglion Cells (ipRGCs) in Late Adult Mice: Implications as a Potential Biomarker for Early Onset Ocular Degenerative Diseases.

Intrinsically photosensitive retinal ganglion cells (ipRGCs) play a crucial role in non-image-forming visual functions. Given their significant loss observed in various ocular degenerative diseases at early stages, this study aimed to assess changes in both the morphology and associated behavioral functions of ipRGCs in mice between 6 (mature) and 12 (late adult) months old. The findings contribute to understanding the preservation of ipRGCs in late adults and their potential as a biomarker for early ocular degenerative diseases. Female and male C57BL/6J mice were used to assess the behavioral consequences of aging to mature and old adults, including pupillary light reflex, light aversion, visual acuity, and contrast sensitivity. Immunohistochemistry on retinal wholemounts from these mice was then conducted to evaluate ipRGC dendritic morphology in the ganglion cell layer (GCL) and inner nuclear layer (INL). Morphological analysis showed that ipRGC dendritic field complexity was remarkably stable through 12months old of age. Similarly, the pupillary light reflex, visual acuity, and contrast sensitivity were stable in mature and old adults. Although alterations were observed in ipRGC-independent light aversion distinct from the pupillary light reflex, aged wild-type mice continuously showed enhanced light aversion with dilation. No effect of sex was observed in any tests. The preservation of both ipRGC morphology and function highlights the potential of ipRGC-mediated function as a valuable biomarker for ocular diseases characterized by early ipRGC loss. The consistent stability of ipRGCs in mature and old adult mice suggests that detected changes in ipRGC-mediated functions could serve as early indicators or diagnostic tools for early-onset conditions such as Alzheimer's disease, Parkinson's disease, and diabetes, where ipRGC loss has been documented.

Use of Absolute Alcohol in Preservation of Cell Morphology in Fluid Cytology at Various Temperatures: A Cross-sectional Study

Introduction: Fluid cytology and the examination of malignant cells play an important role in the treatment of critical cases. The Department of Pathology (Cytopathology laboratory) receives samples for fluid cytology and examines malignant cells, reporting the White Blood Cell (WBC) count in the received fluid and the morphological findings of the cells examined on smears. Degeneration of cells in these body fluid samples, especially if the samples are not stored under proper conditions, is a common problem faced by many laboratories. Even overnight storage (12 hours) of these body fluids at temperatures of 4-8ºC (domestic refrigerators) may still cause some degree of degenerative changes. This becomes problematic in cytopathological reporting of body fluids, particularly when the samples are limited in amount or are from areas that cannot be re-aspirated {e.g., Cerebrospinal Fluid (CSF)}. While alcohol is a common fixative used for cell fixation in cytopathology, it is rarely used for fixing cells in fluid samples. Aim: To test the usage of absolute alcohol as a fixative in body fluid samples received in the cytopathology laboratory, which have been stored for 12 hours at two temperatures: room temperature and 4-8ºC. Materials and Methods: A cross-sectional study was conducted in the Department of Pathology at Subharti Medical College, Meerut, Uttar Pradesh, India. The study was conducted over the periods of two months, from February 2022 to March 2022. A total of 100 body fluid samples (including CSF, ascitic fluid, pleural fluids, etc.) sent for routine workup to the cytopathology laboratory were included in the study. Each body fluid sample (1 mL) was taken in two test tubes, and four drops of 100% (absolute) alcohol were added to both test tubes. The mixture of body fluid and absolute alcohol was then gently shaken, and one set of test tubes was kept at room temperature while the other set was kept at 4-8ºC. The data was tabulated in an MS Excel worksheet, and basic statistical analysis was performed. Results: The age range of cases whose samples were analysed was from one-week-old neonates to 65-year-old adults. The total number of fluid samples was 100, including 42 CSF samples, 23 ascitic fluid samples, 18 pleural fluid samples, and 17 Bronchoalveolar Lavage (BAL) fluid samples. Out of the 42 CSF samples, 15 showed preservation of morphology when the alcohol-body fluid mixture was kept at room temperature, while 22 cases showed preservation of the same mixture at 4-8ºC. Some samples showed no preservation (5 CSF), the reasons for which are discussed below. All the examined body fluids showed better preservation with alcohol at 4-8ºC. Conclusion: The addition of absolute alcohol as a fixative in fluids helps preserve the cytomorphological features of cells and provides better preservation for the diagnosis of fluid samples.

Modulation of Inflammatory Response by Electromagnetic Field Stimulation in Traumatic Brain Injury in Yucatan Swine.

Traumatic brain injury is a leading cause of disability and death worldwide and represents a high economic burden for families and national health systems. After mechanical impact to the head, the first stage of the damage comprising edema, physical damage, and cell loss gives rise to a second phase characterized by glial activation, increased oxidative stress and excitotoxicity, mitochondrial damage, and exacerbated neuroinflammatory state, among other molecular calamities. Inflammation strongly influences the molecular events involved in the pathogenesis of TBI. Therefore, several components of the inflammatory cascade have been targeted in experimental therapies. Application of Electromagnetic Field (EMF) stimulation has been found to be effective in some inflammatory conditions. However, its effect in the neuronal recovery after TBI is not known. In this pilot study, Yucatan miniswine were subjected to TBI using controlled cortical impact approach. EMF stimulation via a helmet was applied immediately or two days after mechanical impact. Three weeks later, inflammatory markers were assessed in the brain tissues of injured and contralateral non-injured areas of control and EMF-treated animals by histomorphometry, immunohistochemistry, RT-qPCR, Western blot, and ELISA. Our results revealed that EMF stimulation induced beneficial effect with the preservation of neuronal tissue morphology as well as the reduction of inflammatory markers at the transcriptional and translational levels. Immediate EMF application showed better resolution of inflammation. Although further studies are warranted, our findings contribute to the notion that EMF stimulation could be an effective therapeutic approach in TBI patients.

Mitochondria-derived vesicles: potential nano-batteries to recharge the cellular powerhouse.

Mitochondria dysfunction is increasingly recognized as a critical factor in various pathogenic processes. The mechanism governing mitochondrial quality control serves as an adaptive response, ensuring the preservation of mitochondrial morphology, quantity, and overall function, crucial for cell survival. The generation of mitochondria-derived vesicles (MDVs) is one of the processes of mitochondrial quality control. Recent literature has suggested MDV heterogeneity; however, the detailed characteristics of various MDV subtypes still need to be studied better. Recent studies have shown that MDVs also play a role in inter-organelle communication for mitochondria besides quality control. For instance, Hazan et al. demonstrated that functional mitochondria from Saccharomyces cerevisiae release vesicles independent of the fission machinery. These vesicles, falling within the typical size range of MDVs, were selectively loaded with mitochondrial proteins, especially with functional ATP synthase subunits. Intriguingly, these MDVs maintained membrane potential and could generate ATP. Moreover, MDVs could fuse with naïve mitochondria, transferring their ATP generation machinery. Lastly, this study revealed a potential delivery mechanism of ATP-producing vesicles, presenting a promising avenue to rejuvenate ATP-deficient mitochondria. Overall, this study unveils a novel mechanism for inter-organelle communication by vesicles, which is crucial for maintaining cellular homeostasis and could also be important in pathological conditions.

Diagnostic Utility of Bone Marrow Aspiration, Trephine Biopsy, and Flow Cytometry in the Evaluation of Various Haematological and Non Haematological Disorders: A Cross-sectional Study from Northern India

Introduction: The spectrum of haematological and non haematological disorders is vast in various age groups. Complete blood counts and other routine laboratory tests are not always sufficient to diagnose these diseases. Bone marrow examination plays an important role in diagnosing their underlying causes. Aim: To analyse the spectrum of various haematological and non haematological disorders reported in Bone Marrow Aspiration (BMA) and compare them with Bone Marrow Trephine Biopsy (BMB) and Flow Cytometry (FCM) findings where applicable. Materials and Methods: A one-year cross-sectional study was conducted in the Department of Clinical Pathology, PGIMS, Rohtak, Haryana, India from April 2022 to March 2023. A total of 518 consecutive BMA samples were morphologically analysed. Comparative evaluations were performed among BMA, BMB, and FCM where applicable. Diagnostic accuracy was calculated, and the findings of discordant cases were tabulated. Definitive diagnosis of lymphoma/leukaemia and Paroxysmal Nocturnal Haemoglobinuria (PNH) cases were made using FCM. Results: The patients’ ages ranged from three months to 86 years, with a mean age of 38.4 years. The male to female ratio was 1.3:1, with a slight male predominance. The highest number of cases were of anaemia {183 (35.3%) and 164 (31.7%), respectively} and leukaemia {128 (24.7%) and 134 (25.9%), respectively}, followed by normal marrow studies {39 (7.5%) and 32 (6.2%), respectively} and megakaryocytic thrombocytopenia {24 (4.6% in each} in both BMA and biopsy. Among anaemia cases {183 and 164 cases in BMA and BMB}, the majority were of the megaloblastic type {62 (33.8%) and 54 (32.9%), respectively}, followed by hypoplastic/aplastic type {40 (21.8%) and 50 (30.5%), respectively}. In cases of leukaemia (128 and 134 cases in BMA and BMB), acute leukaemia cases (76 (59.4%) and 82 (61.2%), respectively} outnumbered chronic leukaemia cases {52 (40.6%) and 52 (38.8%), respectively} in both BMA and BMB. The concordance and discordance rate between BMA and BMB were 87.6% (419 cases) and 12.4% (59 cases), respectively. Diagnosis was exclusively made by BMB in cases of myelofibrosis, granulomatous disease, and Hodgkin’s lymphoma. Conclusion: BMA cytology is a relatively safe and mildly invasive technique for evaluating various haematological and non haematological disorders with better preservation of cellular morphology. However, in cases with dry/blood taps and focal marrow involvement, BMB should be performed, as it shows well-preserved marrow architecture with all its cellular and stromal components. FCM is a definitive diagnostic modality for further categorisation of acute leukaemia and Chronic Lymphoproliferative Disorders (CLPD).

Chemically-Induced Lipoprotein Breakdown for Improved Extracellular Vesicle Purification.

Extracellular vesicles (EVs) are nanosized biomolecular packages involved in intercellular communication. EVs are released by all cells, making them broadly applicable as therapeutic, diagnostic, and mechanistic components in (patho)physiology. Sample purity is critical for correctly attributing observed effects to EVs and for maximizing therapeutic and diagnostic performance. Lipoprotein contaminants represent a major challenge for sample purity. Lipoproteins are approximately six orders of magnitude more abundant in the blood circulation and overlap in size, shape, and density with EVs. This study represents the first example of an EV purification method based on the chemically-induced breakdown of lipoproteins. Specifically, a styrene-maleic acid (SMA) copolymer is used to selectively breakdown lipoproteins, enabling subsequent size-based separation of the breakdown products from plasma EVs. The use of the polymer followed by tangential flow filtration or size-exclusion chromatography results in improved EV yield, preservation of EV morphology, increased EV markers, and reduced contaminant markers. SMA-based EV purification enables improved fluorescent labeling, reduces interactions with macrophages, and enhances accuracy, sensitivity, and specificity to detect EV biomarkers, indicating benefits for various downstream applications. In conclusion, SMA is a simple and effective method to improve the purity and yield of plasma-derived EVs, which favorably impacts downstream applications.

Histologic Evaluation of Effect of Three Wavelengths of Diode Laser on Human Gingival Margins.

Introduction: Available evidence suggests that the response of the intervened tissue is directly linked to the effects generated by the cutting instrument used. To determine the histological findings in gingival tissue margins excised through gingivectomies performed using 450 nm, 940 nm, and 980 nm diode lasers. The present study aimed to determine the histological findings in gingival tissue margins excised through gingivectomies performed using 450 nm, 940 nm, and 980 nm diode lasers. Methods: Gingival tissue samples were collected from 30 patients who had undergone gingivectomy procedures. Each study group comprised 10 patients who willingly provided their samples after providing informed consent. The visualization of histological findings was facilitated through Hematoxylin-Eosin staining. Additionally, variables related to pain and hemostasis were assessed during the intraoperative period. Results: The incision quality was categorized as irregular across all three wavelengths. Histological examination of the epithelial tissue revealed the absence of carbonization and the preservation of cell morphology in over 50% of the resection margin in samples obtained with the 450 nm and 940 nm wavelengths. In the connective tissue, observations included carbonization, collagen coagulation, and basophilia, with the 980 nm wavelength demonstrating the highest percentage of samples displaying collagen coagulation in more than 50% of the resection margin. Conversely, the 450 nm wavelength exhibited the highest degree of preservation of the fibroblast structure. Conclusion: Based on a comprehensive analysis of the study results, it can be inferred that the 450nm and 940nm wavelength lasers tend to produce less thermal damage and better cell preservation when compared to the 980nm wavelength.

Opto-electronic feedback control of membrane potential for real-time control of action potentials

To unlock new research possibilities by acquiring control of action potential (AP) morphologies in excitable cells, we developed an opto-electronic feedback loop-based system integrating cellular electrophysiology, real-time computing, and optogenetic approaches and applied it to monolayers of heart muscle cells. This allowed accurate restoration and preservation of cardiac AP morphologies in the presence of electrical perturbations of different origin in an unsupervised, self-regulatory manner, without any prior knowledge of the disturbance. Moreover, arbitrary AP waveforms could be enforced onto these cells. Collectively, these results set the stage for the refinement and application of opto-electronic control systems to enable in-depth investigation into the regulatory role of membrane potential in health and disease.

Neuroprotective-Neurorestorative Effects Induced by Progesterone on Global Cerebral Ischemia: A Narrative Review.

Progesterone (P4) is a neuroactive hormone having pleiotropic effects, supporting its pharmacological potential to treat global (cardiac-arrest-related) cerebral ischemia, a condition associated with an elevated risk of dementia. This review examines the current biochemical, morphological, and functional evidence showing the neuroprotective/neurorestorative effects of P4 against global cerebral ischemia (GCI). Experimental findings show that P4 may counteract pathophysiological mechanisms and/or regulate endogenous mechanisms of plasticity induced by GCI. According to this, P4 treatment consistently improves the performance of cognitive functions, such as learning and memory, impaired by GCI. This functional recovery is related to the significant morphological preservation of brain structures vulnerable to ischemia when the hormone is administered before and/or after a moderate ischemic episode; and with long-term adaptive plastic restoration processes of altered brain morphology when treatment is given after an episode of severe ischemia. The insights presented here may be a guide for future basic research, including the study of P4 administration schemes that focus on promoting its post-ischemia neurorestorative effect. Furthermore, considering that functional recovery is a desired endpoint of pharmacological strategies in the clinic, they could support the study of P4 treatment for decreasing dementia in patients who have suffered an episode of GCI.

T Cell Ig and ITIM Domains (TIGIT) Expression in Multiple Myeloma

Background Neoplastic myeloma cells show a wide variety of mechanisms that induce the microenvironment, allowing immune evasion and promoting their proliferation, survival, migration and drug resistance. In this context, immunocheckpoints are particularly important, representing the modulators of signal pathways responsible for immune tolerance, a mechanism that prevents the destruction of cancer cells by the immune system. T cell Ig and ITIM domains (TIGIT), a member of the poliovirus (PVR) /nectin receptor family, is a new immune checkpoint that negatively regulates T cell functions. In Multiple Myeloma (MM), TIGIT could play a relevant role leading to immunological escape. TIGIT performs its task mainly through binding ligands: CD155 and CD112, through which it inhibits the cytotoxicity of CD8+ T cells, as well as that of NK. In This Study, we evaluate the presence of TIGIT in bone marrow using instant digital pathology. AIMS Aims of this study are to develop a valid and reproducible workflow for instant digital pathology, to demonstrate the presence of TIGIT-positive T lymphocytes in bone marrow of patients with MM and to evaluate its correlation with clinical features PATIENTS and Methods 18 patients with newly diagnosed MM not previously treated, were evaluated in this study. The sample was enrolled from November 2022 to May 2023. Bone marrow blood was loaded on a porous matrix able to retain cells (Cytomatrix, UCS Diagnostics srl, Rome) for real time assessment of sample adequacy, morphological details preservation, and composition of hematopoietic subpopulations, using an ex-vivo fluorescent confocal microscope (FCM). Expert pathologists compared the digital FCM images with permanent Formalin Fixed Paraffin Embedded (FFPE) sections of the matrix and trephine bone marrow biopsy. Immunohistochemistry for TIGIT (TG-1 Clone, OncoDianova) was performed on matrix FFPE sections. RESULTS Our results showed that 15/17 (88%) patients resulted positive for TIGIT and 2/17 (12%) were negative. TIGIT expression was found mainly on CD8+ T lymphocytes, TIGIT positivity was correlated with parameters that define the aggressiveness of myeloma disease such as PC > 60% or FLC > 100, ISS score, R-ISS score, cytogenetic risk and LDH > 220mU/ml. Among the patients defined as “ultra-High Risk”, 9/10 (90%) resulted positive for TIGIT. Comparing TIGIT with the MM International Staging System (ISS), we observed that all patients with scores II and III were positive for TIGIT (100%) while 78% of them with ISS I results positive. Conclusions Our results suggest that FCM can be applied immediate evaluation of bone marrow aspirates, preserving the cellular sample for final histological analysis. “Furthermore, our data shows the positivity of TIGIT in 88% of patients, demonstrating the presence of this immune checkpoint in the bone marrow microenvironment. These data will need to be confirmed in a larger group of patients, but they open up the possibility of new target treatments in multiple myeloma

Glyoxal acid-free (GAF) histological fixative is a suitable alternative to formalin: results from an open-label comparative non-inferiority study

Formalin, an aqueous solution of formaldehyde, has been the gold standard for fixation of histological samples for over a century. Despite its considerable advantages, growing evidence points to objective toxicity, particularly highlighting its carcinogenicity and mutagenic effects. In 2016, the European Union proposed a ban, but a temporary permission was granted in consideration of its fundamental role in the medical-diagnostic field. In the present study, we tested an innovative fixative, glyoxal acid-free (GAF) (a glyoxal solution deprived of acids), which allows optimal tissue fixation at structural and molecular level combined with the absence of toxicity and carcinogenic activity. An open-label, non-inferiority, multicentric trial was performed comparing fixation of histological specimens with GAF fixative vs standard phosphate-buffered formalin (PBF), evaluating the morphological preservation and the diagnostic value with four binary score questions answered by both the central pathology reviewer and local center reviewers. The mean of total score in the GAF vs PBF fixative groups was 3.7 ± 0.5 vs 3.9 ± 0.3 for the central reviewer and 3.8 ± 0.5 vs 4.0 ± 0.1 for the local pathologist reviewers, respectively. In terms of median value, similar results were observed between the two fixative groups, with a median value of 4.0. Data collected indicate the non-inferiority of GAF as compared to PBF for all organs tested. The present clinical performance study, performed following the international standard for performance evaluation of in vitro diagnostic medical devices, highlights the capability of GAF to ensure both structural preservation and diagnostic value of the preparations.