The traditional recognition of extracellular matrix (ECM) at tissue sections relies on the time-consuming immunofluorescence that could not meet the demand of rapid diagnosis. Herein, we introduce a thickness-resolved electrochemiluminescence (ECL) microscopy to image thin-layer ECM at tissue sections for fast histopathological analysis. The unique surface-confined ECL mechanism enables to unveil the diversity and complexity of multiple tissue structures with varying thicknesses. Notably, the short lifetimes and the limited diffusion of electrogenerated coreactant radicals combined with their chemical reactivity result in a 2-fold increase in ECL intensity on ECM structures compared to the remaining tissue, enabling ECM visualization without specific labeling. The further quantitation of the ECM localization within tissue sections furnishes crucial insights into tumor progression and, more importantly, differentiates carcinoma and paracancerous tissues from patients in less than 30 min. Moreover, the reported electrochemistry-based microscopy is a dynamic approach allowing to investigate the transport, tortuosity, and trafficking properties through the tissues. This thickness-resolved recognition strategy not only opens new avenues for imaging complex samples but also holds promise for expediting tissue pathologic diagnosis, offering a more automated protocol with enhanced quantitative data compared to current intraoperative pathology methods.

The co-occurrence of cyanobacterial blooms and nano-microplastic pollution in the water is becoming an emerging risk. To assess the combined hepatotoxicity of microcystin-LR (MC-LR) and polystyrene microplastics (PSMPs) on zebrafish (Danio rerio), male adult zebrafish were exposed to single MC-LR (0, 1, 5, 25 μg/L) and a mixture of MC-LR and PSMPs (100 μg/L). After 60 d exposure, the results indicated that PSMPs significantly increased the MC-LR bioaccumulation in the livers in contrast to the single 25 μg/L MC-LR treatment group. Moreover, the severity of hepatic pathological lesions was aggravated in the MC-LR + PSMPs treatment groups, which were mainly characterized by cellular vacuolar degeneration, swollen hepatocytes, and pyknotic nucleus. The ultrastructural changes also proved that PSMPs combined with MC-LR could enhance the swollen mitochondria and dilated endoplasmic reticulum. The biochemical results, including increased malondialdehyde (MDA) and decreased glutathione (GSH), indicated that PSMPs intensified the MC-LR-induced oxidative damage in the combined treatment groups. Concurrently, alterations of sod1 and keap1a mRNA levels also confirmed that PSMPs together with MC-LR jointly lead to enhanced oxidative injury. Our findings demonstrated that PSMPs enhanced the MC-LR bioavailability by acting as a vector and exacerbating the hepatic injuries and antioxidant dysfunction in zebrafish.

The high morbidity and mortality of Macrobrachium nipponense occurred in several farms in China, with cardinal symptoms of slow swimming, loss of appetite, empty of intestine, reddening of the hepatopancreas and gills. The pathogen has been confirmed as Decapod Iridescent Virus 1 (DIV1), namely DIV1-mn, by molecular epidemiology, histopathological examination, TEM observation, challenge experiment, and viral load detection. Histopathological analysis showed severe damage in hepatopancreas and gills of diseased prawns, exhibited few eosinophilic inclusions and pyknosis, and TEM of diseased prawns revealed that icosahedral virus particles existed in hepatopancreas and gill, which confirmed the disease of the farmed prawns caused by the DIV1 infection. Besides, challenge tests showed LD50 of DIV1 to M. nipponense was determined to be 2.14 × 104 copies/mL, and real-time PCR revealed that M. nipponense had a very high DIV1 load in the hemocytes, gills and hepatopancreas after infection. Furthermore, qRT-PCR was undertaken to investigated the expression of six immune-related genes in DIV1-infected M. nipponense after different time points, and the results revealed UCHL3, Relish, Gly-Cru2, CTL, MyD88 and Hemocyanin were significantly up-regulated in hemocytes, gills and hepatopancreas, which revealed various expression patterns in response to DIV1 infection. This study revealed that DIV1 infection is responsible for the mass mortality of M. nipponense, one of the important crustacean species, indicating its high susceptibility to DIV1. Moreover, this study will contribute to exploring the interaction between the host and DIV1 infection, specifically in terms of understanding how M. nipponense recognizes and eliminates the invading of DIV1.

BACKGROUND: Mycoplasma ovipneumoniae (M. ovipneumoniae) is one of the main pathogens of sheep pneumonia, causing a series of clinical symptoms, such as depression, anorexia, hyperthermia, cough, dyspnea, and tract secretions. In recent years, the prevalence of M. ovipneumoniae pneumonia has become increasingly serious in sheep farms in Ningxia, China, leading to the death of sheep, and causing significant economic losses. In this study, the pathological organs infected by M. ovipneumoniae were collected to observe histopathological change, to determine the tissue localization of M. ovipneumoniae, and to analyze the cytokine changes, which lays a basis for the diagnosis and pathogenesis of M. ovipneumoniae disease.
RESULTS: In this study, M. ovipneumoniae was detected in 97 of 105 samples collected from 13 large-scale sheep farms for nucleic acid by PCR. One representative isolate per farm was isolated from 13 farms. The lesions caused by M. ovipneumoniae were mainly in the trachea, bronchus, and lung, including necrosis of tracheal mucosal epithelial cells, disintegration of some epithelial cells, edema of mucosal lamina propria, with inflammatory cell infiltration, cytoplasmic vacuolization of epithelial cells of bronchial mucosa, massive infiltration of inflammatory cells in the alveolar space of lung, necrosis and hyperplasia of alveolar epithelial cells. Immunohistochemical analysis showed that the proportion of M. ovipneumoniae positive area in the lung was the largest, followed by that in the bronchus and trachea. Compared to healthy animals, diseased animals exhibited up-regulated gene expression levels of IL-1β, IL-6, and NF-κB in the trachea, bronchus, and lungs. In contrast, the expression of IL-10, IL-12, and IFN-γ was primarily limited to the trachea and bronchus. The expression of IL-1β showed differential patterns across different lung regions, with variations observed among lung lobes. Additionally, other cytokines consistently showed significant up-regulation specifically in the bronchus.
CONCLUSIONS: M. ovipneumoniae is primarily found in the lungs of infected individuals. NF-κB, an essential transcription factor, is involved in the regulation of IL-1β transcription. IL-12 may enhance the cytotoxic function of natural killer cells during M. ovipneumoniae infection. Those findings demonstrate the distinct expression profiles of cytokines in various anatomical sites throughout disease progression, suggesting the potential role of bronchial tissue as a major site of immune response.

Citrobacter freundii, a common pathogen of freshwater fish, causes significant commercial losses to the global fish farming industry. In the present study, a highly pathogenic C. freundii strain was isolated and identified from largemouth bass (Micropterus salmoides). The pathogenicity and antibiotic sensitivity of the C. freundii strain were evaluated, and the histopathology and host immune response of largemouth bass infected with C. freundii were investigated. The results showed that C. freundii was the pathogen causing disease outbreaks in largemouth bass, and the infected fish showed typical signs of acute hemorrhages and visceral enlargement. Antimicrobial susceptibility testing showed that the C. freundii strain was resistant to Kanamycin, Medimycin, Clindamycin, Penicillin, Oxacillin, Ampicillin, Cephalexin, Cefazolin, Cefradine and Vancomycin. Histopathological analysis showed different pathological changes in major tissues of diseased fish. In addition, humoral immune factors such as superoxide dismutase (SOD), catalase (CAT) and lysozyme (LZM) were used as serum indicators to evaluate the immune response of largemouth bass after infection. Quantitative real-time PCR (qRT-PCR) was performed to investigate the expression pattern of immune-related genes (CXCR1, IL-8, IRF7, IgM, CD40, IFN-γ, IL-1β, Hep1, and Hep2) in liver, spleen, and head kidney tissues, which demonstrated a strong immune response induced by C. freundii infection in largemouth bass. The present study provides insights into the pathogenic mechanism of C. freundii and immune response in largemouth bass, promoting the prevention and treatment of diseases caused by C. freundii infection.

For 17 consecutive years, the outbreak of Ulva prolifera in the South Yellow Sea area of China has caused significant negative impacts on coastal ecological environment. However, its specific influence on fish immunity is rare. In this study, the juvenile Paralichthys olivaceus was exposed to fresh U. prolifera algae (FU) and decomposing algal effluent (DU). After short-term stress for 14 days, the histopathological and transcriptome analysis were performed to study the effect of U. prolifera decay on P. olivaceus. Histopathological analysis found that the liver, spleen and head kidneys of P. olivaceus were damaged after the short-term stress. The transcriptome results showed that the steroid biosynthesis signaling pathway and the PI3K-Akt signaling pathway were significantly enriched. Some immune related genes, including c1qc-like, dusp1, dusp16, HSP90 and metabolic related genes serotransferrin, were differentially expressed. These results highlighted the harmfulness of U. prolifera on marine fish, setting a solid foundation for further analyses.

Aeromonas veronii is widespread in aquatic environments and is capable of infecting various aquatic organisms. A. veronii infection is lethal for Chinese soft-shelled turtles (Trionyx sinensis, CSST). We isolated a gram-negative bacterium from the liver of diseased CSSTs, which was named XC-1908. This isolate was identified as A. veronii based on its morphological and biochemical characteristics, and 16S rRNA gene sequence analysis. A. veronii was pathogenic for CSSTs with an LD50 of 4.17 × 105 CFU/g. The symptoms of CSSTs artificially infected with isolate XC-1908 were consistent with those of the naturally infected CSSTs. The levels of total protein, albumin, and white globule in the serum samples of the diseased turtles were decreased, whereas those of aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase were elevated. Moreover, the diseased CSSTs exhibited the following histopathological changes: the liver contained numerous melanomacrophage centers, renal glomerulus were edematous, intestinal villi were shed, and in oocytes, the number of vacuoles increased and red-rounded particles were observed. Antibiotic sensitivity tests revealed that the bacterium was sensitive to ceftriaxone, doxycycline, florfenicol, cefradine, and gentamicin, and resistant to sulfanilamide, carbenicillin, benzathine, clindamycin, erythromycin, and streptomycin. This study provides control strategies to prevent outbreaks of A. veronii infection in CSSTs.

Forceps, clamps, and haemostats are essential surgical tools required for all surgical interventions. While they are widely used to grasp, hold, and manipulate soft tissue, their metallic rigid structure may cause tissue damage due to the potential risk of applying excessive gripping forces. Soft pneumatic surgical grippers fabricated by silicone elastomeric materials with low Young\'s modulus may offer a promising solution to minimize this unintentional damage due to their inherent excellent compliance and compressibility. The goal of this work is to evaluate and compare the grip-induced nerve damage caused by the soft pneumatic elastomeric gripper and conventional haemostats during surgical manipulation. Twenty-four Wistar rats (male, seven weeks) are subjected to sciatic nerve compression (right hind limb) using the soft pneumatic elastomer gripper and haemostats. A histopathological analysis is conducted at different time-points (Day 0, Day 3, Day 7 and Day 13) after the nerve compression to examine the morphological tissue changes between the rats in the \'soft gripper\' group and the \'haemostats\' group. A free walking analysis is also performed to examine the walking function of the rats after recovery from different time points. Comparing the rigid haemostats and soft gripper groups, there is a visible difference in the degree of axonal vacuolar degeneration between the groups, which could suggest the presence of substantial nerve damage in the \'haemostats\' group. The rats in the haemostats group exhibited reduced right hind paw pressure and paw size after the nerve compression. It shows that the rats tend not to exert more force on the affected right hind limb in the haemostats group compared to the soft gripper group. In addition, the stance duration was reduced in the injured right hind limb compared to the normal left hind limb in the haemostats group. These observations show that the soft pneumatic surgical gripper made of silicone elastomeric materials might reduce the severity of grip-induced damage by providing a safe compliant grip compared to the conventional haemostats. The soft pneumatic elastomer gripper could complement the current surgical gripping tool in delicate tissue manipulation.

Owing to the greater absorption affinity for lipo-rich tissue than water, the 1210 nm laser is a promising candidate for transcutaneous lipolysis in the near-infrared band. However, fat reduction is limited because laser therapy may yield thermal injury of normal tissue. A new protocol to incorporate multipulsed cryogen spray cooling is beneficial to improve the lipolysis effect, and the parameters of laser and cooling can be optimized via skin histopathological analysis.
A murine in vivo model of inguinal tissue of SD rats was established to test the effectivity of transcutaneous lipolysis protocol by R134a multipulsed spray cooling assisted 1210 nm laser irradiation. Tissue response of lipolysis with/without cooling 10 days post the treatment was evaluated by histopathological analysis of skin samples stained with hematoxylin-eosin (HE), through which safe and effective parameters for lipolysis were determined.
From histopathological analysis of the inguinal tissue of SD rats irradiated by the 1210 nm laser alone, the optimal durations are respectively 7 and 3 s (seconds) for low-dosage (6 W) and high-dosage (9 W) therapy, with pronounced lipolysis effect and minimum injury of skin tissue. The multipulsed spray cooling by R134a with a pulse duration of 10 ms (milliseconds), a pulse delay of 2000 ms, and a pulse number of 5 can be introduced to assist the 1210 nm laser therapy with a power of 9 W and a duration of 7 s to achieve desirable fat liquefaction while keeping the complete structure of skin tissue as well as esthetic-related beneficial effects of hair removal and skin rejuvenation.
Excellent lipolysis effect can be achieved via R134a multipulsed spray cooling assisted high-dosage 1210 nm laser irradiation with reasonably matched laser and cooling parameters. The protocol is as follows: Start MP-CSC for one cycle, and then fire the laser with specific power and duration, while keeping MP-CSC accordingly. This new protocol may promote the safe and effective clinical implement of transcutaneous laser lipolysis in body contouring.

Edwardsiella tarda is reported as the causative agent of the systemic disease Edwardsiellosis in fish, which lead to huge economic losses in aquaculture. The pathogenicity and immune response to a highly virulent E. tarda isolate responsible for mass mortality in hybrid snakehead were performed. After species identification, morphology and virulence gene detection of Edwardsiella isolated from hybrid snakehead, the pathogenicity of the strain and histopathological changes in infected fish were analyzed. The infected fish exhibited typical acute hemorrhagic symptoms and enlarged internal organs. Histopathology revealed that the liver, spleen, kidney and intestinal tissues of diseased fish exhibited marked inflammatory with vacuolar degeneration and cell necrosis. Subsequently, humoral immune factors such as superoxide dismutase, lysozyme and acid phosphatase activities were detected as serum indicators, and real-time quantitative PCR was used to investigate immune-related genes (STAT1, HSP70, IgM, IL-6, IL-8, TRAF2, CD40, HLA-DMA and LCK) expression patterns in liver, spleen and head kidney. The results showed that these enzyme activity indicators and immune-related gene expression were significantly activated compared with healthy fish. These data provide insight into the pathogenic mechanisms and host immune responses of E. tarda, which could be useful for the future prevention and treatment of Edwardsiellosis in fish.