Ultrastructural Changes Research Articles

Ultrastructural Changes of Neuroendocrine Pheochromocytoma Cell Line PC-12 Exposed In Vitro to Rotenone

Rotenone is a pesticide used in research for its ability to induce changes similar, in vivo and in vitro, to those observed in Parkinson’s disease (PD). This includes a selective death of dopaminergic neurons in the substantia nigra. Nonetheless, the precise mechanism through which rotenone modifies structure and function of neurons remains unclear. The PC12 cells closely resemble dopamine terminal neurons. This makes it a preferred model for studying the morphology of central dopamine neurons and predicting neurotoxicity. In this paper, we investigated the effects of 0.5 µM rotenone for 24–48 h on PC12 cell viability and ultrastructure (TEM), trying to identify primary and more evident alterations that can be related to neuronal damages similar to that seen in animal PD models. Cell viability decreased after 24 h rotenone treatment, with a further decrease after 48 h. Ultrastructural changes included vacuolar degeneration, mitochondrial mild swelling, decrease in the number of neuropeptide granules, and the loss of cell-to-cell adhesion. These findings are in agreement with previous research suggesting that rotenone, by inhibiting energy production and increasing ROS generation, is responsible for significant alterations of the ultrastructure and cell death of PC12 cells. Our data confirm the link between rotenone exposure, neuronal damage, and changes in dopamine metabolism, suggesting its role in the pathogenesis of PD.

Coenzyme Q10 mitigates cadmium cardiotoxicity by downregulating NF-κB/NLRP3 inflammasome axis and attenuating oxidative stress in mice

Coenzyme Q10 (CoQ10) occurs naturally in the body and possesses antioxidant and cardioprotective effects. Cardiotoxicity has emerged as a serious effect of the exposure to cadmium (Cd). This study investigated the curative potential of CoQ10 on Cd cardiotoxicity in mice, emphasizing the involvement of oxidative stress (OS) and NF-κB/NLRP3 inflammasome axis. Mice received a single intraperitoneal dose of CdCl2 (6.5 mg/kg) and a week after, CoQ10 (100 mg/kg) was supplemented daily for 14 days. Mice that received Cd exhibited cardiac injury manifested by the elevated circulating cardiac troponin T (cTnT), CK-MB, LDH and AST. The histopathological and ultrastructural investigations supported the biochemical findings of cardiotoxicity in Cd-exposed mice. Cd administration increased cardiac MDA, NO and 8-oxodG while suppressed GSH and antioxidant enzymes. CoQ10 decreased serum CK-MB, LDH, AST and cTnT, ameliorated histopathological and ultrastructural changes in the heart of mice, decreased cardiac MDA, NO, and 8-OHdG and improved antioxidants. CoQ10 downregulated NF-κB p65, NLRP3 inflammasome, IL-1β, MCP-1, JNK1, and TGF-β in the heart of Cd-administered mice. Moreover, in silico molecular docking revealed the binding potential between CoQ10 and NF-κB, ASC1 PYD domain, NLRP3 PYD domain, MCP-1, and JNK. In conclusion, CoQ10 ameliorated Cd cardiotoxicity by preventing OS and inflammation and modulating NF-κB/NLRP3 inflammasome axis in mice. Therefore, CoQ10 exhibits potent therapeutic benefits in safeguarding cardiac tissue from the harmful consequences of exposure to Cd.

Stress-related physiological responses and ultrastructural changes in Hypoxis hemerocallidea leaves exposed to cadmium and aluminium

AbstractHypoxis hemerocallidea is a medicinal plant containing hypoxoside (a pharmacologically active phytosterol diglucoside). This study evaluated the elemental composition in leaves of H. hemerocallidea treated with cadmium (Cd) and aluminium (Al) using scanning electron microscopy (SEM) combined with energy-dispersive X-ray spectroscopy (EDX). The impact of Cd and Al on photosynthetic pigments and performance, antioxidant activities and ultrastructure were also assessed. Corms of H. hemerocallidea were micropropagated, rooted and then exposed to varying concentrations of Cd, Al, and Cd + Al for six weeks. The SEM/EDX analysis indicated a two-fold increase in carbon content across all treated plants compared to the control. No/little Cd was detected in the leaves compared to a progressive increase in Al concentration with increasing Al treatment levels. This indicted that Al is more readily translocated to the shoots compared to Cd. Plants treated with Cd exhibited a significant decrease in total chlorophyll content accompanied by reduced photosynthetic performance and lower relative electron transport rates. Cd and Al exposure led to higher carotenoid, superoxide dismutase and malondialdehyde levels, indicating oxidative stress. Cd-treated plants displayed increased amylase activity and decreased carbohydrates content. Ultrastructural alterations occurred with exposure to Cd and Al, including abnormal swelling or disintegration of chloroplasts and thylakoid degeneration. An increase in starch grains and a decrease in plastoglobuli were also noted. In conclusion, this investigation provides evidence that both Cd and higher concentrations of Al exert detrimental effects on the ultrastructure, metabolism and photosynthetic performance of H. hemerocallidea, contributing to reduced growth and biological activity when stressed.

Dry eye syndrome associated with traumatic keratitis

Dry eye syndrome (DES) is an urgent problem in healthcare practice worldwide. Various diseases of the eye are of particular importance for the development of this syndrome. A special place among the diseases leading to symptomatic DES with ocular surface lesion belongs to corneal injuries of traumatic origin. The frequency of post-traumatic complications of the eye, especially the cornea, remains high due to its anatomical location. And often these patients are at risk of secondary DES. For the pathogenesis of DES after ke­ratitis, ultrastructural changes in the cells of different corneal layers and the toxic effect on the tear film layers of preservative-containing drugs used in the treatment of keratitis are important. The purpose of the study was to assess the severity of secondary DES in patients after traumatic keratitis and the effectiveness of hyaluronic acid combined with antioxidants in the treatment of this pathology. An ophthalmological examination, functional tests were made, and the Ocular Surface Disease Index was used to evaluate the symptoms and severity of the disease. The criteria for the effectiveness of the therapy with hyaluronic acid in combination with vitamins A and E four times a day were: reduction of corneal syndrome, increase in the tear film break-up time, a decrease in subjective discomfort. Clinical observation of patients with DES after traumatic keratitis showed that the use of hyaluronic acid can result in a significant reduction or complete disappearance of subjective symptoms of the disease, sustained improvement in vision and increased stability of the tear film. The use of these eye drops makes it possible to reach compliance and shorten the treatment period.

Physiological response of chestnuts (Castanea mollissima Blume) infected by pathogenic fungi and their correlation with fruit decay

The postharvest decay of chestnuts (Castanea mollissima Blume) limits their industrial utilization, and pathogenic fungi are the main cause for chestnut decay. In this study, the physiological changes and their correlation with the rotting degree of chestnuts were investigated during single or mixed infection by Fusarium proliferatum, Penicillium crustosum, and Alternaria alternata. During the infection period, the activities of cell wall degrading enzymes (CWDEs) and antioxidant enzymes firstly increased and then decreased, the contents of nutrients decreased, but the levels of physiological indexes increased. The mycelium and spores of pathogenic fungi colonized the intercellular space, and then the mycelium covered the entire tissue surface of chestnuts, leading to the disappearance of cell structure. Notably, the most prominent changes in physiological indexes and ultrastructure were observed in chestnuts infected with three pathogenic fungi. Furthermore, the rotting degree of chestnuts was positively correlated with the levels of CWDEs, antioxidant enzymes and malondialdehyde.

In vitro effects and mathematical modelling of CTCE-9908 (a chemokine receptor 4 antagonist) on melanoma cell survival.

CTCE-9908, a CXC chemokine receptor 4 (CXCR4) antagonist, prevents CXCR4 phosphorylation and inhibits the interaction with chemokine ligand 12 (CXCL12) and downstream signalling pathways associated with metastasis. This study evaluated the in vitro effects of CTCE-9908 on B16 F10 melanoma cells with the use of mathematical modelling. Crystal violet staining was used to construct a mathematical model of CTCE-9908 B16 F10 (melanoma) and RAW 264.7 (non-cancerous macrophage) cell lines on cell viability to predict the half-maximal inhibitory concentration (IC50). Morphological changes were assessed using transmission electron microscopy. Flow cytometry was used to assess changes in cell cycle distribution, apoptosis via caspase-3, cell survival via extracellular signal-regulated kinase1/2 activation, CXCR4 activation and CXCL12 expression. Mathematical modelling predicted IC50 values from 0 to 100 h. At IC50, similar cytotoxicity between the two cell lines and ultrastructural morphological changes indicative of cell death were observed. At a concentration 10 times lower than IC50, CTCE-9908 induced inhibition of cell survival (p = 0.0133) in B16 F10 cells but did not affect caspase-3 or cell cycle distribution in either cell line. This study predicts CTCE-9908 IC50 values at various time points using mathematical modelling, revealing cytotoxicity in melanoma and non-cancerous cells. CTCE-9908 significantly inhibited melanoma cell survival at a concentration 10 times lower than the IC50 in B16 F10 cells but not RAW 264.7 cells. However, CTCE-9908 did not affect CXCR4 phosphorylation, apoptosis,\ or cell cycle distribution in either cell line.

Significance of erythropoesis correction in preoperative preparation in the surgical treatment of patients with colorectal cancer

Background. Anemic syndrome, which accompanies the development of pathology in patients with colorectal cancer, can cause serious early postoperative complications. The question of how preoperative preparation activities influence the state of the intestinal structures and the clinical situation in general is relevant. Of particular importance in this situation is the correction of anemic syndrome before surgery, using hematopoietic stimulants. Objective. The purpose of the study is to analyze the dynamics of erythropoiesis and changes in tissue and cell ultrastructure in intraoperative colon biopsy specimens after stimulation of erythropoiesis in preoperative preparation in the surgical treatment of patients with colorectal cancer. Methods. The study of red bone marrow and the wall of the large intestine using transmission electron microscopy. Results. Most patients with colorectal cancer are characterized by the development of signs of ineffective erythropoiesis with a moderate slowdown in the differentiation of erythroblasts and a delay in the release of mature erythrocytes into the systemic circulation. Stimulation of erythropoiesis with the help of epoetin leads to an increase in the functional activity of the central macrophages of the erythropoietic islets of the red bone marrow and the enhancement of their intercellular association with maturing erythroid cells. This is accompanied by the formation of a large number of full-fledged erythrocytes, an increase in the numerical density of cells of the erythroid series and the normalization of their ultrastructure. Conclusion. Stimulation of erythropoiesis with the help of epoetin in the preoperative period causes the inhibition of endothelial dysfunction due to the restriction of dystrophic and destructive changes in endothelial cell organelles, stabilization of their membranes, prevention of arteriole spasm, erythrocyte stasis and erythrocyte phenomena. As a result, the phenomena of infiltration, perivascular and interstitial edema in the composition of the membranes of the intestinal wall are much less pronounced than in the group of patients without preoperative activation of erythropoiesis.

Effects of High-Dose Cyclophosphamide on Ultrastructural Changes and Gene Expression Profiles in the Cardiomyocytes of C57BL/6J Mice

The pathogenesis of cyclophosphamide (CY)-induced cardiotoxicity remains unknown, and methods for its prevention have not been established. To elucidate the acute structural changes that take place in myocardial cells and the pathways leading to myocardial damage under high-dose CY treatments, we performed detailed pathological analyses of myocardial tissue obtained from C57BL/6J mice subjected to a high-dose CY treatment. Additionally, we analysed the genome-wide cardiomyocyte expression profiles of mice subjected to the high-dose CY treatment. Treatment with CY (400 mg/kg/day intraperitoneally for two days) caused marked ultrastructural aberrations, as observed using electron microscopy, although these aberrations could not be observed using optical microscopy. The expansion of the transverse tubule and sarcoplasmic reticulum, turbulence in myocardial fibre travel, and a low contractile protein density were observed in cardiomyocytes. The high-dose CY treatment altered the cardiomyocyte expression of 1210 genes (with 675 genes upregulated and 535 genes downregulated) associated with cell–cell junctions, inflammatory responses, cardiomyopathy, and cardiac muscle function, as determined using microarray analysis (|Z-score| > 2.0). The expression of functionally important genes related to myocardial contraction and the regulation of calcium ion levels was validated using real-time polymerase chain reaction analysis. The results of the gene expression profiling, functional annotation clustering, and Kyoto Encyclopedia of Genes and Genomes pathway functional-classification analysis suggest that CY-induced cardiotoxicity is associated with the disruption of the Ca2+ signalling pathway.

In vitro and in vivo activities of a trithiolato-diRuthenium complex conjugated with sulfadoxine against the apicomplexan parasite Toxoplasma gondii

Organometallic compounds, including Ruthenium complexes, have been widely developed as anti-cancer chemotherapeutics, but have also attracted much interest as potential anti-parasitic drugs. Recently hybrid drugs composed of organometallic Ruthenium moieties that were complexed to different antimicrobial agents were synthesized. One of these compounds, a trithiolato-diRuthenium complex (RU) conjugated to sulfadoxine (SDX), inhibited proliferation of Toxoplasma gondii tachyzoites grown in human foreskin fibroblast (HFF) monolayers with an IC50 < 150 nM, while SDX and the non-modified RU complex applied either individually or as an equimolar mixture were much less potent. In addition, conjugation of SDX to RU lead to decreased HFF cytotoxicity. RU-SDX did not impair the in vitro proliferation of murine splenocytes at concentrations ranging from 0.1 to 0.5 μM but had an impact at 2 μM, and induced zebrafish embryotoxicity at 20 μM, but not at 2 or 0.2 μM. RU-SDX acted parasitostatic but not parasiticidal, and induced transient ultrastructural changes in the mitochondrial matrix of tachyzoites early during treatment. While other compounds that target the mitochondrion such as the uncouplers FCCP and CCCP and another trithiolato-Ruthenium complex conjugated to adenine affected the mitochondrial membrane potential, no such effect was detected for RU-SDX. Evaluation of the in vivo efficacy of RU-SDX in a murine T. gondii oocyst infection model comprised of non-pregnant outbred CD1 mice showed no effects on the cerebral parasite burden, but reduced parasite load in the eyes and in heart tissue.

Electroacupuncture improves peripheral neuropathy by up-regulating Sirt1/PGC-1α/TFAM pathway in type 2 diabetes rats with peripheral neuropathy.

To observe the effect of electroacupuncture (EA) on activation of silent information regulator 1 (Sirt1)/peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α)/mitochondrial transcription factor A (TFAM) pathway in type 2 diabetes (T2DM) rats with peripheral neuropathy (DPN) , so as to explore its possible mechanisms underlying improvement of DPN. Thirty male SD rats were randomly divided into blank control group (n=8) and DPN model group (n=22) which were further divided into model group (n=8) and EA group (n=8) after successful modeling. The model of T2DM was established by high-fat diet and low-dose intraperitoneal injection of streptozocin (35 mg/kg). For rats of the EA group (anesthetized with isoflurane), EA stimulation (2 Hz/15 Hz, 2 mA) was applied to "Tianshu"(ST25) for 20 min, once daily, 6 times a week for 6 weeks. The blood glucose level, body weight, area under curve (AUC) of glucose tolerance test, and hind-paw mechanical pain threshold and thermal pain threshold were observed. The intra-epidermal nerve fiber density (IENFD) of the hind-foot pad was observed by immunofluorescence staining. The motor nerve conduction velocity (MNCV) of the sciatic nerve was measured by using electrophysiological method. H.E. staining was used to observe the histopathological changes of the sciatic nerve after modeling. Transmission electron microscopy (TEM) was used to observe the ultrastructural changes of the sciatic nerve. The protein expressions of energy-related Sirt1, PGC-1α and TFAM in the sciatic nerve was detected by Western blot. Compared with the blank control group, the model group had a higher blood glucose contents and AUC (P<0.001), a slower MNCV (P<0.01), and a decrease in the body weight and in the mechanical and thermal pain thresholds (P<0.001) and IENFD (P<0.001), and in the expression levels of Sirt1, PGC-1α and TFAM (P<0.05, P<0.01). In contrast to the model group, the EA group had a decrease in the blood glucose contents and AUC (P<0.05, P<0.01), and an increase in mechanical and thermal pain thresholds, MNCV, IENFD, and expression levels of Sirt1, PGC-1α and TFAM proteins (P<0.01, P<0.05). In addition, results of histopathological and ultrastructural changes of the sciatic nerve showed more fragmented and disordered distribution of axons on the transverse section, and extensive separation of myelin and axons, uneven myelin thickness, axonal degeneration and irregular shape in the model group, whereas in the EA group, the axons on the transverse section were relatively more dense and more complete, the myelin sheath of the sciatic nerve was relatively uniform, and the axonal shape was relatively regular with relatively milder lesions. EA up-regulates the expressions of Sirt1, PGC-1α, TFAM in T2DM rats with DPN, which may be associated with its functions in improving and repairing the injured peripheral nerves in rats with DPN.

Chikungunya virus infection in human microglial C20 cells induces mitochondria-mediated apoptosis.

Chikungunya virus (CHIKV) infection is associated with acute clinical manifestations and chronic joint inflammation. CHIKV has emerged as a significant causative agent of central nervous system (CNS) complications, including encephalitis and related sequelae. Microglial cells, crucial for immune responses and tissue repair in the CNS, play a vital role in the host response to viral infections, with their activation potentially leading to either protection or pathology. In this study, the infection biology of CHIKV in the C20 human microglial cell line was investigated. The permissiveness of C20 cells to CHIKV infection was assessed, and viral replication kinetics were compared to Vero E6 cells. Cytopathic effects of CHIKV infection on C20 cells were examined, along with ultrastructural changes using transmission electron microscopy. Additionally, apoptosis induction, mitochondrial membrane potential, and alterations in cell surface marker expression were evaluated by flow cytometry. CHIKV infection demonstrated permissiveness in C20 cells, similar to Vero cells, resulting in robust viral replication and cytopathic effects. Ultrastructural analysis revealed viral replication, mature virion formation, and distinctive cytoplasmic and nuclear changes in infected C20 cells. CHIKV infection induced significant apoptosis in C20 cells, accompanied by mitochondrial membrane depolarization and altered expression of cell surface markers such as CD11c, CD14, and HLA-DR. Notably, decreased CD14 expression was observed in CHIKV-infected C20 cells. The study findings suggest that CHIKV infection induces apoptosis in C20 microglial cells via the mitochondrial pathway, with significant alterations in cell surface marker expression, particularly CD14 that is linked with apoptosis induction. These observations provide valuable insights into the role of human microglial cells in the host response to CHIKV infection and contribute to the knowledge on the neuropathogenesis of this virus.

Ultrastructural, molecular and haemato-immunological changes: Multifaceted toxicological effects of microcystin-LR in rohu, Labeo rohita

No water body is resilient to afflicts of algal bloom, if goes unmanaged. With the increasing trend of intensification, eutrophication and climate change, Labeo rohita (rohu) is highly anticipated to suffer from the deleterious effects of bloom and eventually its toxins. A comprehensive study was conducted to understand the toxicopathological effects of microcystin-LR (MC-LR) in rohu following intraperitoneal injection of 96 h-LD50 dose i.e., 713 μg kg−1. Substantial changes in micro- and ultrastructural level were evident in histopathology and transmission electron microscope (TEM) study. The haematological, biochemical, cellular and humoral innate immune biomarkers were significantly altered (p < 0.05) in MC-LR treated fish. The mRNA transcript levels of IL-1β, IL-10, IgM and IgZ in liver and kidney tissues were significantly up-regulated in 12 hpi and declined in 96 hpi MC-LR exposed fish. The relative mRNA expression of caspase 9 in the liver and kidney indicates mitochondrial-mediated apoptosis which was strongly supported by TEM study. In a nutshell, our study illustrates for the first time MC-LR induced toxicological implications in rohu displaying immunosuppression, enhanced oxidative stress, pathophysiology, modulation in mRNA transcription, genotoxicity, structural and ultrastructural alterations signifying it as a vulnerable species for MC-LR intoxication.

Evaluation of histological and ultrastructural changes provoked by prenatal tramadol on postnatal cortical cerebellar neuronal development in rats: possible implication of Ki67, GFAP and MicroRNA-7/P53 signalling trajectories.

Tramadol is a novel centrally acting analgesic. Despite, its implementation during pregnancy may impair neuronal survival and synaptic development in neonatal cerebella. The current investigation assessed the histological and ultrastructural alterations in postnatal cortical cerebellar neuronal development induced by prenatal tramadol. 30 offsprings were divided to control group I: fifteen pups born to mothers given saline from D10 till D21 of gestation. Tramadol-treated group II: fifteen pups born to mothers received tramadol HCL (50mg/kg/day) from D10 till D21 of gestation. Pups were categorized into three subgroups (a, b, and c) and offered for sacrifice on the seventh, fourteenth and twenty-first post-natal days. Light microscopic examination revealed the overcrowding and signs of red degeneration affecting purkinje cell layer. Neurodegenerative signs of both purkinje and granule cell neurons were also confirmed by TEM in form of chromatin condensation, dilated Golgi channels, disrupted endoplasmic reticulum, marked infolding of the nuclear envelope and decrease in granule cell precursors. In addition, the astrocytic processes and terminal nerve axons appeared with different degrees of demyelination and decreased number of oligodendrocytes and degenerated mitochondria. Furthermore, group II exhibited an increase in P53 immune expression. The area percentage of apoptotic cells detected by TUNEL assay was significantly increased. Besides to the significant decrease of Ki67 immunoreactivity in the stem neuronal cell progenitors. Quantitative PCR results showed a significant decline in micro RNA7 gene expression in tramadol treated groups resulting in affection of multiple target genes in P53 signaling pathways, improper cortical size and defect in neuronal development.

“Ultrastructural changes of platelets in COVID-19 and chronic viral hepatitis patients “

ABSTRACT Platelet-viral interactions are evolving as a new concern. Coagulation disorder is a major consequence of the COVID-19 infection. In chronic hepatitis virus infections, defect in coagulation factors, thrombocytopenia and platelet function abnormalities are common. A SARS-CoV-2 infection on top of chronic viral hepatitis infection can be common in areas where viral hepatitis is endemic. Here, we investigate the platelet ultrastructural changes and estimate the serum platelet factor-4 (PF-4), ferritin, CRP, and D-dimer in COVID-19 patients (n = 60), COVID-19 patients with associated chronic viral hepatitis (n = 20), and healthy subjects (n = 20). Ultrastructural changes were demonstrated in all test groups, denoting platelet activation. In chronic viral hepatitis patients, Platelet ultrastrustural apoptotic changes were also seen. Significantly high levels of PF-4 were confirmed in moderate and severe COVID-19 patients (P.value <0.001), with a cut off value of 17 ng/ml for predicting disease severity. A positive correlation of PF-4 with the level of serum ferritin, CRP, and D-dimer (p value < 0.001) was noted, while negatively correlated with platelet count and platelet granule count (p value < 0.001). In our study, chronic viral hepatitis patients presented mild COVID-19 signs, and their PF-4 level was comparable with the subgroup of mild COVID-19 infection. The platelet’s critical role in COVID-19 coagulopathy and chronic viral hepatitis is evidenced by the ultrastructural changes and the high levels of PF4. Moreover, a dual viral infection poses a substantial burden on the platelets, necessitating close monitoring of the patient’s coagulation profile.

Brainendothelial GSDMD activation mediates inflammatory BBB breakdown.

The blood-brain barrier (BBB) protects the central nervous system from infections or harmful substances1; its impairment can lead to or exacerbate various diseases of the central nervous system2-4. However, the mechanisms of BBB disruption during infection and inflammatory conditions5,6 remain poorly defined. Here we find that activation of the pore-forming protein GSDMD by the cytosolic lipopolysaccharide (LPS) sensor caspase-11 (refs. 7-9), but not by TLR4-induced cytokines, mediates BBB breakdown in response to circulating LPS or during LPS-induced sepsis. Mice deficient in the LBP-CD14 LPS transfer and internalization pathway10-12 resist BBB disruption. Single-cell RNA-sequencing analysis reveals that brain endothelial cells (bECs), which express high levels of GSDMD, have a prominent response to circulating LPS. LPS acting on bECs primes Casp11 and Cd14 expression and induces GSDMD-mediated plasma membrane permeabilization and pyroptosis in vitro and in mice. Electron microscopy shows that this features ultrastructural changes in the disrupted BBB, including pyroptotic endothelia, abnormal appearance of tight junctions and vasculature detachment from thebasement membrane. Comprehensive mouse genetic analyses, combined with a bEC-targeting adeno-associated virus system, establish that GSDMD activation in bECs underlies BBB disruption by LPS. Delivery of active GSDMD into bECs bypasses LPS stimulation and opens the BBB. In CASP4-humanized mice, Gram-negative Klebsiella pneumoniae infection disrupts the BBB; this is blocked by expression of a GSDMD-neutralizing nanobody in bECs. Our findings outline a mechanism for inflammatory BBB breakdown, and suggest potential therapies for diseases of the central nervous system associated with BBB impairment.

Phytochemical characterization and antifungal activity of Marchantia polymorpha L. against Rhizoctonia solani

IntroductionMarchantia polymorpha L. is a common liverwort in the family Marchantiaceae. It is used in traditional Chinese herbal medications to heal cuts, scalds, snake bites, fractures, burns and open wounds. Chinese people used it to treat jaundice and inflammation also. Polyphenols (bis-bibenzyls and flavonoids), long-chain polyunsaturated fatty acids and terpenoids have been identified as the primary metabolites in M. polymorpha by phytochemical studies. The present study was aimed to explore the phytochemicals and the fungicidal properties of M. polymorpha extracts against Rhizoctonia solani by Poisoned Food Technique. MethodsThe plant was extracted in four different solvents (Acetone, Methanol, Hexane and Di-ethyl ether) for assessing antifungal activity by Poisoned Food Technique and for phytochemical characterization by GC–MS and LC-MS. The extract with best antifungal activity was further examined for its mode of action using FE-SEM and Fluorescence microscopy. ResultsSignificant fungal growth inhibition was caused by M. polymorpha extracts. The highest inhibition was shown by di-ethyl ether extract (PI= 77.25±0.68). Field Emission Scanning electron microscopy and Fluorescence microscopy depicted the ultrastructural changes in the fungal species induced after treatment with the plant extract. GC–MS analysis revealed the presence of various bioactive compounds in di –ethyl ether extracts such as n- Hexadecanoic acid, Stigmasterol and Phytol with reported pharmacological properties. LC-MS revealed the presence of three antifungal compounds: Marchantin A, Marchantin B. and Plagiochin E. DiscussionThe antifungal properties of M. polymorpha were equivalent to the standard, Amphotericin, which had significant effects against R. solani, indicating good efficacy of the plant extract. The damaging effect of plant extract was further confirmed by FE-SEM and Confocal microscopy. Thus, the present study potentially introduces a solution to the existing dilemma caused by antifungal agrochemicals.

Influence of dark deprivation on the ultrastructure and mitochondrial apparatus of rat hepatocytes

Background&#x0D; Melatonin (MT) is a hormone with a wide range of biological activities. The diversity of biological regulatory effects inherent in MT involves this hormone in the formation of adaptive reactions and in the pathogenesis of various diseases. А decrease оf MT secretion due to exposure to light at night is observed in a significant proportion of people. A number of previous studies have shown that melatonin deficiency, causes significant changes in the structure of the liver of laboratory animals. The state of ultrastructural features of hepatocytes, and in particular their mitochondria, under conditions of dark deprivation remains poorly understood.&#x0D; Aim of study&#x0D; To study the ultrastructural features of liver hepatocytes of male Wistar rats under conditions of 21-day dark deprivation.&#x0D; Materials and methods&#x0D; The study was performed on 40 male Wistar rats, divided into 2 groups. Group 1 was kept under a fixed light regime. Group 2 was kept under dark deprivation conditions. Liver samples, were analyzed using a transmission electron microscope. Micromorphometric methods were used to assess the mitochondrial apparatus of hepatocytes. Statistical processing of the results were performed in the GraphPad Prism v8.41 program (USA).&#x0D; Results&#x0D; In hepatocytes of rats of II group, dark deprivation causes a transformation in the shape of the nuclei, accompanied by swelling of the cytoplasm and the presence of a significant number of lipid-containing vacuoles. Mitochondria are characterized by pronounced hyperplasia, size polymorphism, high electron density, and disordered cristae orientation. In the cytoplasm, the phenomenon of shedding of ribosomes from the endoplasmic reticulum is observed. The number of glycogen granules is significantly reduced. The studied micromorphometric parameters of mitochondria are significantly reduced relative to the control.&#x0D; Conclusion&#x0D; The study suggests that melatonin deficiency, resulting from dark deprivation, leads to a number of significant ultrastructural changes in hepatocytes, especially their mitochondrial apparatus.

Effects of 4-Hydroxybenzaldehydeon on Interactions With Neurovascular Unit-Related Cells

4-hydroxybenzaldehyde (4-HBd) is one of the active compounds with neuroprotective effects, which has been confirmed to have anti cerebral ischemical reperfusion injury (CIRI) effect in previous study. In this study, we explored the protective effect of 4-HBd on the neurovascular unit (NVU) after CIRI and its mechanism through in vivo and in vitro experiments. Establish rat of middle cerebral artery occlusion/reproduction (MCAO/R) model, transwell chamber was used to establish an in vitro primary cell co-culture model to simulate NVU, causing oxygen glucose deprivation/reperfusion (OGD/R) injury, simulating the pathological of CIRI in vivo. Longa 5-point method was used to evaluate the neurological function of the rats, and transmission electron microscope was used to observe the ultrastructural changes of NVU. Western Blot was used to detect the expression of neuronal protein in rat brain. The mRNA expressions of ang-1/tie-2 signaling pathway and bdnf/trkb signaling pathway were detected by qPCR. In vivo results showed that 4-HBd reduced neurological function scores and improved the ultrastructure of NVU after MCAO/R rats. 4-HBd could up-regulate the expression of microtubule associated protein-2 (Map-2), glial fibrillary acidic protein (GFAP) and occludin. In vitro results showed that 4-HBd could activate ang-1/tie-2 signaling pathway, increased occludin mRNA expression and protect the blood brain barrier (BBB). 4-HBd can activate bdnf/trkb signaling pathway, up-regulate map-2 mRNA expression, and promote neuronal repair. In vitro and in vivo results indicated 4-HBd can affect the ang-1/tie-2 and bdnf/trkb signaling pathways, and BBB damage is alleviated and NVU homeostasis is maintained to improve CIRI.

Late Effects of Ionizing Radiation on the Ultrastructure of Hepatocytes and Activity of Lysosomal Enzymes in Mouse Liver Irradiated In Vivo.

The study aimed to investigate late radiation-induced changes in the histology, ultrastructure, and activity of lysosomal enzymes in mouse liver exposed to ionizing radiation. The experiment was conducted on C57BL/6J male mice whose distal part of the liver was exposed occasionally to single doses of radiation (6 MV photons) during targeted heart irradiation; estimated doses delivered to analyzed tissue were 0.025 Gy, 0.25 Gy, 1 Gy, and 2 Gy. Tissues were collected 40 weeks after irradiation. We have observed that late effects of radiation have an adaptive nature and their intensity was dose-dependent. Morphological changes in hepatocytes included an increased number of primary lysosomes and autophagic vacuoles, which were visible in tissues irradiated with 0.25 Gy and higher doses. On the other hand, a significant increase in the activity of lysosomal hydrolases was observed only in tissues exposed to 2 Gy. The etiology of these changes may be multifactorial and result, among others, from unintentional irradiation of the distal part of the liver and/or functional interaction of the liver with an irradiated heart. In conclusion, we confirmed the presence of late dose-dependent ultrastructural and biochemical changes in mouse hepatocytes after liver irradiation in vivo.

Cerebral venous congestion alters CNS homeostatic plasticity, evoking tinnitus-like behavior

BackgroundBrain function and neuronal activity depend on a constant supply of blood from the cerebral circulation. The cerebral venous system (CVS) contains approximately 70% of the total cerebral blood volume; similar to the cerebral arterial system, the CVS plays a prominent role in the maintenance of central nervous system (CNS) homeostasis. Impaired venous autoregulation, which can appear in forms such as cerebral venous congestion, may lead to metabolic abnormalities in the brain, causing severe cerebral functional defects and even chronic tinnitus. However, the role of cerebral venous congestion in the progression of tinnitus is underrecognized, and its pathophysiology is still incompletely understood. This study elucidated the specific pathogenetic role of cerebral venous congestion in the onset and persistence of tinnitus and the possible neurophysiological mechanisms.ResultsWe found that a rat model of cerebral venous congestion exhibited tinnitus-like behavioral manifestations at 14 days postoperatively; from that point onward, they showed signs of persistent tinnitus without significant hearing impairment. Subsequent neuroimaging and neurochemical findings showed CNS homeostatic plasticity disturbance in rats with cerebral venous congestion, reflected in increased neural metabolic activity, ultrastructural synaptic changes, upregulated synaptic efficacy, reduced inhibitory synaptic transmission (due to GABA deficiency), and elevated expression of neuroplasticity-related proteins in central auditory and extra-auditory pathways.ConclusionCollectively, our data suggest that alternations in CNS homeostatic plasticity may play a vital role in tinnitus pathology caused by cerebral venous congestion. These findings provide a new perspective on tinnitus related to cerebral venous congestion and may facilitate the development of precise interventions to interrupt its pathogenesis.