Animal Tissue Sections Research Articles

5-Phenyl valeric acid attenuates α-synuclein aggregation and endoplasmic reticulum stress in rotenone-induced Parkinson’s disease rats: A molecular mechanistic study

The abnormal accumulation of fibrillar α-synuclein in the substantia nigra contributes to Parkinson’s disease (PD). Chemical chaperones like 4-phenyl butyric acid (4PBA) show neuroprotective potential, but high doses are required. A derivative, 5-phenyl valeric acid (5PVA), has reported therapeutic potential for PD by reducing Pael-R expression. This study assessed 5PVA’s efficacy in PD animals and its molecular mechanism. In vitro studies revealed 5PVA’s anti-aggregation ability against alpha-synuclein and neuroprotective effects on SHSY5Y neuroblastoma cells exposed to rotenone. PD-like symptoms were induced in SD rats with rotenone, followed by 5PVA treatment at 100 mg/kg and 130 mg/kg. Behavioral analysis showed significant improvement in memory and motor activity with 5PVA administration. Histopathological studies demonstrated normal neuronal histoarchitecture in mid-brain tissue sections of 5PVA-treated animals compared to the PD group. mRNA studies revealed significant suppression in the expression of various protein folding and heat-shock protein markers in the 5PVA-treated group. In conclusion, 5PVA, with its anti-aggregation ability against alpha-synuclein, acts as a chemical chaperone, showing potential as a therapeutic candidate for PD treatment.

Evaluation optimum ratio of synthetic bone graft material and platelet rich fibrin mixture in a metal 3D printed implant to enhance bone regeneration

BackgroundThis study aims to evaluate the optimal ratio of synthetic bone graft (SBG) material and platelet rich fibrin (PRF) mixed in a metal 3D-printed implant to enhance bone regeneration.MethodsSpecialized titanium hollow implants (5 mm in diameter and 6 mm in height for rabbit; 6 mm in diameter and 5 mm in height for pig) were designed and manufactured using 3D printing technology. The implants were divided into three groups and filled with different bone graft combinations, namely (1) SBG alone; (2) PRF to SBG in 1:1 ratio; (3) PRF to SBG in 2:1 ratio. These three groups were replicated tightly into each bone defect in distal femurs of rabbits (nine implants, n = 3) and femoral shafts of pigs (fifteen implants, n = 5). Animal tissue sections were obtained after euthanasia at the 8th postoperative week. The rabbit specimens were stained with analine blue, while the pig specimens were stained with Masson–Goldner’s trichrome stain to perform histologically examination. All titanium hollow implants were well anchored, except in fracture specimens (three in the rabbit and one fracture in the pig).ResultRabbit specimens under analine blue staining showed that collagen tissue increased by about 20% and 40% in the 1:1 ratio group and the 2:1 ratio group, respectively. Masson–Goldner's trichrome stain results showed that new bone growth increased by 32% in the 1:1 ratio PRF to SBG, while − 8% in the 2:1 ratio group.ConclusionThis study demonstrated that placing a 1:1 ratio combination of PRF and SBG in a stabilized titanium 3D printed implant resulted in an optimal increase in bone growth.

Clinical Confirmation of Pan-Amyloid Reactivity of Radioiodinated Peptide 124I-p5+14 (AT-01) in Patients with Diverse Types of Systemic Amyloidosis Demonstrated by PET/CT Imaging.

There are at least 20 distinct types of systemic amyloidosis, all of which result in the organ-compromising accumulation of extracellular amyloid deposits. Amyloidosis is challenging to diagnose due to the heterogeneity of the clinical presentation, yet early detection is critical for favorable patient outcomes. The ability to non-invasively and quantitatively detect amyloid throughout the body, even in at-risk populations, before clinical manifestation would be invaluable. To this end, a pan-amyloid-reactive peptide, p5+14, has been developed that is capable of binding all types of amyloid. Herein, we demonstrate the ex vivo pan-amyloid reactivity of p5+14 by using peptide histochemistry on animal and human tissue sections containing various types of amyloid. Furthermore, we present clinical evidence of pan-amyloid binding using iodine-124-labeled p5+14 in a cohort of patients with eight (n = 8) different types of systemic amyloidosis. These patients underwent PET/CT imaging as part of the first-in-human Phase 1/2 clinical trial evaluating this radiotracer (NCT03678259). The uptake of 124I-p5+14 was observed in abdominothoracic organs in patients with all types of amyloidosis evaluated and was consistent with the disease distribution described in the medical record and literature reports. On the other hand, the distribution in healthy subjects was consistent with radiotracer catabolism and clearance. The early and accurate diagnosis of amyloidosis remains challenging. These data support the utility of 124I-p5+14 for the diagnosis of varied types of systemic amyloidosis by PET/CT imaging.

Comparative study of the pathogenicity of SARS-CoV-2 B.1 AND B.1.617.2 lineages for syrian hamsters

BACKGROUND: Syrian hamsters are the most sensitive model for studying the pathogenesis of a new coronavirus infection and testing prophylactic and therapeutic drugs against SARS-CoV-2. Accordingly, it is important to identify pathomorphological indicators of tissue damage in coronavirus-infected animals, which would correlate with the severity of the disease.
 AIM: Comprehensive assessment of the pathogenicity of SARS-CoV-2 viruses of B.1 and B.1.167.2 lineages on the model of Syrian hamsters to identify the most sensitive criteria that correlate with the clinical manifestation of the disease.
 MATERIALS AND METHODS: Intranasal infection of animals with SARS-CoV-2, followed by the assessment of the clinical picture of the disease and detailed pathomorphological studies of various organs collected on the 5th day after infection.
 RESULTS: The SARS-CoV-2 Delta virus (B.1.617.2) was shown to be less pathogenic for Syrian hamsters compared to the ancestral strain that circulated during the first wave of the COVID-19 pandemic (B.1). The histopathological characterization of lung tissue sections of infected animals revealed the most sensitive morphometric indicator that correlates with the severity of SARS-CoV-2-induced pathology, namely, the alveolar wall thickness.
 CONCLUSIONS: The use of this indicator makes it possible to determine even slight differences in the severity of virus-induced pathology in the Syrian hamster model, which can be critical in the preclinical evaluation of prophylactic and therapeutic drugs for COVID-19.

Development and Application of DropletProbe Mass Spectrometry for Examining Biodistribution of Therapeutics.

dropletProbe mass spectrometry is a novel technique for molecular characterization of surfaces. It can be used for rapid ex vivo analysis of therapeutics from thin animal tissue sections and has been shown to improve understanding of a drug's absorption, distribution, metabolism and excretion (ADME) properties. Here, we describe the tissue distribution analysis of diclofenac from a dosed whole-body mouse thin tissue section using a dropletProbe mass spectrometry system.

Construction of a Drug Delivery System and Photodynamic Therapy Reagent Based on the Biotin-HSA-DDA-TCPP Molecules and the Application of Synergistic Antitumor Effect.

A biotin-HSA-DDA-TCPP molecule, which can be used as a photodynamic therapeutic agent and drug carrier, was synthesized. The molecule can self-assemble into spherical aggregates, which can be loaded with Dox to form biotin-HSA-DDA-TCPP-Dox nanoparticles in aqueous solution, and the Dox loading efficiency was 86.6 ± 1.76%. The Dox's release behavior was pH responsive and has a sustained release. Cell experiments showed that biotin-HSA-DDA-TCPP-Dox nanoparticles could effectively induce cancer cell apoptosis to exert anticancer and photodynamic therapy effects. The results of animal experiments, tissue sections, and blood biochemistry tests showed that the biotin-HSA-DDA-TCPP-Dox nanoparticles could exert the effect of photodynamic therapy and antitumor, which is similar to Dox after laser irradiation, and achieve a synergistic antitumor effect. The nanoparticles can significantly reduce the Dox toxicity and increase the circulation time of the drug in the body. In summary, the biotin-HSA-DDA-TCPP molecule, which combines the advantages of photodynamic therapy and drug carrier, has great potential in clinical application.

Medica-Plus: a novel Micromegas detector for high-resolution β imaging for improved pharmacological applications

For many years 3H and 14C labelling of molecules of pharmaceutical interest has been performed to study their in vivo biodistribution on animal tissue sections through β-particles detection. Film autoradiography has progressively been replaced by digital β-imagers capable of high sensitivity, real-time imaging and activity counting for absolute quantification of radioactive compounds in tissue sections. After the discovery of the tumor heterogeneity phenomenon, research efforts for characterizing cell heterogeneity have been at the heart of oncology research, aiming at a better understanding of the causes and progression of the disease. This new perspective has also allowed for cell-targeting drugs, and radically changed both sample sizes and radiotracer activities. In this context, Medica-Plus, a transversal project gathering biologists, microfluidics specialists and detector developers, intends to perform quantification of low dose 3H- or 14C-labelled drugs inside single cells. This article reports preliminary results obtained with a prototype detector measuring tritium-generated signal.

Масс-спектрометрическая визуализация для исследования поверхности стали

Визуализация масс-спектрометрическими методами исследования поверхности широко используется для построения карт распределения различных соединений. В настоящей работе данныйинструмент впервые применён для исследования морфологии и состояния различных промышленных сталей. Ранее метод применялся только для визуализации с тонких поверхностей. Авторами показана принципиальная возможность оценки состояния поверхности стали и скорости протекания на ней коррозионных процессов. Показано, что при образовании продуктов коррозии последние легко и на низком уровне обнаруживаются в виде кластерных ионов, что позволяет легко определять начинающуюся коррозию. Визуализация наглядно показывает степень локализации и распространения коррозии, которая на сталях проходит сразу почти по всей поверхности. Также на поверхности стали обнаружены следы адсорбции хлора, который является потенциальным активатором коррозии и показана его локализация. Также возможно обнаружить продукты коррозии и адсорбированные на поверхности соединения, построить диаграмму распределения по широкой области поверхности любого обнаруженного соединения, провести локализацию исследуемой области для изучения её в сочетании с классическими методами исследования поверхности, проводить общий контроль состояния поверхности сплава во времени при его эксплуатации. Метод является уникальным, не имеет аналогов в мире и может быть распространён на любые другие поверхности и материалы

Synthesis of β-cyclodextrin-PEG-G molecules to delay tumor growth and application of β-cyclodextrin-PEG-G aggregates as drug carrier

The clinical use of many chemotherapeutic drugs is considerably greatly limited due to their serious side effects. This problem can be solved by using a low-toxic or nontoxic drug carrier that exhibits excellent performance in entrapping chemotherapeutic drugs. Accordingly, β-cyclodextrin-PEG-guanosine (β-CD-PEG-G) molecule was first synthesized. The molecules can self-assemble into negatively charged spherical aggregates (called β-CD-PEG-G aggregates) that can stably exist in an aqueous solution and entrap doxorubicin (Dox) to form β-CD-PEG-G-Dox nanomedicine. Dox encapsulation efficiency is approximately 79 ± 6.3%. Dox from β-CD-PEG-G-Dox nanomedicine exhibits sustained release and pH responsiveness. Cell and animal experiments showed that β-CD-PEG-G-Dox nanomedicine could effectively induce cancer cell apoptosis to exert antitumor activity. Unexpectively, the animal experiment and tissue sections demonstrated that β-CD-PEG-G aggregates exhibit certain antitumor activity that could delay the tumor growth. Therefore, the β-CD-PEG-G molecule has high potential as a drug carrier candidate.

Evaluation of antiviral effect and toxicity of total flavonoids extracted from Robinia pseudoacacia cv. idaho

In this study, we aimed to evaluate the antiviral effect of total flavonoids extracted from Robinia pseudoacacia cv. idaho (RPTF) in vivo and its toxicity on rats with oral gavage. RPTF was prepared by percolation with 70% ethanol for 24 h and its antiviral effect on different kinds of viruses was evaluated in vitro by MTT staining. The long-term toxicity of RPTF on rats was evaluated through the detection of general behavior, body weight, food intake and related organ tissue sections of experimental animals. We found that RPTF produced significantly inhibitory effects on HSV-1 and EV-71 viruses with the therapeutic index TI values 113.8 and 46.2, respectively. Moreover, toxicity evaluation in vivo showed no significantly adverse effects in rats, indicating that RPTF was safe in use. In conclusion, we demonstrated that RPTF, natural compounds in the Chinese traditional medicine, could act as promising and effective antiviral therapeutics with relative safety in use.

Upregulation of MUC5AC production and deposition of LEWIS determinants by HELICOBACTER PYLORI facilitate gastric tissue colonization and the maintenance of infection

BackgroundHelicobacter pylori bacteria colonize human gastric mucosa, cause chronic inflammation, peptic ulcers and gastric cancer. Colonization is mediated by H. pylori adhesins, which preferentially bind mucin 5 (MUC5AC) and Lewis (Le) determinants. The aim of this study was to evaluate the influence of H. pylori and their components on MUC5AC production and deposition of LeX/LeY in gastric epithelial cells in relation to bacterial adhesion using Caviae porcellus primary gastric epithelial cells and an in vivo model of experimental H. pylori infection in these animals.MethodsMUCA5C and LeX/LeY were induced in vitro by live H. pylori reference strain CCUG 17874 (2 × 107 CFU/ml), H. pylori glycine acid extract (GE), 10 μg/ml; cytotoxin associated gene A (CagA) protein, 1 μl/ml; UreA urease subunit, 5 μg/ml; lipopolysaccharide (LPS) 25 ng/ml and imaged by fluorescence microscopy after anti-MUC5AC or anti-LeX/LeY FITC antibody staining. Bacterial adhesion was imaged by using anti-H. pylori FITC antibodies. The animals were inoculated per os with H. pylori (3 times in 2 days intervals, 1 × 1010 CFU/ml). After 7 or 28 days an infection and inflammation were assessed by histological, serological and molecular methods. Gastric tissue sections of infected and control animals were screend for MUCA5C and LeX, and H. pylori adhesion as above.ResultsMUC5AC production and deposition of Lewis determinants, especially LeX were upregulated in the milieu of live H. pylori as well as GE, CagA, UreA or LPS in vitro and in vivo during infection, more effectively in the acute (7 days) than in the chronic (28 days) phase of infection. This was related to enhanced adhesion of H. pylori, which was abrogated by anti-MUC5AC and anti-LeX or anti-LeY antibody treatment.ConclusionsModulation of MUCA5C production and LeX/LeY deposition in the gastric mucosa by H. pylori can significantly increase gastric tissue colonization during H. pylori infection.

Optical density analysis in autoradiographic images from BNCT protocols

In Boron Neutron Capture Therapy (BNCT) research, information on spatial distribution and concentration of boron in tissues is essential for the treatment planning and for dose determination. Neutron autoradiography technique offers the possibility to gain insight into boron biodistribution in tissues. The irradiation of tissue sections deposited on nuclear track detectors produces damages in the latter, which can be visualized by microscope imaging and that are spatially correlated with the boron localization in the biological section. We had previously developed a methodology for the quantification of boron concentration in tissue samples by nuclear track density determination (QTA).In this work we analyzed the optical density (OD) in autoradiographic images where track density is too high to be quantified by track counting. OD was determined from grey level measurements on low magnification micrographs and proved to be a suitable parameter to quantify boron concentration. The images were originated by placing polycarbonate detectors in contact with samples containing 10B atoms, irradiated with thermal neutrons and finally chemically attacked. Reference standards were developed from aqueous solutions prepared with known boron concentrations in order to construct calibration curves. The obtained optical density values were compared with curves proposed by other authors, showing similar tendencies. The calibration curve was used to make preliminary boron concentration determinations in histological tissue sections of animals infused with boron compounds, which agreed with measurements realized by the QTA approach.The methodology proposed in this work would allow a fast preliminary analysis of histological and autoradiographic images in samples of interest for BNCT.

Recent advances in matrix-assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI) for in situ analysis of endogenous molecules in plants.

Mass spectrometry imaging (MSI) as a label-free and powerful imaging technique enables in situ evaluation of a tissue metabolome and/or proteome, becoming increasingly popular in the detection of plant endogenous molecules. The characterisation of structure and spatial information of endogenous molecules in plants are both very important aspects to better understand the physiological mechanism of plant organism. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) is a commonly-used tissue imaging technique, which requires matrix to assist in situ detection of a variety of molecules on the surface of a tissue section. In previous studies, MALDI-MSI was mostly used for the detection of molecules from animal tissue sections, compared to plant samples due to cell structural limitations, such as plant cuticles, epicuticular waxes, and cell walls. Despite the enormous progress that has been made in tissue imaging, there is still a challenge for MALDI-MSI suitable for the imaging of endogenous compounds in plants. This review summarises the recent advances in MALDI-MSI, focusing on the application of in situ detection of endogenous molecules in different plant organs, i.e. root, stem, leaf, flower, fruit, and seed. Further improvements on instrumentation sensitivity, matrix selection, image processing and sample preparation will expand the application of MALDI-MSI in plant research.

Tracing the fate and transport of secondary plant metabolites in a laboratory mesocosm experiment by employing mass spectrometric imaging

Mass spectrometric imaging (MSI) has received considerable attention in recent years, since it allows the molecular mapping of various compound classes, such as proteins, peptides, glycans, secondary metabolites, lipids, and drugs in animal, human, or plant tissue sections. In the present study, the application of laser-based MSI analysis of secondary plant metabolites to monitor their transport from the grass leaves of Dactylis glomerata, over the crop of the grasshopper Chorthippus dorsatus to its excrements, and finally in the soil solution is described. This plant-herbivore-soil pathway was investigated under controlled conditions by using laboratory mesocosms. From six targeted secondary plant metabolites (dehydroquinic acid, quinic acid, apigenin, luteolin, tricin, and rosmarinic acid), only quinic acid, and dehydroquinic acid, an in-source-decay (ISD) product of quinic acid, could be traced in nearly all compartments. The tentative identification of secondary plant metabolites was performed by MS/MS analysis of methanol extracts prepared from the investigated compartments, in both the positive and negative ion mode, and subsequently compared with the results generated from the reference standards. Except for tricin, all secondary metabolites could be tentatively identified by this approach. Additional liquid-chromatography mass spectrometry (LC-MS) experiments were carried out to verify the MSI results and revealed the presence of quinic acid only in grass and chewed grass, whereas apigenin-hexoside-pentoside and luteolin-hexoisde-pentoside could be traced in the grasshopper body and excrement extracts. In summary, the MSI technique shows a trade-off between sensitivity and spatial resolution.Graphical abstractMonitoring quinic acid in a mesocosm experiment by mass spectrometric imaging (MSI).

MALDI imaging mass spectrometry of Pacific White Shrimp L. vannamei and identification of abdominal muscle proteins.

MALDI imaging mass spectrometry (IMS) has been applied to whole animal tissue sections of Pacific White Shrimp, Litopenaeus vannamei, in an effort to identify and spatially localize proteins in specific organ systems. Frozen shrimp were sectioned along the ventral-dorsal axis and methods were optimized for matrix application. In addition, tissue microextraction and homogenization was conducted followed by top-down LC-MS/MS analysis of intact proteins and searches of shrimp EST databases to identify imaged proteins. IMS images revealed organ system specific protein signals that highlighted the hepatopancreas, heart, nervous system, musculature, and cuticle. Top-down proteomics identification of abdominal muscle proteins revealed the sequence of the most abundant muscle protein that has no sequence homology to known proteins. Additional identifications of abdominal muscle proteins included titin, troponin-I, ubiquitin, as well as intact and multiple truncated forms of flightin; a protein known to function in high frequency contraction of insect wing muscles. The combined use of imaging mass spectrometry and top-down proteomics allowed for identification of novel proteins from the sparsely populated shrimp protein databases.

Analysis of Forensic Casework Utilizing Infrared Spectroscopic Imaging.

A search of the current scientific literature yields a limited number of studies that describe the use of Fourier transform infrared (FT-IR) spectroscopic imaging for the analysis of forensic casework, which is likely due to the fact that these instruments are fairly new commodities to the field of analytical chemistry and are therefore not yet commonplace in forensic laboratories. This report describes recent forensic case studies that have used the technique for determining the composition of a wide variety of multi-component sample types, including animal tissue sections for toxic inclusions, drugs/dietary supplements, an antibiotic with an active pharmaceutical ingredient (API) present as several different salt forms, an adulterated bulk API, unknown trace powders for illicit drugs and an ophthalmic solution suspected of being adulterated with bleach.

Dual isotopes imaging in whole-body autoradiography (WBARG): distribution of 14C-benzo-a-pyrene and 113Sn-tributyltin in mummichog (Fundulus heteroclitus)

Tissue distributions of 14C-BaP and 113Sn-TBT, individually and together, were compared in mummichog by WBARG, after forced-feeding or intraperitoneal injection. Electron differential absorption (EDA) by metal foil was tested to visualize the distribution of 14C and 113Sn in tissue sections of animals exposed to both radiochemicals. Distribution of 14C and 113Sn in fish did not differ as a function of exposure route, but 14C liver/blood and bile/blood concentration ratios were lower in fish dosed with BaP + TBT. EDA allowed a clear differentiation of 14C and 113Sn signals and could be used to image other radionuclides pairs.

Novel methods of automated quantification of gap junction distribution and interstitial collagen quantity from animal and human atrial tissue sections.

BackgroundGap junctions (GJs) are the principal membrane structures that conduct electrical impulses between cardiac myocytes while interstitial collagen (IC) can physically separate adjacent myocytes and limit cell-cell communication. Emerging evidence suggests that both GJ and interstitial structural remodeling are linked to cardiac arrhythmia development. However, automated quantitative identification of GJ distribution and IC deposition from microscopic histological images has proven to be challenging. Such quantification is required to improve the understanding of functional consequences of GJ and structural remodeling in cardiac electrophysiology studies.Methods and ResultsSeparate approaches were employed for GJ and IC identification in images from histologically stained tissue sections obtained from rabbit and human atria. For GJ identification, we recognized N-Cadherin (N-Cad) as part of the gap junction connexin 43 (Cx43) molecular complex. Because N-Cad anchors Cx43 on intercalated discs (ID) to form functional GJ channels on cell membranes, we computationally dilated N-Cad pixels to create N-Cad units that covered all ID-associated Cx43 pixels on Cx43/N-Cad double immunostained confocal images. This approach allowed segmentation between ID-associated and non-ID-associated Cx43. Additionally, use of N-Cad as a unique internal reference with Z-stack layer-by-layer confocal images potentially limits sample processing related artifacts in Cx43 quantification. For IC quantification, color map thresholding of Masson's Trichrome blue stained sections allowed straightforward and automated segmentation of collagen from non-collagen pixels. Our results strongly demonstrate that the two novel image-processing approaches can minimize potential overestimation or underestimation of gap junction and structural remodeling in healthy and pathological hearts. The results of using the two novel methods will significantly improve our understanding of the molecular and structural remodeling associated functional changes in cardiac arrhythmia development in aged and diseased hearts.

Minnelide: A Novel Therapeutic That Promotes Apoptosis in Non-Small Cell Lung Carcinoma In Vivo

BackgroundMinnelide, a pro-drug of triptolide, has recently emerged as a potent anticancer agent. The precise mechanisms of its cytotoxic effects remain unclear.MethodsCell viability was studied using CCK8 assay. Cell proliferation was measured real-time on cultured cells using Electric Cell Substrate Impedence Sensing (ECIS). Apoptosis was assayed by Caspase activity on cultured lung cancer cells and TUNEL staining on tissue sections. Expression of pro-survival and anti-apoptotic genes (HSP70, BIRC5, BIRC4, BIRC2, UACA, APAF-1) was estimated by qRTPCR. Effect of Minnelide on proliferative cells in the tissue was estimated by Ki-67 staining of animal tissue sections.ResultsIn this study, we investigated in vitro and in vivo antitumor effects of triptolide/Minnelide in non-small cell lung carcinoma (NSCLC). Triptolide/Minnelide exhibited anti-proliferative effects and induced apoptosis in NSCLC cell lines and NSCLC mouse models. Triptolide/Minnelide significantly down-regulated the expression of pro-survival and anti-apoptotic genes (HSP70, BIRC5, BIRC4, BIRC2, UACA) and up-regulated pro-apoptotic APAF-1 gene, in part, via attenuating the NF-κB signaling activity.ConclusionIn conclusion, our results provide supporting mechanistic evidence for Minnelide as a potential in NSCLC.

An inexpensive alternative to routine section adhesives for histology: the mucilaginous substance of the Assyrian plum

We found that the mucilaginous substance of the Assyrian plum, Cordia myxa, can be used as an adhesive for attaching sections of animal tissues to slides. Unlike Mayer's albumen, this material left no stainable residue and had no noticeable effect on the histological structure of the tissue sections. The mucilaginous substance of C. myxa is a useful and inexpensive alternative to standard adhesives.