Cryostat Sections Research Articles

EM Optimized Cryostat Sections (EMOCS) for Wide Scale Ultrastructural Imaging of Tissue Morphology Combined with Antigen Detection by Immunohistochemistry

EM Optimized Cryostat Sections (EMOCS) for Wide Scale Ultrastructural Imaging of Tissue Morphology Combined with Antigen Detection by Immunohistochemistry

Molecular Cloning and Functional Identification of a Pericarp- and Testa-Abundant Gene's (AhN8DT-2) Promoter from Arachis hypogaea.

Cultivated peanut (Arachis hypogaea L.) is a key oil- and protein-providing legume crop of the world. It is full of nutrients, and its nutrient profile is comparable to that of other nuts. Peanut is a unique plant as it showcases a pegging phenomenon, producing flowers above ground, and after fertilization, the developing peg enters the soil and produces seeds underground. This geocarpic nature of peanut exposes its seeds to soil pathogens. Peanut seeds are protected by an inedible pericarp and testa. The pericarp- and testa-specific promoters can be effectively used to improve the seed defense. We identified a pericarp- and testa-abundant expression gene (AhN8DT-2) from available transcriptome expression data, whose tissue-specific expression was further confirmed by the qRT-PCR. The 1827bp promoter sequence was used to construct the expression vector using the pMDC164 vector for further analysis. Quantitative expression of the GUS gene in transgenic Arabidopsis plants showed its high expression in the pericarp. GUS staining showed a deep blue color in the pericarp and testa. Cryostat sectioning of stained Arabidopsis seeds showed that expression is only limited to seed coat (testa), and staining was not present in cotyledons and embryos. GUS staining was not detected in any other tissues, including seedlings, leaves, stems, and roots, except for some staining in flowers. Under different phytohormones, this promoter did not show an increase in expression level. These results indicated that the AhN8DT-2 promoter drives GUS gene expression in a pericarp- and testa-specific manner. The identified promoter can be utilized to drive disease resistance genes, specifically in the pericarp and testa, enhancing peanut seed defense against soil-borne pathogens. This approach has broader implications for improving the resilience of peanut crops and other legumes, contributing to sustainable agricultural practices and food security.

Excessive sucrose consumption reduces synaptic density and increases cannabinoid receptors in Göttingen minipigs

Diets high in sucrose and fat are becoming more prevalent the world over, accompanied by a raised prevalence of cardiovascular diseases, cancers, diabetes, obesity, and metabolic syndrome. Clinical studies link unhealthy diets with the development of mental health disorders, particularly depression. Here, we investigate the effects of 12 days of sucrose consumption administered as 2 L of 25% sucrose solution daily for 12 days in Göttingen minipigs on the function of brain receptors involved in reward and motivation, regulating feeding, and pre- and post-synaptic mechanisms. Through quantitative autoradiography of cryostat sections containing limbic brain regions, we investigated the effects of sucrose restricted to a 1-h period each morning, on the specific binding of [3H]raclopride on dopamine D2/3 receptors, [3H]UCB-J at synaptic vesicle glycoprotein 2A (SV2A), [3H]MPEPγ at metabotropic glutamate receptor subtype 5 (mGluR5) and [3H]SR141716A at the cannabinoid receptor 1 (CB1). Compared to control diet animals, the sucrose group showed significantly lower [3H]UCB-J and [3H]MPEPγ binding in the prefrontal cortex. The sucrose-consuming minipigs showed higher hippocampal CB1 binding, but unaltered dopamine D2/3 binding compared to the control group. We found that the sucrose diet reduced the synaptic density marker while increasing CB1 binding in limbic brain structures, which may subserve maladaptive changes in appetite regulation and feeding. Further studies of the effects of diets and lifestyle habits on brain neuroreceptor and synaptic density markers are warranted.

Localization of sensory nerve terminals containing calcitonin gene-related peptide (CGRP) on striated muscle fibers in the rat esophagus: Evidence for triple innervation via motor endplates

BackgroundMany esophageal striated muscles of mammals are dually innervated by the vagal and enteric nerves. Recently, substance P (SP)-sensory nerve terminals with calcitonin gene-related peptide (CGRP) were found on a few striated muscle fibers in the rat esophagus, implying that these muscle fibers are triply innervated. In this study, we examined the localization and origin of CGRP-nerve endings in striated muscles to consider their possible roles in the esophagus regarding triple innervation. MethodsWholemounts of the rat esophagus were immunolabeled to detect CGRP-nerve endings in striated muscles. Also, retrograde tracing was performed by injecting Fast Blue (FB) into the esophagus, and cryostat sections of the medulla oblongata, nodose ganglion (NG), and the tenth thoracic (T10) dorsal root ganglion (DRG) were immunostained to identify the origin of the CGRP-nerve endings. ResultsCGRP-fine, varicose nerve endings were localized in motor endplates on a few esophageal striated muscle fibers (4 %), most of which received nitric oxide (NO) synthase nerve terminals, and most of the CGRP nerve endings were SP- and transient receptor potential vanilloid member 1 (TRPV1)-positive. Retrograde tracing showed many FB-labeled CGRP-neurons positive for SP and TRPV1 in the NG and T10 DGR. ConclusionsThis study suggests that the CGRP-varicose nerve endings containing SP and TRPV1 in motor endplates are sensory, and a few esophageal striated muscle fibers are triply innervated. The nerve endings may detect acetylcholine-derived acetic acid from the vagal motor nerve endings and NO from esophageal intrinsic nerve terminals in the motor endplates to regulate esophageal motility.

Effect of a water-soluble form of dihydroquercetin on age-dependent LPS-induced gliovascular remodeling of the substantia nigra in rats

Background. The molecular and cellular mechanisms of action of the water-soluble form of dihydroquercetin (DHA-VF) on neurogliovascular units, age-related disturbances of intrasystem connections that may underlie neuroinflammatory and neurodegenerative diseases, remain unclear.The aim: to study structural changes of microcirculatory vessels and functional responses of micro- and astroglial cells in the substantia nigra of young and old rats in response to intranigral injection of lipopolysaccharide (LPS) and subsequent oral administration of DHA-VF.Methods. Young (250-320 g) and old (390-450 g) Wistar rats were injected with 2 μL LPS solution at a concentration of 0.01 μL/mL (experimental group; n=24) or 2 μL sterile physiological solution (control group; n=12) into the substantia nigra area of the brain using a stereotaxic device. Half of the animals in the experimental groups (6 animals of each age group) were given 2 ml of a solution containing DHA-VF at a concentration of 3 mg/mL daily by gavage with a probe. After 8 weeks, the animals were decapitated and cryostat sections were obtained for histochemical (FITC-labeled tomato lectin) and immunohistochemical (antibodies against GFAP and CD-11β) staining of vascular endothelium and glial cells, respectively. Results. 8 weeks after LPS administration to old rats in the CS, a significant excess of areas occupied by cell bodies and processes of microglial and astroglial cells as well as the number of vessels at standard sites was found compared to both young animals exposed to similar exposure and old control animals. Oral administration of DHA-VF to rats significantly reduced LPS-induced glial activation in young and old animals. In addition, administration of DHA-VF to old animals reduced the intensity of LPS-induced microvascular remodeling of the CS. Conclusions. LPS administration in the rat CS induces neuroinflammation and vascular angiogenesis, which are maximally expressed in old animals. Administration of DHA-VF for 8 weeks significantly reduced these LPS-induced changes.

Survival and Functional Integration of Human Embryonic Stem Cell-Derived Retinal Organoids After Shipping and Transplantation into Retinal Degeneration Rats.

Because derivation of retinal organoids (ROs) and transplantation are frequently split between geographically distant locations, we developed a special shipping device and protocol capable of the organoids' delivery to any location. Human embryonic stem cell (hESC)-derived ROs were differentiated from the hESC line H1 (WA01), shipped overnight to another location, and then transplanted into the subretinal space of blind immunodeficient retinal degeneration (RD) rats. Development of transplants was monitored by spectral-domain optical coherence tomography. Visual function was accessed by optokinetic tests and superior colliculus (SC) electrophysiology. Cryostat sections through transplants were stained with hematoxylin and eosin; or processed for immunohistochemistry to label human donor cells, retinal cell types, and synaptic markers. After transplantation, ROs integrated into the host RD retina, formed functional photoreceptors, and improved vision in rats with advanced RD. The survival and vision improvement are comparable with our previous results of hESC-ROs without a long-distance delivery. Furthermore, for the first time in the stem cell transplantation field, we demonstrated that the response heatmap on the SC showed a similar shape to the location of the transplant in the host retina, which suggested the point-to-point projection of the transplant from the retina to SC. In conclusion, our results showed that using our special device and protocol, the hESC-derived ROs can be shipped over long distance and are capable of survival and visual improvement after transplantation into the RD rats. Our data provide a proof-of-concept for stem cell replacement as a therapy for RD patients.

Expression of NMU and NMUR1 in tryptase-positive mast cells and PBLs in allergic rhinitis patients' nasal mucosa.

The neuropeptide U (NMU) has been proven to elicit the release of mediators from mast cells (MCs) through its receptor NMUR1 in allergic inflammatory models. However, little is known about the correlations between NMU and MCs in human allergic rhinitis (AR). The objective of this study is to investigate the expressions of NMU and NMUR1 in the tryptase + MCs and the peripheral blood leukocytes (PBLs) in human nasal mucosa with AR. Specimens of nasal mucosa from patients with AR (n = 10) and control patients without AR (n = 8) were collected and soaked in frozen tissue liquid solution (OCT) in tum. Cryostat sections were prepared for immunofluorescence staining. Tryptase was used as a marker to detect mast cells and other tryptase + immune cells. The expression of NMU and NMUR1 was respectively determined by double staining using a confocal microscope. Neither NMU nor NMUR1 were detected in the tryptase + mast cells in the human nasal mucosa. To our surprise, both NMU and NMUR1 were co-expressed with tryptase in the PBLs within peripheral blood vessels in AR and controls. Our findings showed that NMU could not influence human nasal tryptase + mast cells directly through NMUR1 in AR. The co-expression of both NMU and NMUR1 with tryptase in the PBLs provided new insight into the potential roles of NMU and tryptase in the circulation PBLs, and the infiltrated PBLs may promote nasal allergic inflammation by producing tryptase and NMU.

A Water-soluble Form of Dihydroquercetin Reduces LPS-induced Astrogliosis, Vascular Remodeling, and mRNA VEGF-A Levels in the Substantia Nigra of Aged Rats

Background: The age-dependent sporadic form of PD is characterized by the degeneration of dopaminergic (DA) neurons in the Substantia Nigra (SN), gliosis, and vascular changes. Vascular changes may contribute to the onset of the disease and exacerbate the neurodegenerative process, as some vascular changes occur before the onset of neuronal loss. To demonstrate the anti-neuroinflammatory efficacy of a new compound, a water-soluble form of dihydroquercetin (DHQ-WF), we studied the structural changes of microcirculatory vasculature, astroglial GFAP, and vascular endothelial growth factor –A (VEGF-A) mRNA expression in the SN of young and old rats after unilateral nigral treatment by lipopolysaccharide (LPS) and oral administration of DHQ-WF. Materials and methods: The experiments were performed on 18 young (8 weeks - 10 weeks old; 250 g - 320 g) and 18 old (18 months - 19 months old; 390 g - 450 g) male Vistar rats. Young and adult rats from the experimental groups were stereotactically injected with 2 μL LPS solution (LPS from Escherichia coli; 0,01 μL/mL) into one side of the SN. Control young and old rats were similarly injected with 2 μL sterile saline. Half of the animals in both the control and experimental groups (6 animals in each group) received a 2 ml solution containing DHQ-WF at a concentration of 3 mg/ml orally every day. After 8 weeks, brains were harvested and serial cryostat sections were prepared for histochemical (FITC-labeled tomato lectin), immunohistochemical (anti-GFAP Antibody, Cy3 Conjugate) staining, and real-time PCR (mRNA VEGF-A). Results: Eight weeks after LPS injection into the SN, a significant excess of areas occupied by cell bodies and processes of astroglial cells, the density of microcirculatory vessels, and mRNA VEGF-A expression was observed in old animals compared to control old animals and young LPS-treated rats. Oral administration of DHQ-WF to LPS-treated rats resulted in a significant reduction of these parameters in old animals. Conclusion: Injection of LPS into rat SN induces neuroinflammation and vascular angiogenesis, maximally expressed in old animals. Administration of DHQ-WF for 8 weeks significantly reduces these LPS-induced changes. DHQ-WF may be an effective treatment for reducing the effects of neuroinflammation in the aging brain.

Phenotype-Genotype Correlations in Three Different Cases of Adult-Onset Genetic Focal Segmental Glomerulosclerosis.

This study highlights the importance of a combined diagnostic approach in the diagnosis of rare diseases, such as adult-onset genetic FSGS. We present three adult patient cases evaluated with kidney biopsy for proteinuria, chronic kidney disease, and hypertension, which were suggestive of adult-onset genetic FSGS. Renal biopsy samples and formalin-fixed, paraffin-embedded fetal kidneys were evaluated using standard light microscopical stainings, direct immunofluorescence on cryostat sections, and electron microscopy. Clinical exome sequencing was performed for each case, and 45 FSGS-related genes were analyzed. Identifying mutations in the PAX2, ACTN4, and COL4A5 genes have prompted a re-evaluation of the previous histopathological examinations. The PAX2 mutation led to a thinner nephrogenic zone and decreased number of glomeruli, resulting in oligohydramnios during fetal development and oligomeganephronia and adaptive focal-segmental glomerulosclerosis in adulthood. The ACTN4 mutation caused distinct electron-dense aggregates in podocyte cell bodies, while the COL4A5 mutation led to segmental sclerosis of glomeruli with marked interstitial fibrosis and tubular atrophy. The identification of specific mutations and their histopathological consequences can lead to a better understanding of the disease and its progression, as well as potential treatment options.

The unappreciated roles of oligodendrocytes in the etiology of Alzheimer’s disease

AbstractBackgroundDementias such as Alzheimer’s disease are characterized as neurodegenerative diseases. While researchers accept that the etiology of dementia involves multiple cell types, the use of “neuro” as a prefix to “degeneration” biases our mechanistic thinking towards the nerve cell. The cell type that has been most marginalized by this bias is the oligodendrocyte and its myelin ensheathment of the larger axons of the brain. As early as the 1960’s the involvement of myelin pathology had been recognized in the Alzheimer’s disease (AD) brain. Later, with advances in imaging technology, a strong argument was advanced for the mechanistic involvement of myelin, a cholesterol rich membrane, in the etiology of dementia.MethodFixed frozen human prefrontal cortex (BA9), hippocampus, and cerebellum were received from both the University of Pittsburgh ADRC and the NIH‐sponsored NeuroBioBank. Mouse brain from wild type and AD models (R1.40 and APP/PS1) were also obtained. Cryostat sections were immunostained for APP and various myelin‐related peptides.ResultThis myelin hypothesis will be reviewed and put in the AD context by summarizing recent results on the impact of APOE, a cholesterol carrier and a major risk factor gene for sporadic AD, on the cells of the oligodendrocyte lineage. Observations on the presence of APP and its processing secretases in oligodendrocytes will deepen the connection to the etiology of ADConclusionBoth past and present data support the assignment of an important role for oligodendrocytes in the initiation and progression of Alzheimer’s disease. Broadening our current “neurocentric” view will speed progress towards meaningful therapeutic approaches.

Features of neuroglia at the epicenter of spinal cord contusion injury and at distant areas in mini-pigs

Aim. To determine the delayed (after 2 months) effect of spinal cord injury (SCI) in the lower thoracic region in the mini-pigs on the morphologic state of macro- and microglia in nearby and remote caudal areas. Materials and methods. Sexually mature female Vietnamese pot-bellied pigs were randomly divided into two groups: SCI (n = 3) and intact (n = 3). Dosed contusion SCI was modelled at the level of the Th8–Th9 vertebrae, and transverse cryostat sections of the caudal segment adjacent to the epicenter of injury and the lumbar thickening (L4–S2) were examined 2 months later. The expression of astrocyte markers (glial fibrillary acidic protein, GFAP) and microglial markers (ionized calcium-binding adapter molecule 1, Iba1) was assessed as the relative immunopositive area occupied by cells. When counting the number of oligodendroglial cells (oligodendrocyte transcription factor 2, Olig2), the presence of nuclei detectable with 4’,6-diamidino-2-phenylindole (DAPI) was taken into account.Results. After SCI, an increase in the relative areas occupied by GFAP-positive astrocytes and Iba1-positive microglia and a decrease in Olig2-positive oligodendrocytes were detected in both the lesion area and lumbar thickening. In both regions, 2 months after SCI, the proportion of astrocytes was not significantly different in the anterior horns and doubled in the posterior horns. Microglia cells with SCI were 2.5 times more in the anterior horns of both regions and in the posterior horns of the lumbar thickening, while the presence of microglia increased slightly (1.2 times) in the posterior horns in the SCI region. The number of oligodendrocytes decreased in the area of the epicenter of SCI in the anterior and posterior horns by 1.5–1.75 times, and in the lumbar thickening more significantly: the number decreased by 2.5 times in the anterior horn and 5.5 times in the posterior horn. Conclusion. The results of the study revealed a similar pattern of macro- and microglial cell distribution both in the SCI region and in remote areas. The obtained data testify to the necessity to take into account the state of the areas of nervous tissue remote from the epicenter of SCI when stimulating neuroregeneration in such patients

Hepatocellular Ballooning is Due to Highly Pronounced Glycogenosis Potentially Associated with Steatosis and Metabolic Reprogramming.

Hepatocellular ballooning is a common finding in chronic liver disease, mainly characterized by rarefied cytoplasm that often contains Mallory-Denk bodies (MDB). Ballooning has mostly been attributed to degeneration but its striking resemblance to glycogenotic/steatotic changes characterizing preneoplastic hepatocellular lesions in animal models and chronic human liver diseases prompts the question whether ballooned hepatocytes (BH) are damaged cells on the path to death or rather viable cells, possibly involved in neoplastic development. Using specimens from 96 cirrhotic human livers, BH characteristics were assessed for their glycogen/lipid stores, enzyme activities, and proto-oncogenic signaling cascades by enzyme- and immunohistochemical approaches with serial paraffin and cryostat sections. BH were present in 43.8% of cirrhotic livers. Particularly pronounced excess glycogen storage of (glycogenosis) and/or lipids (steatosis) were characteristic, ground glass features and MDB were often observed. Decreased glucose-6-phosphatase, increased glucose-6-phosphate dehydrogenase activity and altered immunoreactivity of enzymes involved in glycolysis, lipid metabolism, and cholesterol biosynthesis were discovered. Furthermore, components of the insulin signaling cascade were upregulated along with insulin dependent glucose transporter glucose transporter 4 and the v-akt murine thymoma viral oncogene homolog/mammalian target of rapamycin signaling pathway associated with de novo lipogenesis. BH are hallmarked by particularly pronounced glycogenosis with facultative steatosis, many of their features being reminiscent of metabolic aberrations documented in preneoplastic hepatocellular lesions in experimental animals and chronic human liver diseases. Hence, BH are not damaged entities facing death but rather viable cells featuring metabolic reprogramming, indicative of a preneoplastic nature.

Marine pollutant Phenanthrene (PHE) exposure causes immunosuppression of hemocytes in crustacean species, Scylla paramamosain

Phenanthrene (PHE), a representative polycyclic aromatic hydrocarbons (PAHs), is widely found in aquatic environments, which exhibits high toxicity to aquatic organisms and has a substantial impact on overall health. In order to investigate the immunosuppressive effects of PHE exposure on marine crustacean species, the Scylla paramamosain was exposure to different concentrations of PHE, which was 0 μg/L (control group), 0.7 μg/L, 7 μg/L, or 70 μg/L PHE with 35 individuals in every group, respectively. The results showed that the color of hemocytes gradually deepened with increasing PHE concentration, and the total hemocyte count (THC) was activated and increased after PHE exposure analyzed by Flow cytometry. Meanwhile, compared with the control group, cryostat sections of hepatopancreas showed cell infiltration, cell steatosis, eosinophilic masses and vacuolization in PHE groups. The superoxide dismutase (SOD) activity was decreased immensely in PHE exposure groups, meanwhile, the acid phosphatase (ACP) activity and glutathione (GSH) activities were increased after PHE exposure compared with control group. Moreover, the expression profile of Crustin, TLR, MCM7, JAK, caspase, Moyosin and P53 were up-regulated significantly after 7th day PHE exposure in all treatment groups by Q-PCR. Those data illustrated that PHE exposure could inhibit the immune function of mud crab by causing hepatopancreas damage, induce the activity of antioxidant enzymes and the expression of immune genes. These data provide a scientific basis for evaluating the impact of PAH pollution on marine organisms.

Evaluating the optimal tissue thickness for mass spectrometry imaging using infrared matrix-assisted laser desorption electrospray ionization.

Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) utilizes a 2970 nm mid-IR laser to desorb samples with depth resolutions (Z) on the order of micrometers. Conventionally, 5-20 μm thick tissue sections are used to characterize different applications of the IR-MALDESI source, but an optimal thickness has not been systematically investigated. Mouse liver was sectioned to various thicknesses and analyzed using IR-MALDESI mass spectrometry imaging (MSI). Height profiles of tissue sections of various cryosectioned thicknesses were acquired to affirm tissue thickness. Tissue sections of each thickness were measured using a Keyence microscope. Paraffin wax was cryosectioned, mounted on microscope slides, and measured using a chromatic confocal sensor system to determine the cryostat sectioning accuracy. Analyzing sectioned tissues at higher thickness (>10μm) leads to lower ion abundance, a decrease in signal over long analysis times, and more frequent instrument cleaning. Additionally, increasing tissue thickness above the optimum (7μm) does not result in a significant increase in lipid annotations. This work defines an optimal sample thickness for IR-MALDESI-MSI and demonstrates the utility of optimizing tissue thickness for MSI platforms of comparable Z resolution.

Phrenic Nerve Transfer to Musculocutaneous Nerve: An Anatomical and Histological Study.

To restore elbow flexor muscle function in case of traumatic brachial plexus avulsion, the phrenic nerve transfer to the musculocutaneous nerve has become part of clinical practice. The nerve transfer can be done by means of video-assisted thoracic surgery without nerve graft or via supraclavicular approach in combination with an autograft. This study focuses on a detailed microscopic and macroscopic examination of the phrenic nerve. It will allow a better interpretation of existing clinical results and, thus, serve as a basis for future clinical studies. An anatomical study was conducted on 28 body donors of Caucasian origin (female n = 14, male n = 14). A sliding caliper and measuring tape were used to measure the diameter and length of the nerves. Sudan black staining was performed on 15 µm thick cryostat sections mounted on glass slides and the number of axons was determined by the ImageJ counting tool. In 23 individuals, the phrenic nerve could be examined on both sides. In 5 individuals, however, only one side was examined. Thus, a total of 51 nerves were examined. The mean length of the left phrenic nerves (33 cm (29-38 cm)) was significantly longer compared to the mean length of the right phrenic nerves (30 cm (24-33 cm)) (p < 0.001). Accessory phrenic nerves were present in 9 of 51 (18%) phrenic nerves. The mean number of phrenic nerves axons at the level of the first intercostal space in body donors with a right accessory phrenic nerve was significantly greater compared to the mean number of phrenic nerves axons at the same level in body donors without a right accessory phrenic nerve (3145 (range, 2688-3877) vs. 2278 (range, 1558-3276)), p = 0.034. A negative correlation was registered between age and the nerve number of axons in left (0.742, p < 0.001) and right (-0.273, p = 0.197) phrenic nerves. The mean distance from the upper edge of the ventral ramus of the fourth cervical spinal nerve to the point of entrance of the musculocutaneous nerve between the two parts of the coracobrachialis muscle was 19 cm (range, 15-24 cm) for the right and 20 cm (range, 15-25 cm) for the left arm. If an accessory phrenic nerve is available, it presumably should be spared. Thus, in that case, a supraclavicular approach in combination with a nerve graft would probably be of advantage.

Histochemical mapping of the duration of action of photobiomodulation on cytochrome c oxidase in the rat brain.

This is the first study mapping the duration of action of in vivo photobiomodulation (PBM) on cytochrome-c-oxidase (CCO). In cellular bioenergetics, CCO is the terminal rate-limiting enzyme in the mitochondrial electron transport chain, which catalyzes oxygen utilization for aerobic energy production. PBM using transcranial infrared laser stimulation (TILS) is a promising intervention for non-invasively modulating CCO in the brain. TILS of the human prefrontal cortex directly causes CCO photo-oxidation, which is associated with increased cerebral oxygenation and improved cognition. This experiment aimed to map the duration of action of in vivo PBM on CCO activity in discrete neuroanatomic locations within rat brains up to 4 weeks after a single TILS session (50 s, 1064 nm CW, 250 mW/cm2). Control brains from rats treated with a sham session without TILS (laser off) were compared to brains from TILS-treated rats that were collected 1 day, 2 weeks, or 4 weeks post-TILS. Cryostat sections of the 36 collected brains were processed using quantitative enzyme histochemistry and digitally imaged. Densitometric readings of 28 regions of interest were recorded and converted to CCO activity units of oxygen utilization using calibration standards. Data analysis (ANCOVA) compared each laser-treated group to sham with whole-brain average as a covariate. The prefrontal infralimbic cortex showed the earliest significant increase in CCO activity between 1-day post-TILS and sham groups, which continued elevated for 2-4 weeks post-TILS. Significant differences in CCO activity between 2-weeks and sham groups were also found in the lateral septum, accumbens core, CA3 of the hippocampus, and the molecular layer of the hippocampus. The medial amygdala showed a significant decrease in CCO activity between 4-weeks and sham. Further analyses showed significant inter-regional CCO activity correlations among the brain regions as the result of TILS, with the most pronounced changes at 4-weeks post-stimulation. The time course of changes in CCO activity and network connectivity suggested that TILS caused different neuroplasticity types of bioenergetic changes at different time scales, depending on brain region and its depth from the cortex. In conclusion, this controlled CCO histochemical study demonstrated a long-lasting duration of action of PBM in the rat brain.

Improved syntheses of P2X7 ligands based on substituted benzyl amide of pyroglutamic acid motif labelled with iodine-123 or iodine-125

P2X7 sites are emerging targets for molecular imaging research, notably in the context of neurodegeneration and inflammatory conditions. Therefore, we prepared a precursor for (radio)iodination of the P2X7 ligand VPGIS191. We then developed a radioiodination method with 123I for SPECT with a radiochemical yield of 71 ± 13% and 125I for autoradiography with a radiochemical yield of 85 ± 6% Autoradiography of [125I]VPGIS191 in mouse brain cryostat sections demonstrated approximately 36 nM binding affinity and Bmax of approximately 400 pmol/gram tissue for P2X7 binding sites. VPGIS191 (cis) had a two-fold lower affinity compared to its geometric trans-isomer TZ6019.Graphical abstract

Interrelation of HLA-I and class II major histocompatibility complex molecules with clinical and morphological signs of breast cancer: A retrospective cohort study

Background. For a long time, interest in the HLA peptide complex is unabated, the clinical significance of which in cancer is still the subject of intense debate. Through the presentation of HLA antigens, tumor cells become available for recognition and destruction by effector cells of the immune system. A detailed analysis of the expression status of HLA molecules by breast cancer cells is of both scientific and important practical value. It can provide additional information about the immune system to determine a further strategy for treating breast cancer.&#x0D; Aim. To evaluate the frequency of expression of HLA-I and class II molecules by breast cancer cells and to determine its relationship with the morphological and clinical characteristics of the tumor.&#x0D; Materials and methods. This study included 82 patients with breast cancer who received treatment at the Blokhin National Medical Research Center of Oncology. Immunophenotyping of the primary tumor was performed by the immunohistochemical method (immunofluorescent staining) on cryostat sections. The reaction was evaluated using a ZEISS luminescent microscope (AXIOSKOP, Germany). The frequency of expression of HLA-I and class II molecules was studied depending on the clinical and morphological characteristics of breast cancer.&#x0D; Results. It was found that the frequency of expression of HLA I and II class molecules by breast cancer cells differed. HLA class I antigens are preserved in almost half of the cases 54.5%, while HLA class II antigens are preserved in 22.0%. Associations of molecules of the major histocompatibility complex with clinical and morphological signs of breast cancer were revealed. The frequency of HLA-DR negative cases increases in the stage advanсed (p=0.029). The frequency of monomorphic expression of HLA class II with T1 tumor was 50% versus 0% at T4 tumor (p=0.032). Estrogen receptor-negative tumors in most cases did not express HLA-II class (85.2% vs 64%; p=0.034). No connection with other clinical and morphological features of the tumor has been established.&#x0D; Conclusion. In most cases of breast cancer, the expression of HLA class II molecules is lost, while the expression of HLA class I is preserved in half of the cases. Monomorphic expression of HLA class II is characteristic of the early stage of breast cancer development and predominantly of receptor-positive tumors.

A Procedure for Mouse Dorsal Root Ganglion Cryosectioning.

High-quality mouse dorsal root ganglion (DRG) cryostat sections are crucial for proper immunochemistry staining and RNAscope studies in the research of inflammatory and neuropathic pain, itch, as well as other peripheral neurological conditions. However, it remains a challenge to consistently obtain high-quality, intact, and flat cryostat sections onto glass slides because of the tiny sample size of the DRG tissue. So far, there is no article describing an optimal protocol for DRG cryosectioning. This protocol presents a step-by-step method to resolve the frequently encountered difficulties associated with DRG cryosectioning. The presented article explains how to remove the surrounding liquid from the DRG tissue samples, place the DRG sections on the slide facing the same orientation, and flatten the sections on the glass slide without curving up. Although this protocol has been developed for cryosectioning the DRG samples, it can be applied for the cryosectioning of many other tissues with a small sample size.

GFP labeling of a Bradyrhizobium strain and an attempt to track the crack entry process during symbiosis with peanuts.

Compared to the well-studied model legumes, where symbiosis is established via root hair entry, the peanut is infected by Bradyrhizobium through the crack entry, which is less common and not fully understood. Crack entry is, however, considered a primitive symbiotic infection pathway, which could be potentially utilized for engineering non-legume species with nitrogen fixation ability. We utilized a fluorescence-labeled Bradyrhizobium strain to help in understanding the crack entry process at the cellular level. A modified plasmid pRJPaph-bjGFP, harboring the codon-optimized GFP gene and tetracycline resistance gene, was created and conjugated into Bradyrhizobium strain Lb8, an isolate from peanut nodules, through tri-parental mating. Microscopic observation and peanut inoculation assays confirmed the successful GFP tagging of Lb8, which is capable of generating root nodules. A marking system for peanut root potential infection sites and an optimized sample preparation protocol for cryostat sectioning was developed. The feasibility of using the GFP-tagged Lb8 for observing crack entry was examined. GFP signal was detected at the nodule primordial stage and the following nodule developmental stages with robust GFP signals observed in infected cells in the mature nodules. Spherical bacteroids in the root tissue were visualized at the nodules' inner cortex under higher magnification, reflecting the trace along the rhizobial infection path. The GFP labeled Lb8 can serve as an essential tool for plant-microbe studies between the cultivated peanut and Bradyrhizobium, which could facilitate further study of the crack entry process during the legume-rhizobia symbiosis.