INFLUENCE OF DIFFERENT LIGHTING REGIMEN ON THE MORPHOMETRIC CHARACTERISTICS OF NEURONS IN THE PARAVENTRICULAR NUCLEI OF THE RAT HYPOTHALAMUS

Paraventricular nuclei (PVN) of the hypothalamus are the autonomic center forcoordination of functions and consists of a number of neuronal populations - sub-nuclei,which differ in structural and functional features and the nature of nerve connectionswith various parts of the nervous and neuroendocrine systems. When studying stressreactions and actions of the stress-limiting factors, it is important to study subpopulationsof PVN neurons of the hypothalamus, synthesizing stress-releasing hormones, whichinitiate stress reactions of the body. One of the main factors that show a pronouncedeffect in the regulation of ACTH secretion is corticotropin-releasing factor (CRF). TheCRF-immunoreactive tracer was found mainly in the medial small-cell subnucleus of theparaventricular nuclei of the rat hypothalamus. Despite deep and comprehensive studiesof the hypothalamus, until now there are no unifed ideas about its individual reactivityand the degree of involvement of these structures into the stress reaction caused by theprolonged change in the lighting regime.Purpose - to fnd out the effect of different illumination regimes on the morphometriccharacteristics of the medial small-cell subnuclei of the paraventricular nuclei of thehypothalamus of mature and old rats.Material and methods. The experiments were conducted on 72 nonlinear white malerats, divided into 6 series of studies, in each of which the biomaterial sampling wascarried out at 14.00 and 02.00 h using morphometric and statistical research methods.Results. Morphometric assessment of the medial small-cell sub-nuclei of theparaventricular nuclei (msPVN) of the hypothalamus of rats revealed the diurnaldynamics of indices. It has been established that in both old and mature rats, theiraverage volume (p <0.05) signifcantly decreases at 2.00 compared to 14.00 undernormal lighting. The nuclear-cytoplasmatic ratio at 02.00 increases in comparison with14.00 respectively.When the animals were kept under constant illumination, more pronounced changesin the structures under study were established. In particular, the volume of neurons inthe msPVN of the hypothalamus decreases both mature and old rats compared withrats under standard illumination and even more in comparison with rats under lightdeprivation. In accordance with this, the NCR also changes. The average number ofneurons on the standard plane of the histological section also decreases in comparisonwith other illumination regimes. It is also necessary to point out that under conditionsof light stimulation in mature rats in msPVN, the average volume of neurons (p <0.05)noticeably decreases at 02:00 in comparison with 14:00, when the volume of theirnuclei does not change on average during the indicated study periods. This is reflected,respectively, in the NCR change, which increases at 02.00 in comparison with 14.00.As to the old rats, the average neuron volume does not decrease at 2.00 in comparisonwith 14.00 in msPVN neurons at light stimulation, as well as the volume of their neucleichanges, on average, is barely noticeable ,and according to it, NCR at 02.00 remainsstable in comparison with 14.00, and the average quantity of neurocytes in msPVN doesnot change on a standard plane of the histological section during various periods ofthe twenty four hours. At the same time, with various modifcations of the light regime,cytometric parameters of neurons under study in old rats, on average, are signifcantlylower (p <0.001) than those in mature rats.Conclusions. 1. The duration of the photoperiod signifcantly affects the 24 hoursactivity of msPVN in the hypothalamus of mature and old rats. In particular, in bothinvestigated groups of rats, the average volume of neurons (p <0.05) reliably decreasesat 2.00 under normal illumination in comparison with 14.00, and, accordingly, the NCR at 02.00 increases compared to 14.00. All cytometric parameters of their neurons in oldrats are, on average, signifcantly lower (p <0.001) than in mature rats.2. Constant illumination causes more pronounced changes in the morphometricparameters of the msPVN of the hypothalamus than light deprivation. Thus, in maturerats in the msPVN, the average volume of neurons (p <0.05) decreases noticeably at02:00 in comparison with 14:00, which is reflected, respectively, in the change in NCR,which increases at 02:00 compared to 14:00. For old rats, under light stimulation, allthe studied cytometric parameters of their neurons, on average, are signifcantly lower(p <0.001) than those in mature rats.

Cyperus rotundus root extract inhibits progress of lymphedema in mouse tail model

Lymphedema is clinically manifested as swelling due to abnormal accumulation of interstitial fluid attributed to inefficient fluid uptake and reduced lymphatic flow. Here we have evaluated the effect of Cyperus rotundus root (CRR) ethanolic extract in a mouse tail model of lymphedema and hypothesized that the Cyperus rotundus plant is known for its anti-inflammatory effect through inhibition of TNF alpha will be effective in managing this condition. The skin was removed was done after leaving 1cm of distance from the base of the trunk. The cut was introduced in a sterile condition. The animals were divided into Experimental control(EC) and Cyperus rotundus (CRR) treated groups. A change in tail volume around the wound was monitored for up to 20 days. The kinetics of the swelling was calculated for statistical significance. Further TS of the upper part of the wound was stained with H&E stain and documented for histological changes.RESULTS: In the EC group, a gradual rise in swelling was recorded, which peaked on the 10th day and continued up to the 15th day, followed by a gradual decrease. In CR extract-treated group, the swelling was significantly low and the peak was obtained on the 8th day, while in the EC group the swelling was significantly higher and the peak was obtained 11th day. The histological section show, fibrous band intersection the lobules of adipocyte and lymphatic vascular channel and muscles in the sub epithelium region in the EC group, which was very less in CRR group.

scoMorphoFISH: A Deep-Learning enabled toolbox for single-cell single-mRNA quantification and correlative (ultra-)morphometry

Increasing the information depth of single kidney biopsies can improve diagnostic precision, personalized medicine and accelerate basic kidney research. Until now, information on mRNA abundance and morphologic analysis has been obtained from different samples, missing out on the spatial context and single-cell correlation of findings. Herein, we present scoMorphoFISH, a modular toolbox to get spatial single-cell single-mRNA expression data optimized for routinely generated kidney biopsies. Deep-Learning was used to virtually dissect tissue sections in tissue compartments and cell types to which single-cell expression data was assigned. Furthermore, we show correlative and spatial single-cell expression quantification with super-resolved podocyte foot process morphometry on the same histological section. In contrast to bulk analysis methods, this approach will help to identify local transcription changes even in less frequent kidney cell types on a spatial single-cell level with single-mRNA resolution. As this method performs well with standard formalin-fixed paraffin-embedded samples and we provide pretrained DL-networks embedded in a comprehensive image analysis workflow, this method can be applied immediately in a variety of settings.

Histological criteria of maturity of the uterine-placental area of manure

In order to improve the procedure of morphological diagnosis of the degree of maturity of the uterine-placental area of the placenta, the authors conducted special studies of the structures of the placental bed of the uterus at different stages of gestation during physiological pregnancy and under various pathologies of the mother or placenta. Criteria for maturity of the uterine-placental area of manure in gestational segments 13-27, 28-36 and 37-40 weeks based on the percentage of spiral arteries with complete gestational remodeling of the walls due to the destructive effect of invasive trophoblast and the first parameters of the norm for the number of veins per unit area in the uterine-placental area. To morphologically determine the degree of maturity of the uteroplacental area, a table of the percentage of spiral arteries with complete gestational adjustment and the number of venous vessels per 1 mm2 area of histological section according to gestational age. For morphological diagnosis of the degree of maturity of the placenta at different stages of gestation during physiological pregnancy and under conditions of different pathology of the mother or placenta, it is recommended to conduct a comprehensive morphological assessment of placental structures using maturity criteria of placental chorionic tree and maturity criteria of placental placenta.

Repair of Critical Size Bone Defects Using Synthetic Hydroxyapatite or Xenograft with or without the Bone Marrow Mononuclear Fraction: A Histomorphometric and Immunohistochemical Study in Rat Calvaria

Bone defects are a challenging clinical situation, and the development of hydroxyapatite-based biomaterials is a prolific research field that, in addition, can be joined by stem cells and growth factors in order to deal with the problem. This study compares the use of synthetic hydroxyapatite and xenograft, used pure or enriched with bone marrow mononuclear fraction for the regeneration of critical size bone defects in rat calvaria through histomorphometric (Masson’s staining) and immunohistochemical (anti-VEGF, anti-osteopontin) analysis. Forty young adult male rats were divided into five groups (n = 8). Animals were submitted to critical size bone defects (Ø = 8 mm) in the temporoparietal region. In the control group, there was no biomaterial placement in the critical bone defects; in group 1, it was filled with synthetic hydroxyapatite; in group 2, it was filled with xenograft; in group 3, it was filled with synthetic hydroxyapatite, enriched with bone marrow mononuclear fraction (BMMF), and in group 4 it was filled with xenograft, enriched with BMMF. After eight weeks, all groups were euthanized, and histological section images were captured and analyzed. Data analysis showed that in groups 1, 2, 3 and 4 (received biomaterials and biomaterials plus BMMF), a significant enhancement in new bone matrix formation was observed in relation to the control group. However, BMMF-enriched groups did not differ from hydroxyapatite-based biomaterials-only groups. Therefore, in this experimental model, BMMF did not enhance hydroxyapatite-based biomaterials’ potential to induce bone matrix and related mediators.

Novel approach for quantification of multiple immunofluorescent signals using histograms and 2D plot profiling of whole-section panoramic images

We describe a novel approach for quantification and colocalization of immunofluorescence (IF) signals of multiple markers on high-resolution panoramic images of serial histological sections utilizing standard staining techniques and readily available software for image processing and analysis. Human gingiva samples stained with primary antibodies against the common leukocyte antigen CD45 and factors related to heparan sulfate glycosaminoglycans (HS GAG) were used. Expression domains and spatial gradients of IF signals were quantified by histograms and 2D plot profiles, respectively. The importance of histomorphometric profiling of tissue samples and IF signal thresholding is elaborated. This approach to quantification of IF staining utilizes pixel (px) counts and comparison of px grey value (GV) or luminance. No cell counting is applied either to determine the cellular content of a given histological section nor the number of cells positive to the primary antibody of interest. There is no selection of multiple Regions-Of-Interest (ROIs) since the entire histological section is quantified. Although the standard IF staining protocol is applied, the data output enables colocalization of multiple markers (up to 30) from a given histological sample. This can serve as an alternative for colocalization of IF staining of multiple primary antibodies based on repeating cycles of staining of the same histological section since those techniques require non standard staining protocols and sophisticated equipment that can be out of reach for small laboratories in academic settings. Combined with the data from ontological bases, this approach to quantification of IF enables creation of in silico virtual disease models.

Correlation between two-dimensional micro-CT and histomorphometry for assessment of the implant osseointegration in rabbit tibia model

Background Histology is considered as a gold standard for analyzing bone architecture. However, histomorphometry is a destructive method and only offers the bone information of a limited location. Micro-computed tomography (μCT) is a non-destructive technology and provides a slice at any site. The aim of this study was to compare the correlation of the Bone-to-Implant Contact ratio (BIC) between 2D micro-CT (μCT) and histomorphometry and to investigate a method for assessing the osseointegration of the implant by 2D μCT. Methods A total of 18 implants were divided into three groups (6 implants per group), and inserted into the rabbit tibia defects as follow: implant only (Implant group), implant with β-TCP/hydrogel (TCP group), implant with rhBMP-2 loaded β-TCP/hydrogel composite (BMP-2 group). After 4 weeks of implantation, the specimens were collected to take the micro-CT scan with an aluminum filter and performed H&E staining on the undecalcified sections. The 2D μCT slices were chosen at an angle of 0°, 45°, 90° and 135° with the representative histological section to measure BIC. And the correlations between BICs of 2D μCT and BICs of histology were evaluated. Results In each group, BICs at the same sites measured by histomorphometry and corresponding 2D μCT presented the same trend and shown no significant difference between the two methods (P > 0.05). BICs of histological sections and BICs of corresponding 2D μCT slices presented a strong correlation in the implant group (γ = 0.74, P = 0.09), a moderate correlation in the TCP group (γ = 0.46, P = 0.35), a weak correlation in the BMP-2 group (γ = 0.30, P = 0.56). In the implant group, the relationship between BIC-Mean-μCTs and BICs-Histology has presented a significant linear correlation (γ = 0.84, P = 0.04). Conclusions Integrating bone information of several 2D μCT slices in different sites to measure BIC is a feasible method for assessing the implant osseointegration.

Hepatoprotective Effect of Supercritical Carbon Dioxide Extracted Dabai Pulp Oil and Its Defatted Pulp

All food scientists must utilize plants for their application as functional foods to reduce hypercholesterolemia incidence through diet. Canarium odontophyllum (dabai) is a novel source for new healthy oil and functional foods. In this work, we evaluate the hepatoprotective effects of supercritical carbon dioxide (SC-CO2) extracted dabai pulp oil (DPO) and defatted dabai pulp (DDP) against hypercholesterolemia elicited by a high-cholesterol diet in rats. Our results show that DPO and DDP supplementation exerted beneficial hypocholesterolemic effects against the high-cholesterol diet-fed rat. Nevertheless, supplementation with DDP revealed superior total cholesterol, low-density lipoprotein, and HMG-CoA reductase lowering efficacy (p < 0.05). Supplementation of either DPO or DDP did not significantly affect AST and ALT levels than normal rats (p > 0.05). Therefore, DDP and DPO are considered as having no toxicological significance. The histological section of rats treated with DPO and DDP showed improved steatosis in hepatocytes. HPLC analysis revealed that DPO and DDP contained syringic acid, which plays an important role in the beneficial effect. In conclusion, our results support the hypocholesterolemic and hepatoprotective effects of DPO and DDP in the hypercholesterolemic rats model.

Growth performance, organ weight and histology of rabbits fed graded levels of bitter kola (Gracinia kola)

The effect of varying levels (0%, 2.5%, 5% and 7.5%) of Garcinia kola on the carcass, organ weight and growth performance of growing rabbits were investigated using twenty four rabbits of mixed breed (New Zealand white and Chinchilla) and mixed sexes of average weight1+ 0.49kg. The rabbits were randomly assigned to four dietary treatments comprising Garcinia kola: T1 (0%), T2 (2.5%), T3 (5%) and T4 (7.5%) for 56 day period of the experiment. The experiment was based on Completely Randomized Design with each treatment having six rabbits in three replicates of two rabbits per replicate. During the 56 days period of the experiment, the rabbits were subjected to similar managerial and sanitation conditions such that the only source of variation is the level of Garcinia kola inclusion. At the end of the 56 days treatments, three rabbits were randomly selected from each treatment, weighed, slaughtered, skinned and eviscerated. The carcass weight and organ weight were also taken. Thereafter, the internal organs precisely the liver and kidney were prepared for histological analysis and taken to Nnamdi Azikiwe University, Awka Teaching hospital histological section for analysis. Results showed no significant differences (P > 0.05) in most of the parameters measured except for the carcass weight and kidney weight but the differences did not take any particular trend that could be attributed to the effect of the bitter kola (G. kola). There was also no significant difference (P<0.05) for feed intake level, feed conversion ratio and specific growth rate. Weight gain however differed significantly (P<0.05) withtreatments (T4 < T3 < T2 < T1) meaning that body weight gain decreased as the level of bitter kola inclusion increased. The kidneys of rabbits fed bitter kola all had different degrees of damage. Similarly, the histology of the liver organs of rabbits fed different levels of bitter kola showed that rabbits on T1 (0%) bitter kola was normal, while the liver of those that had the test ingredient bitter kola all had defects. We therefore recommend large doses of G. kola should be given to rabbits else the negative effects will outweigh the gains of its phytobiotic properties.

Reproduction and reproductive tract morphology of male and female pigs whose mothers were heat stressed during the second month of gestation

The primary objective was to assess the development of fetal gonads and measure the subsequent reproductive capacity of boars and gilts whose mother was either subjected to gestational heat stress (GHS) or thermoneutral (GTN; control) conditions during pregnancy. Gilts were subjected to either GHS (28 to 38 °C; 65% to 88% relative humidity [RH]; n = 30) or GTN (17 to 22 °C; 56% to 65% RH; n = 29) for the second month of gestation (a period that coincides with a critical window of gonadal development). A subset of GHS (n = 12) and GTN (n = 11) gilts was sacrificed immediately following treatment for the collection of pregnancy data. The remaining gilts (n = 18 GHS and n = 18 GTN) were allowed to farrow. Female offspring from the farrowed gilts were studied through puberty, first insemination, and early pregnancy when fetal tissues were again collected. During the treatment period, GHS gilts had greater (P &lt; 0.001) rectal temperature and respiration rate at both measurement time points (morning and afternoon) compared with GTN gilts. When assessed at the end of the second month of gestation, the total number of viable fetuses did not differ (P &gt; 0.10) for GHS vs. GTN. Likewise, the weight of the fetus, placenta, fetal testes, and fetal ovaries were similar (P &gt; 0.10) for GHS and GTN pregnancies. There was a tendency for an effect of treatment (63.3 ± 2.3 vs. 70.1 ± 2.6; GHS vs. GTN; P &lt; 0.073) on the number of oogonia per histological section in the fetal ovaries. There was no effect of treatment on the number of prespermatogonia per histological section in the fetal testis. For gilts farrowing after treatment, litter size, piglet birth weight, and weaning weight were similar (P &gt; 0.10) for the GHS and GTN gilts. Testes collected from castrated GHS boars had fewer prespermatogonia per seminiferous tubule cross section (P &lt; 0.049). Female offspring from the GHS (n = 30) or GTN (n = 37) sows reached puberty at a similar age, and their pregnancies (ninth week of gestation) had fewer corpora lutea (15.6 ± 0.5 vs. 17.1 ± 0.4; GHS vs. GTN; P &lt; 0.038) but the number of fetuses was similar for GHS and GTN. In summary, compared with GTN, GHS during a critical window of gonadal development tended to reduce the number of oogonia in the fetal ovary, reduced the number of prespermatogonia in the neonatal testes, and reduced ovulation rate at first pregnancy in gilts.