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Background In the horizon of therapeutic restrictions in intracerebral hemorrhage (ICH), recently, non-invasive transcranial electrical stimulation (tES) has achieved considerable prosperities. Translational studies have postulated that transcranial direct current stimulation (tDCS) and the other types of tES remain potentially a novel therapeutic option to reverse or stabilize cognitive and motor impairments. Objective The aim of this study was to comparatively evaluate the effects of the four main paradigms of tES, including tDCS, transcranial alternating (tACS), pulsed (tPCS), and random noise (tRNS) stimulations on collagenase-induced sensorimotor impairments and striatum tissue damage in male rats. Methods To induce ICH, 0.5 μl of collagenase was injected into the right striatum of male Sprague Dawley rats. One day after surgery, tES, was applied to the animals for seven consecutive days. Motor functions were appraised by neurological deficit score, rotarod, and wire hanging tests on the day before surgery and postoperative days 3, 7, and 14. After behavioral tests, brain tissue was prepared appropriately to perform the stereological evaluations. Results The results indicated that the application of the four tES paradigms (tDCS, tACS, tRNS, and tPCS) significantly reversed motor disorders in collagenase-induced ICH groups. Further, the motor function improvement of tACS and tRNS receiving rats in wire-hanging and rotarod tests were higher than the other two tES receiving groups. Structural changes and stereological assessments also confirmed the results of behavioral functions. Conclusion Our findings suggest that in addition to tDCS application in the treatment of ICH, other tES paradigms, especially tACS and tRNS may be considered as add-on therapeutic strategies in stroke.

Background Ferula persica is one of the most important traditional medicinal plants that is used to treat various diseases such as diabetes, backache and rheumatism. The aim of the present study was to evaluate the anti‐inflammatory and wound healing potential of F. persica using stereological and molecular methods in experimental models. Methods In the present study, two wound models (circular excision and linear incision) were used. Male Wistar rats were divided into four groups (n = 16), including control, vehicle treated, treated with F. persica extract ointment (5% w/w) and treated with the reference drug (Madecassol). All the animals were treated topically once a day. The circular and linear wounds were treated for 9 and 17 days, respectively. At the end of the study, samples from wounds area were harvested for histology, stereology, immunohistochemistry and molecular assessments to determine the in vivo healing potential and anti‐inflammatory activity. Results We observed significant recovery in macroscopic evaluation of wound healing in the F. persica extract treated group compared with the control and vehicle treated groups (p 0.05, p, Stereological and molecular studies on the effects of Ferula persica extract on wound healing in rats.

Employing orexin-A immunohistochemistry, we describe the distribution, morphology, and nuclear parcellation of orexinergic neurons within the hypothalami of an Asiatic lion (Panthera leo subsp. persica), an African lion (Panthera leo subsp. melanochaita), and a Southeast African cheetah (Acinonyx jubatus subsp. jubatus). In all three felids, the clustering of large, bipolar, and multipolar hypothalamic orexinergic neurons primarily follows the pattern observed in other mammals. The orexinergic neurons were found, primarily, to form three distinct clusters-the main, zona incerta, and optic tract clusters. In addition, large orexinergic neurons were observed in the ventromedial supraoptic region of the hypothalamus, where they are not typically observed in other species. As has been observed in cetartiodactyls and the African elephant, a cluster of small, multipolar orexinergic neurons, the parvocellular cluster, was observed in the medial zone of the hypothalamus in all three felids, although this parvocellular cluster has not been reported in other carnivores. In both subspecies of lions, but not the cheetah, potential orexin-immunopositive neurons were observed in the paraventricular hypothalamic nucleus, supraoptic nucleus, the lateral part of the retrochiasmatic area, and the inner layer of the median eminence. The distribution and parcellation of orexinergic neurons in the hypothalami of the three felids studied appear to be more complex than observed in many other mammals and for the two subspecies of lion may be even more complex. These findings are discussed in terms of potential technical concerns, phylogenetic variations of this system, and potentially associated functional aspects of the orexinergic system.

Only a fraction of specimens under study are usually selected for quantification in histology. Multilevel sampling or tissue probes, slides and fields of view (FOVs) in the regions of interest (ROIs) are required. In general, all parts of the organs under study should be given the same probability to be taken into account; that is, the sampling should be unbiased on all levels. The objective of our study was to provide an overview of the use of virtual microscopy in the context of developing sampling strategies of FOVs for stereological quantification. We elaborated this idea on 18 examples from multiple fields of histology, including quantification of extracellular matrix and muscle tissue, quantification of organ and tumour microvessels and tumour-infiltrating lymphocytes, assessing osseointegration of bone implants, healing of intestine anastomoses and osteochondral defects, counting brain neurons, counting nuclei in vitro cell cultures and others. We provided practical implications for the most common situations, such as exhaustive sampling of ROIs, sampling ROIs of different sizes, sampling the same ROIs for multiple histological methods, sampling more ROIs with variable intensities or using various objectives, multistage sampling and virtual sampling. Recommendations were provided for pilot studies on systematic uniform random sampling of FOVs as a part of optimizing the efficiency of histological quantification to prevent over- or undersampling. We critically discussed the pros and cons of using virtual sections for sampling FOVs from whole scanned sections. Our review demonstrated that whole slide scans of histological sections facilitate the design of sampling strategies for quantitative histology.

Histochemistry of tumor sections is a widely employed technique utilized to examine cell death in preclinical xenograft animal models of cancer. However, this is under the assumption that tumors are homogeneous, leading to practices such as automatic cell counting across the entire section. We have noted that in our experiments the core of the tumor is largely or partially necrotic, and lacks evidence of vascularization (in contrast to the outer areas of the tumor). We note that this can bias and confound immunohistochemical analyses that do not take care to sample areas of interest in a way to take this into account. Design-based stereology with image analysis techniques is an alternative process that could be used to measure the volume of the necrotic region compared to the volume of the whole tumor.

The development of metanephros is a complex and gradual process. The number, size and distribution of nephrons provide important information about the organization of the kidney. Stereology is the current gold-standard technique for the morphometrical evaluation of kidney structures. This study describes morphometric features of the kidney development in sheep using design-based stereological techniques aimed to introduce the sheep as a translational model in human nephrogenesis. Left kidneys of 16 sheep fetuses in four groups at 9–11, 12–14, 15–17, and 18–20 weeks of gestation were used in the present study. Systematic uniform random sections were obtained. The kidney volume, volume fraction of nephrogenic zone, cortex and medulla, and glomerular volume were estimated using point counting and Cavalieri's estimator. The total glomerular number was estimated using a physical disector/fractionator technique. The kidney and its compartments presented gradual changes with aging, with differences found in the last fetal ages. The kidney volume increased from 0.94 ± 0.22 cm3 to 8.6 ± 0.88 cm3 during development. The volume of cortex increased from 406 ± 85 mm3 to 5,151 ± 309 mm3 and the volume of medulla showed increase from 301 ± 91 mm3 to 3,426 ± 599 mm3. The total glomerular volume increased from 13.8 ± 1.6 mm3 to 235 ± 44 mm3. The total glomerular number increased from 4,683 ± 757 to 639 × 103 ± 11.6 × 103. Our data might contribute to the knowledge of embryological urology and promote future experimental investigations in this field.

Chronic sleep restriction (CSR) is common in modern society, adversely affecting cognitive performance and health. Yet how it impacts neurons regulating sleep remains unclear. Several studies using mice reported substantial losses of wake-active orexin/hypocretin and locus coeruleus (LC) noradrenergic neurons, but not rapid eye movement sleep-active melanin-concentrating hormone (MCH) neurons, following CSR. Here, we used immunohistochemistry and stereology to examine orexin, MCH and LC noradrenergic neurons in a rat model of CSR that uses programmed wheel rotation (3 h on/1 h off; '3/1' protocol). Adult male Wistar rats underwent one or four cycles of the 4-day 3/1 CSR protocol, with 2-day recovery between cycles in home cages. Time-matched control rats were housed in locked wheels/home cages. We found no significant differences in the numbers of orexin, MCH and LC noradrenergic neurons following either one- or four-cycle CSR protocol compared to respective controls. Similarly, the four-cycle CSR protocol had no effect on the densities of orexin axon terminals in the LC, noradrenergic dendrites in the LC and noradrenergic axon terminals in the frontal cortex. Body weights, however, decreased after one cycle of CSR and then increased with diminishing slope over the next three cycles. Thus, we found no evidence for loss of orexin or LC noradrenergic neurons following one and four cycles of the 4-day 3/1 CSR protocol in rats. Differences in CSR protocols and/or possible species differences in neuronal vulnerability to sleep loss may account for the discrepancy between the current results in rats and previous findings in mice.

Background: Aberrant cell cycle re-entry is a well-documented process occurring early in Alzheimer’s disease (AD). This is an early feature of the disease and may contribute to disease pathogenesis. Objective: To assess the effect of forced neuronal cell cycle re-entry in mice expressing humanized Aβ, we crossed our neuronal cell cycle re-entry mouse model with AppNLF knock-in (KI) mice. Methods: Our neuronal cell cycle re-entry (NCCR) mouse model is bitransgenic mice heterozygous for both Camk2a-tTA and TRE-SV40T. The NCCR mice were crossed with AppNLF KI mice to generate NCCR-AppNLF animals. Using this tet-off system, we triggered NCCR in our animals via neuronal expression of SV40T starting at 1 month of age. The animals were examined at the following time points: 9, 12, and 18 months of age. Various neuropathological features in our mice were evaluated by image analysis and stereology on brain sections stained using either immunofluorescence or immunohistochemistry. Results: We show that neuronal cell cycle re-entry in humanized Aβ plaque producing AppNLF KI mice results in the development of additional AD-related pathologies, namely, pathological tau, neuroinflammation, brain leukocyte infiltration, DNA damage response, and neurodegeneration. Conclusion: Our findings show that neuronal cell cycle re-entry enhances AD-related neuropathological features in AppNLF mice and highlight our unique AD mouse model for studying the pathogenic role of aberrant cell cycle re-entry in AD.

Positively charged oligo(poly(ethylene glycol) fumarate) (OPF+) hydrogel scaffolds, implanted into a complete transection spinal cord injury (SCI), facilitate a permissive regenerative environment and provide a platform for controlled observation of repair mechanisms. Axonal regeneration after SCI is critically dependent upon nutrients and oxygen from a newly formed blood supply. Our objective was to investigate fundamental characteristics of revascularization in association with the ingrowth of axons into hydrogel scaffolds, thereby defining spatial relationships between axons and the neovasculature. A novel combination of stereologic estimates and precision image analysis techniques quantitate neurovascular regeneration in rats. Multichannel hydrogel scaffolds containing Matrigel-only (MG), Schwann cells (SCs), or SCs with rapamycin-eluting poly(lactic co-glycolic acid) microspheres (RAPA) were implanted for 6 weeks following complete spinal cord transection. Image analysis of 72 scaffold channels identified a total of 2494 myelinated and 4173 unmyelinated axons at 10 μm circumferential intervals centered around 708 individual blood vessel profiles. Blood vessel number, density, volume, diameter, intervessel distances, total vessel surface and cross-sectional areas, and radial diffusion distances were compared. Axon number and density, blood vessel surface area, and vessel cross-sectional areas in the SC group exceeded that in the MG and RAPA groups. Individual axons were concentrated within a concentric radius of 200–250 μm from blood vessel walls, in Gaussian distributions, which identified a peak axonal number (Mean Peak Amplitude) corresponding to defined distances (Mean Peak Distance) from each vessel, the highest concentrations of axons were relatively excluded from a 25–30 μm zone immediately adjacent to the vessel, and from vessel distances >150 μm. Higher axonal densities correlated with smaller vessel cross-sectional areas. A statistical spatial algorithm was used to generate cumulative distribution F- and G-functions of axonal distribution in the reference channel space. Axons located around blood vessels were definitively organized as clusters and were not randomly distributed. A scoring system stratifies 5 direct measurements and 12 derivative parameters influencing regeneration outcomes. By providing methods to quantify the axonal-vessel relationships, these results may refine spinal cord tissue engineering strategies to optimize the regeneration of complete neurovascular bundles in their relevant spatial relationships after SCI. IMPACT STATEMENT: Vascular disruption and impaired neovascularization contribute critically to the poor regenerative capacity of the spinal cord after injury. In this study, hydrogel scaffolds provide a detailed model system to investigate the regeneration of spinal cord axons as they directly associate with individual blood vessels, using novel methods to define their spatial relationships and the physiologic implications of that organization. These results refine future tissue engineering strategies for spinal cord repair to optimize the re-development of complete neurovascular bundles in their relevant spatial architectures.

Busulfan (BSU) is a chemotherapeutic drug that can cause subfertility or sterility in males. We investigated the effects of adipose tissue-derived mesenchymal stem cells (AT-MSC) conditioned medium (CM) (AT-MSC-CM) on histopathological and molecular characteristics of mouse testes exposed to BSU using stereology. We used adult male mice divided randomly into five groups: control, Dulbecco's modified Eagle's medium (DMEM), dimethyl sulfoxide (DMSO), BSU, and BSU + CM. Thirty-five days following BSU injection, sperm and testis tissues were harvested for stereological and molecular studies. The BSU group exhibited significantly reduced testis volume, interstitium and tubules compared to the other groups, although the volume of the testis remained unchanged for BSU and CM groups. The number of testis cells was reduced in the BSU group compared to the other groups. The CM group exhibited a significantly increased number of testis cells compared to the BSU group. Sperm count and motility, and length density of seminiferous tubules were increased in CM group compared to the BSU group. AT-MSC-CM exhibited ameliorative effects on histopathologic changes of mouse testes exposed to BSU.

Background: Oxaliplatin (OX) has been widely used for treatment of colorectal and other cancers. Adverse effect of OX and other anticancer agents on cognition have been reported, but studies on the effects of chemotherapy on brain structure are scarce. This study describes the morphometrical features of the hippocampus structures in rat following OX treatment using design-based stereological methods. Materials and methods: Ten male Wistar rats were randomized into two groups. The rats from OX group received 2.4 mg/kg OX in vehicle for five consecutive days every week for 2 weeks intraperitoneally (IP). Controls received vehicle only. Cavalieri 's method and the optical fractionator method were used for volume and neuron estimation, respectively. Results: Cavalieri 's method was used for to estimate volume and showed that the volume of the hippocampus was significantly decreased in OX group (31.84 ± 1.24 mm3) compared with the vehicle control group (36.95 ± 3.48 mm3). The optical fractionator method was used to estimate neuron number and showed that the number of neurons in dentate gyrus, cornu ammonis 1 and 3 in OX group (8.147 ± 2.84 × 105, 4.257 ± 0.59 × 105 and 2.133 ± 0.22 × 105, respectively) did not differ from those of vehicle control group (7.36 ± 1.42 × 105, 3.521 ± 0.54 × 105 and 1.989 ± 0.46 × 105, respectively). Conclusions: These findings suggested that OX treatment induce loss of hippocampal volume without neuronal loss which might help to clarify the mechanism by which OX affects cognition and to improve preventive treatment strategies.

Neural system development is one of the most important stages of embryogenesis. Perturbations in this crucial process due to genetic and environmental risk factors cause neural tube defects and other central nervous system diseases. We investigated the effects of prenatal exposure to 900-MHz electromagnetic field (EMF) on the spinal cord. Pregnant rats were exposed to 900-MHz EMF for 1 h/day from E13.5 until birth. Six pups from the control and EMF groups were sacrificed at postnatal day 32, and the upper thoracic region of the spine was removed and processed for histological procedures. For histopathological analyses, hematoxylin&eosin staining and, for stereological analyses and the quantitation of motor neurons, cresyl violet staining was performed. H3K27me3 levels were determined via immunofluorescence staining. Histopathological analysis identified structural alterations of ependymal cells, enlarged central canals, as well as degenerated and shrunken motor neurons in the EMF group, while the control group tissues had normal appearances. We also observed enrichment of H3K27me3 in the ependymal cells and the motor neurons in the spinal cord of the control group rats, while the EMF group had low levels of H3K27me3 staining. Our results suggest that the loss of H3K27me3 signals might correlate with reduced neuronal stem cell potential in the EMF group and result in anatomical and structural differences in the spinal cord. This study provided a comprehensive histopathological analysis of the spinal cord after prenatal EMF exposure and offered an H3K27me3-dependent molecular explanation for the detrimental effects of EMF exposure on the spine.

Canine cutaneous mast cell tumors (ccMCTs) are currently graded according to Patnaik and Kiupel grading schemes. The qualitative and semiquantitative parameters applied in these schemes may lead to inter- and intraobserver variability. This study investigates the prognostic value of volume-weighted mean nuclear volume ([Formula: see text]), a stereological estimation that provides information about nuclear size and its variability. [Formula: see text] of 55 ccMCTs was estimated using the “point-sampled intercept” method and compared with histological grade and clinical outcome. The clinical history of dogs treated with surgical excision alone was available for 30 ccMCTs. Statistical differences in [Formula: see text] were found between grade II ([Formula: see text]= 115 ± 29 µm3) and grade III ccMCTs ([Formula: see text]= 197 ± 63 µm3), as well as between low-grade ([Formula: see text]= 113 ± 28 µm3) and high-grade ccMCTs ([Formula: see text]= 184 ± 63 µm3). An optimal cutoff value of [Formula: see text] ≥ 150 µm3 and [Formula: see text] ≥ 140 µm3 was determined for grade III and high-grade ccMCTs, respectively. In terms of prognosis, [Formula: see text] was not able to predict the clinical outcome in 42% of the cases; however, cases with [Formula: see text]3 had a favorable outcome. These results indicate that, despite having limited prognostic value when used as a solitary parameter, [Formula: see text] is highly reproducible and is associated with histological grade as well as with benign behavior.

The morphometric analysis of lung structure using the principles of stereology has emerged as a powerful tool to describe the structural changes in lung architecture that accompany the development of lung disease that is experimentally modelled in adult mice. These stereological principles are now being applied to the study of the evolution of the lung architecture over the course of prenatal and postnatal lung development in mouse neonates and adolescents. The immature lung is structurally and functionally distinct from the adult lung, and has a smaller volume than does the adult lung. These differences have raised concerns about whether the inflation fixation of neonatal mouse lungs with the airway pressure (Paw) used for the inflation fixation of adult mouse lungs may cause distortion of the neonatal mouse lung structure, leading to the generation of artefacts in subsequent analyses. The objective of this study was to examine the impact of a Paw of 10, 20 and 30 cmH2O on the estimation of lung volumes and stereologically assessed parameters that describe the lung structure in developing mouse lungs. The data presented demonstrate that low Paw (10 cmH2O) leads to heterogeneity in the unfolding of alveolar structures within the lungs, and that high Paw (30 cmH2O) leads to an overestimation of the lung volume, and thus, affects the estimation of volume-dependent parameters, such as total alveoli number and gas-exchange surface area. Thus, these data support the use of a Paw of 20 cmH2O for inflation fixation in morphometric studies on neonatal mouse lungs.

Hypospadias repair involves correcting urethra defects and improving the wound healing process. Zinc has been well accepted as an effective agent in wound healing. This study aimed to investigate the effect of zinc on corpus spongiosum after experimental hypospadias in rats. The animals were divided into three groups. The control group rats underwent general anesthesia, but did not receive any surgeries and treatments. The second and third groups underwent surgeries and respectively received Distilled Water (DW, 2 ml) and zinc sulfate solution (2 ml, containing 4 mg zinc sulfate) by gavages twice a day for 14 days. Stereological methods were used to quantify the corpus spongiosum tissue. The volumes of corpus spongiosum, spongy tissue, urethral lumens, urethral epithelium, and collagen bundles and the number of fibroblasts were respectively amplified by 28%, 40%, 36%, 48%, 40%, and 29% in the surgery + zinc sulfate group in comparison to the surgery + DW group (p 0.02). It can be concluded that consumption of 4 mg/day zinc sulfate for 14 days could improve the healing of hypospadias through increasing the population of fibroblasts, producing collagen bundles, and building a wider lumen and more epithelized urethra.

The purpose of this study was to investigate the potential side-effects of lead acetate (LA), which is toxic to the nerves, blood and muscles, in the rat brain. The neuroprotective effects of pomegranate juice (PJ) against LA exposure were also observed. The experiment involved 28 male Wistar albino rats aged 12 weeks. These were divided into four groups: Control, PJ, LA and LA+PJ. Stereological techniques were employed to determine hippocampal volume in each rat brain. Biochemical investigations and histopathological examinations were also performed. Analysis demonstrated a significant decrease in hippocampal volume in the LA group compared to the control group (p < .05). The stereology results also indicated that PJ has protective effects when compared with the LA and LA+PJ groups. A significant increase was also determined in malondialdehyde (MDA) levels and glutathione S-transferase (GST) activity in the LA group compared to the control group, in contrast to glutathione (GSH) levels and carboxylesterase (CaE) and acetylcholinesterase (AchE) activities. MDA and GST activity decreased significantly in the LA+PJ group compared to the LA group in contrast to GSH levels and CaE and AchE activities. Histopathological examination revealed a number of degenerative changes in the LA group. Exposure to LA adversely affects the hippocampus on the male rat brain. It might also be suggested that PJ may ameliorate these deleterious effects.

© 2022. Turkish Neurosurgery.All Rights ReservedAIM: To evaluate the impact of carnosine on Purkinje neurons in rats exposed to a 900 Mhz electromagnetic field. MATERIAL and METHODS: This study evaluated 24 rats divided into the following three different groups: a control group, a group exposed to the electromagnetic field, and a group that was injected with carnosine while being exposed to the electromagnetic field. The electromagnetic field group was exposed to a 900 Mhz electromagnetic field for an hour daily over 28 days. Thereafter, stereological analysis was performed histologically on cerebellar sections, and the number of Purkinje cells were counted. RESULTS: The electromagnetic field group had remarkably fewer Purkinje cell compared to control. The electromagnetic field group plus 20 mg of carnosine had significantly more total Purkinje cells compared to the electromagnetic field group (p

BACKGROUND: Modulation of serotonergic signaling by treatment with selective serotonin reuptake inhibitors (SSRIs) has been suggested to mitigate amyloid-β (Aβ) pathology in Alzheimer's disease, in addition to exerting an anti-depressant action.OBJECTIVE: To investigate the efficacy of chronic treatment with the SSRI paroxetine, in mitigating Aβ pathology and Aβ plaque-induced microgliosis in the hippocampus of 18-month-old APPswe/PS1 ΔE9 mice.METHODS: Plaque-bearing APPswe/PS1 ΔE9 and wildtype mice were treated with paroxetine per os at a dose of 5 mg/kg/day, from 9 to 18 months of age. The per os treatment was monitored by recording of the body weights and serum paroxetine concentrations, and by assessment of the serotonin transporter occupancy by [3H]DASB-binding in wildtype mice. Additionally, 5,7-dihydroxytryptamine was administered to 9-month-old APPswe/PS1 ΔE9 mice, to examine the effect of serotonin depletion on Aβ pathology. Aβ pathology was evaluated by Aβ plaque load estimation and the Aβ 42/Aβ 40 ratio by ELISA.RESULTS: Paroxetine treatment led to > 80% serotonin transporter occupancy. The treatment increased the body weight of wildtype mice, but not of APPswe/PS1 ΔE9 mice. The treatment had no effect on the Aβ plaque load (p = 0.39), the number and size of plaques, or the Aβ plaque-induced increases in microglial numbers in the dentate gyrus. Three months of serotonin depletion did not significantly impact the Aβ plaque load or Aβ 42/Aβ 40 ratio in APPswe/PS1 ΔE9 mice at 12 months.CONCLUSION: Our results show that chronic treatment with the SSRI paroxetine does not mitigate Aβ pathology and Aβ plaque-induced microgliosis in the hippocampus of APPswe/PS1 ΔE9 mice.

The cognitive dysfunction associated with type 2 diabetes mellitus (T2DM) has been widely studied, and many structures in the hippocampus, such as neurons and synapses, have been shown to play a crucial role in the cognitive decline. However, the mechanism of these changes remains unknown. To further explore this issue, we investigated the changes in the blood supply of the hippocampus in transgenic T2DM mice. In the current study, histochemistry, immunohistochemistry, and unbiased stereological methods were utilized to research the effects of T2DM on hippocampal capillaries of transgenic db/db mice. Twenty (Leprdb ) mut/mut mice and twenty (Leprdb ) wt/wt mice were used in this study. The learning and memory ability was appraised by Morris water maze test.

Mechanical ventilation triggers the manifestation of lung injury and pre-injured lungs are more susceptible. Ventilation-induced abnormalities of alveolar surfactant are involved in injury progression. The effects of mechanical ventilation on the surfactant system might be different in healthy compared to pre-injured lungs. In the present study, we investigated the effects of different positive end-expiratory pressure (PEEP) ventilations on the structure of the blood–gas barrier, the ultrastructure of alveolar epithelial type II (AE2) cells and the intracellular surfactant pool (= lamellar bodies, LB). Rats were randomized into bleomycin-pre-injured or healthy control groups. One day later, rats were either not ventilated, or ventilated with PEEP = 1 or 5 cmH2O and a tidal volume of 10 ml/kg bodyweight for 3 h. Left lungs were subjected to design-based stereology, right lungs to measurements of surfactant proteins (SP−) B and C expression. In pre-injured lungs without ventilation, the expression of SP-C was reduced by bleomycin; while, there were fewer and larger LB compared to healthy lungs. PEEP = 1 cmH2O ventilation of bleomycin-injured lungs was linked with the thickest blood–gas barrier due to increased septal interstitial volumes. In healthy lungs, increasing PEEP levels reduced mean AE2 cell size and volume of LB per AE2 cell; while in pre-injured lungs, volumes of AE2 cells and LB per cell remained stable across PEEPs. Instead, in pre-injured lungs, increasing PEEP levels increased the number and decreased the mean size of LB. In conclusion, mechanical ventilation-induced alterations in LB ultrastructure differ between healthy and pre-injured lungs. PEEP = 1 cmH2O but not PEEP = 5 cmH2O ventilation aggravated septal interstitial abnormalities after bleomycin challenge.

Dexamethasone is a common medicine that is capable of causing malformation in the male reproductive system. The aim of this study was to investigate the effect of melatonin on testis histological changes and Spermatogenesis indexes in adult mice following treatment with dexamethasone. Adult male NMRI mice were divided randomly into four groups: control, dexamethasone (i.p injections, 7 mg/kg/day), dexamethasone + melatonin and melatonin (i.p injections, 20 mg/kg/day). After 7 days of treatment, the right testes were studied stereologically and the left testes were used to measure the daily sperm production (DSP). The serum levels of malondialdehyde (MDA), testosterone and total antioxidant capacity (TAC) were also measured. The left caudal epididymis was used to analyze sperm parameters. Data were analyzed using one way ANOVA and means were considered significantly different at

Stereology is the method of choice for the quantitative assessment of biological objects in microscopy. It takes into account the fact that, in traditional microscopy such as conventional light and transmission electron microscopy, although one has to rely on measurements on nearly two-dimensional sections from fixed and embedded tissue samples, the quantitative data obtained by these measurements should characterize the real three-dimensional properties of the biological objects and not just their “flatland” appearance on the sections. Thus, three-dimensionality is a built-in property of stereological sampling and measurement tools. Stereology is, therefore, perfectly suited to be combined with 3D imaging techniques which cover a wide range of complementary sample sizes and resolutions, e.g. micro-computed tomography, confocal microscopy and volume electron microscopy. Here, we review those stereological principles that are of particular relevance for 3D imaging and provide an overview of applications of 3D imaging-based stereology to the lung in health and disease. The symbiosis of stereology and 3D imaging thus provides the unique opportunity for unbiased and comprehensive quantitative characterization of the three-dimensional architecture of the lung from macro to nano scale.

Obesity due to high calorie intake induces cardiac hypertrophy and dysfunction, thus contributing to cardiovascular morbidity and mortality. Recent studies in aging suggest that oral supplementation with the natural polyamine spermidine has a cardioprotective effect. Here, the hypothesis was tested that spermidine or voluntary activity alone or in combination protect the heart from adverse effects induced by obesity. Therefore, C57Bl/6 mice (n = 8–10 per group) were subjected to control or high fat diet (HFD) and were left untreated, or either received spermidine via drinking water or were voluntarily active or both. After 30 weeks, the mice were killed and the left ventricle of the hearts was processed for light and electron microscopy. Design-based stereology was used to estimate parameters of hypertrophy, fibrosis, and lipid accumulation. HFD induced cardiac hypertrophy as demonstrated by higher volumes of the left ventricle, cardiomyocytes, interstitium, myofibrils and cardiomyocyte mitochondria. These changes were not influenced by spermidine or voluntary activity. HFD also induced myocardial fibrosis and accumulation of lipid droplets within cardiomyocytes. These HFD effects were enhanced in spermidine treated animals but not in voluntarily active mice. This was even the case in voluntarily active mice that received spermidine. In conclusion, the data confirm the induction of left ventricular hypertrophy by high-fat diet and suggest that—under high fat diet—spermidine enhances cardiomyocyte lipid accumulation and interstitial fibrosis which is counteracted by voluntary activity.

Background Accurate evaluation of primordial follicle numbers in mouse ovaries is an essential endpoint for studies investigating how endogenous and exogenous insults, such as maternal aging and chemotherapy, impact the ovarian reserve. In this study, we compared and contrasted two methods for counting healthy primordial follicles following exposure to cyclophosphamide (75 mg/kg), a well-established model of follicle depletion. The first was the fractionator/optical dissector technique, an unbiased, assumption-free stereological approach for quantification of primordial follicle numbers. While accurate, highly reproducible and sensitive, this method relies on specialist microscopy equipment and software, requires specific fixation, embedding and sectioning parameters to be followed, and is largely a manual process that is tedious and time-consuming. The second method was the more widely used serial section and direct count approach, which is relatively quick and easy. We also compared the impacts of different fixatives, embedding material and section thickness on the overall results for each method. Results Direct counts resulted in primordial follicle numbers that were significantly lower than those obtained by stereology, irrespective of fixation and embedding material. When applied to formalin fixed tissue, the direct count method did not detect differences in follicle numbers between saline and cyclophosphamide treated groups to the same degree of sensitivity as the gold standard stereology method (referred to as the Reference standard). However, when Bouin’s fixative was used, direct counts and stereology were comparable in their ability to detect follicle depletion caused by cyclophosphamide. Conclusions This work indicates that the direct count method can produce similar results to stereology when Bouin’s fixative is used instead of formalin. The findings presented here will assist others to select the most appropriate experimental approach for accurate follicle enumeration, depending on whether the primary objective of the study is to determine absolute primordial follicle numbers or relative differences between groups.

The aim of this study is to investigate the effects of metformin treatment at different dosage levels on the ovaries and uteruses of rat offspring in the course of the intrauterine period. Saline, metformin (100 mg/kg/day), and metformin (200 mg/kg/day) were administeredThere were no statistically significant differences among the groups regarding the number of oocytes, the volumes of ovarian cortex, medulla, primary and secondary follicles (The gestational exposure to high dose metformin may result in decreased estradiol production and subsequently decreased endometrial thickness of offspring rats.

The hippocampal formation is important for higher brain functions such as spatial navigation and the consolidation of memory, and it contributes to abilities thought to be uniquely human, yet little is known about how the human hippocampal formation compares to that of our closest living relatives, the chimpanzees. To gain insight into the comparative organization of the hippocampal formation in catarrhine primates, we quantified neurons stereologically in its major subdivisions - the granular layer of the dentate gyrus, CA4, CA2–3, CA1, and the subiculum – in archival brain tissue from six chimpanzees ranging from 29 to 43 years of age. We also sought evidence of Aβ deposition and hyperphosphorylated tau in the hippocampus and adjacent neocortex. A 42-year-old animal had moderate cerebral Aβ-amyloid angiopathy and tauopathy, but Aβ was absent and tauopathy was minimal in the others. Quantitatively, granule cells of the dentate gyrus were most numerous, followed by CA1, subiculum, CA4, and CA2–3. In the context of prior investigations of rhesus monkeys and humans, our findings indicate that, in the hippocampal formation as a whole, the proportions of neurons in CA1 and the subiculum progressively increase, and the proportion of dentate granule cells decreases, from rhesus monkeys to chimpanzees to humans. Because CA1 and the subiculum engender key hippocampal projection pathways to the neocortex, and because the neocortex varies in volume and anatomical organization among these species, these findings suggest that differences in the proportions of neurons in hippocampal subregions of catarrhine primates may be linked to neocortical evolution.

Noninvasive determination of the severity of parenchymal injury in acute kidney injury remains challenging. Edema is an early pathological process following injury, which may correlate with changes in kidney volume. The goal of the present study was to test the hypothesis that “increases in kidney volume measured in vivo using ultrasound correlate with the degree of renal parenchymal injury.” Ischemia-reperfusion (IR) of varying length was used to produce graded tissue injury. We first determined 1) whether regional kidney volume in rats varied with the severity (0, 15, 30, and 45 min) of warm bilateral IR and 2) whether this correlated with tubular injury score. We then determined whether these changes could be measured in vivo using three-dimensional ultrasound. Finally, we evaluated cumulative changes in kidney volume up to 14 days post-IR in rats to determine whether changes in renal volume were predictive of latent tubular injury following recovery of filtration. Experiments concluded that noninvasive ultrasound measurements of change in kidney volume over 2 wk are predictive of tubular injury following IR even in animals in which plasma creatinine was not elevated. We conclude that ultrasound measurements of volume are a sensitive, noninvasive marker of tissue injury in rats and that the use of three-dimensional ultrasound measurements may provide useful information regarding the timing, severity, and recovery from renal tissue injury in experimental studies.

Obesity is associated with lung function impairment and respiratory diseases; however, the underlying pathophysiological mechanisms are still elusive, and therapeutic options are limited. This study examined the effects of prolonged excess fat intake on lung mechanics and microstructure and tested spermidine supplementation and physical activity as intervention strategies. C57BL/6N mice fed control diet (10% fat) or high-fat diet (HFD; 60% fat) were left untreated or were supplemented with 3 mM spermidine, had access to running wheels for voluntary activity, or a combination of both. After 30 wk, lung mechanics was assessed, and left lungs were analyzed by design-based stereology. HFD exerted minor effects on lung mechanics and resulted in higher body weight and elevated lung, air, and septal volumes. The number of alveoli was higher in HFD-fed animals. This was accompanied by an increase in epithelial, but not endothelial, surface area. Moreover, air-blood barrier and endothelium were significantly thicker. Neither treatment affected HFD-related body weights. Spermidine lowered lung volumes as well as endothelial and air-blood barrier thicknesses toward control levels and substantially increased the endothelial surface area under HFD. Activity resulted in decreased volumes of lung, septa, and septal compartments but did not affect vascular changes in HFD-fed mice. The combination treatment showed no additive effect. In conclusion, excess fat consumption induced alveolar capillary remodeling indicative of impaired perfusion and gas diffusion. Spermidine alleviated obesity-related endothelial alterations, indicating a beneficial effect, whereas physical activity reduced lung volumes apparently by other, possibly systemic effects.

Doxorubicin (Dox) is an anthracycline antibiotic with antineoplastic activity. Acetylsalicylic acid (Asa) is recommended for use as a prophylactic for thromboembolism during treatment of cancers. We investigated liver toxicity due to combined use of Dox and Asa in chemotherapy regimens. We used 140 Swiss albino mice divided into four main groups: control, Dox, Asa, and Dox + Asa. Each group was subdivided into seven subgroups based on time of sacrifice, i.e., 6, 12, 24, 48 h and 7, 14, 21 days. Quantitative and histopathological changes in liver were assessed by light microscopy and stereology. The portal triad area of the Dox and Dox + Asa groups was increased significantly compared to controls at 6 h, whereas in the Asa group, the means were similar to controls. Assessment of histopathology indicated an increased time-dependent degeneration and necrosis of liver tissues in mice in the Dox and Dox + Asa groups. The protective effects of Asa were not evident in Dox + Asa group. When Dox and Asa were administered together, degenerative changes were greater than for in the group that was given Dox alone. We found that Asa and Dox combined therapy increased tissue damage.

The present study examines cortical neuronal morphology in the African lion (Panthera leo leo), African leopard (Panthera pardus pardus), and cheetah (Acinonyx jubatus jubatus). Tissue samples were removed from prefrontal, primary motor, and primary visual cortices and investigated with a Golgi stain and computer-assisted morphometry to provide somatodendritic measures of 652 neurons. Although neurons in the African lion were insufficiently impregnated for accurate quantitative dendritic measurements, descriptions of neuronal morphologies were still possible. Qualitatively, the range of spiny and aspiny neurons across the three species was similar to those observed in other felids, with typical pyramidal neurons being the most prominent neuronal type. Quantitatively, somatodendritic measures of typical pyramidal neurons in the cheetah were generally larger than in the African leopard, despite similar brain sizes. A MARsplines analysis of dendritic measures correctly differentiated 87.4% of complete typical pyramidal neurons between the African leopard and cheetah. In addition, unbiased stereology was used to compare the soma size of typical pyramidal neurons (n = 2,238) across all three cortical regions and gigantopyramidal neurons (n = 1,189) in primary motor and primary visual cortices. Both morphological and stereological analyses indicated that primary motor gigantopyramidal neurons were exceptionally large across all three felids compared to other carnivores, possibly due to specializations related to the felid musculoskeletal systems. The large size of these neurons in the cheetah which, unlike lions and leopards, does not belong to the Panthera genus, suggests that exceptionally enlarged primary motor gigantopyramidal neurons evolved independently in these felid species.

Fetal alcohol spectrum disorders (FASD) constitute a prevalent, yet preventable, developmental disorder worldwide. While a wealth of research demonstrates that altered function of hippocampus and prefrontal cortex may underlie behavioral impairments in FASD, only one published paper to date has examined the impact of developmental alcohol exposure on the region responsible for coordinated prefrontal-hippocampal activity: thalamic nucleus reuniens (Re). In the current study, we used a rodent model of human third trimester alcohol exposure to examine both the acute and lasting impact of a single-day alcohol exposure on Re. We administered 5.25 g/kg of ethanol to male and female Long Evans rat pups on postnatal day (PD) 7. We used unbiased stereological estimation to evaluate cell death or cell loss at three time points: 12 hours after alcohol administration; 4 days after alcohol administration (i.e., PD11); in adulthood (i.e.,postnatal day 72). Alcohol exposure on PD7 increased apoptotic cell death in Re on PD7, and caused short-term cell loss on PD11. This relationship between short-term cell death versus cell number suggests that alcohol-related cell loss is driven by induction of apoptosis. In adulthood, alcohol-exposed animals displayed permanent cell loss (mediating volume loss in the Re), which included a reduction in neuron number (relative to procedural controls). Both procedural controls and alcohol exposed animals displayed a deficit in non-neuronal cell number relative to typically-developing controls, suggesting that Re cell populations may be vulnerable to early life stress as well as alcohol exposure in an insult- and cell type-dependent manner.

The integrity of the structural cerebral cortex is disrupted after stroke either at the macroscopic or microscopic levels. Therefore, many attempts have been gathered to circumvent stroke-associated problems in the brain tissue. The current study was aimed to design an animal model of photochemical stroke using rose bengal (RB) plus laser irradiation (L) after 10, 15, and 20 min (´) and evaluate its effect on the cerebral tissue using unbiased stereological quantitative methods and morphometric histological analysis. Photochemical stroke was induced by intraperitoneal injection of RB dye and further activation through the exposure of the right sensorimotor cortex with the green laser radiation (100 mW; 532 nm). Mice were randomly allocated into 4 groups (each in 15) as follows: control (10 μg/gbw RB), RB + 10’L, RB + 15’L, and RB + 20’L. Target irradiation site was adjusted to 2 mm lateral to the bregma. Vernier caliper morphometric evaluation, cresyl violet staining, and unbiased stereological assays including Cavalier’s principle and point counting techniques were used to monitor the pathological changes and lesion volume on days 1, 3, and 7 after the ischemia induction. Our data showed that the mean diameter of the lesion site and lesion infarct volume in the group RB + 20’L) was significantly increased relative to the other groups (P

Painful diabetic neuropathy may associate with nerve morphological plasticity in both peripheral and central nervous system. The aim of this study was to determine numerical changes of myelinated fibers in the spinothalamic tract region and oligodendrocytes in the spinal dorsal horn of rats with painful diabetic neuropathy and the effects of metformin on the above changes. Male Sprague–Dawley rats were randomly allocated into the control group (n = 7), the painful diabetic neuropathy group (n = 6) and the painful diabetic neuropathy treated with metformin group (the PDN + M group, n = 7), respectively. Twenty-eight days after medication, numbers of myelinated fibers in the spinothalamic tract and oligodendrocytes in the spinal dorsal horn were estimated by the optical disector (a stereological technique). Compared to the control group, number of myelinated fibers in the spinothalamic tract increased significantly in the painful diabetic neuropathy and PDN + M group, compared to the painful diabetic neuropathy group, number of myelinated fibers decreased in the PDN + M group (P < 0.05). As the oligodendrocyte in the spinal dorsal horn was considered, its number increased significantly in the painful diabetic neuropathy group compared to the control and the PDN + M group (P < 0.05), there was no significant difference between the control and the PDN + M group (P > 0.05). Our results indicate that painful diabetic neuropathy is associated with a serial of morphometric plasticity in the rat spinal cord including the numerical increase of the myelinated fibers in the spinothalamic tract and the oligodendrocytes in the spinal dorsal horn. The analgesic effect of metformin against painful diabetic neuropathy might be related to its adverse effects on the above morphometric plasticity.

The glycoprotein reelin has been implicated in both memory-related synaptic plasticity and Alzheimer's disease pathogenesis. Aged rats with memory impairment display decreased reelin expression in layer II of the entorhinal cortex (EC) relative to memory-intact subjects, and here we tested whether this effect extends to the primate brain. Seven young adult (8–10 years) and 14 aged (27–38 years) rhesus monkeys (Macaca mulatta) were examined, including 7 old animals classified as impaired based on their scores from a delayed nonmatching-to-sample recognition memory test. Histological sections spanning the rostrocaudal extent of the intermediate and caudal divisions of EC were processed by immunohistochemistry and the total number of reelin-positive neurons in layer II was estimated using design-based stereological techniques. The main finding was that the number of reelin-expressing neurons in EC layer II is decreased selectively in aged monkeys with memory deficits relative to young adult and aged subjects with intact memory. The results add to evidence implicating EC-hippocampal integrity in neurocognitive aging, and they suggest that disrupted reelin signaling may be among the mechanisms that mediate the associated vulnerability of this circuitry in Alzheimer's disease.