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The efficiency of sampling in stereology and its prediction
Abstract
The superior efficiency of systematic sampling at all levels in stereological studies is emphasized and various commonly used ways of implementing it are briefly described. Summarizing recent theoretical and experimental studies a set of very simple estimators of efficiency are presented and illustrated with a variety of biological examples. In particular, a nomogram for predicting the necessary number of points when performing point counting is provided. The very efficient and simple unbiased estimator of the volume of an arbitrary object based on Cavalieri's principle is dealt with in some detail. The efficiency of the systematic fractionating of an object is also illustrated.
... The coefficient of error is 0.05 and below this value is an important parameter for the reliability of the research [24]. The pilot study determined the number of the required sections and sampling technique to obtain an approximately 0.05 or a lower coefficient of error (CE) in the stereological studies. ...
... The values were recorded in the Microsoft Office Excel program. Following, volumetric measurements were performed by Cavalieri's Principle(Figure 1)[24]. ...
... present only once in each cell, only cells containing this specific point will be counted (Gundersen & Jensen, 1987). ...
... Q -: points counted. Ideally variance due to systematic random sampling should be low as s 2 , if this value is too high, more sections should be added to sampling, the lowest number of sections for this number to be statistically relevant is 3 sections (Gundersen & Jensen, 1987). ...
Different types of interneurons in the striatum are still relatively poorly understood, especially compared to other parts of the brain such as the cortex or hippocampus. One of the lesser-known types of striatal interneurons that has received increasing attention recently, are neuropeptide Y-expressing interneurons (NPY+ interneurons). Available work indicates that these interneurons are controlled by striatal cholinergic interneurons and thus they may serve as mediators of cholinergic modulation in the striatum. The first aim of this thesis is to determine the absolute number of NPY+ interneurons in the mouse dorsal striatum by stereology, using a transgenic mouse model that shows GFP expression in NPY+ neurons. The second aim of this thesis is to use stereology to determine the volume of the dorsal striatum that we are able to target with viral expression when using a standard stereotactic injection protocol commonly used in our laboratory. The third aim of this study is to test the behavioural effect of knock down of the beta2 subunit of the nicotinic acetylcholine receptor expressed by NPY+ interneurons in the dorsal striatum. The work should serve as a first step in a study that will ultimately allow us to develop a quantitative idea of the absolute and relative number of NPY+ interneurons in the striatum that must be affected in order to expect a significant effect on striatal neural circuits and, consequently, on striatum-driven behaviour.
... The selected areas were similar for all animals. Then, the outlined region was overlaid with a random series of counting frames [96,97]. The TH-ir cell nuclei counts were performed under high magnification (40X). ...
... TH-ir cell nuclei were counted if they were found entirely within a counting frame or transected by at least one of the inclusion lines of a counting frame but not any of the exclusion lines of the same counting frame [98]. The area TH-ir dopaminergic neuron density (N A ) in the ROI was calculated using the following formula: N V = N/ A ROI where N is the total number of TH-ir neurons and A ROI is the total area analyzed (number of counting frame x area of counting frame) [97,99]. ...
Background:
Attention deficit hyperactivity disorder (ADHD) is one of the most prevalent neuropsychiatric disorders with morphological brain abnormalities. There is a growing body of evidence that abnormalities in the dopaminergic system may account for ADHD pathogenesis. However, it is not clear whether the dopaminergic system is hyper or hypoactive. To determine whether the DA neurons and/or axons deficiency might be the cause of the postulated dopaminergic hypofunction in spontaneously hypertensive rats (SHR, animal model of ADHD), this study examined the dopaminergic neurons and fibers in the brain tissues of SHRs and Wistar Kyoto rats (WKY, control animals). Here, we performed immunohistochemical tyrosine hydroxylase (TH) and dopamine-beta-hydroxylase (DBH) staining on brain sections collected on juveniles from SHR and WKY. Moreover, behavioral testing to examine the hyperactivity in the open field area was also elucidated.
Results:
The mesocortical dopaminergic system appears to be normal in juvenile SHR, as suggested by (i) no alteration in the area density of TH-immunoreactive (TH-ir) dopaminergic neurons in the ventral tegmental area (VTA), (ii) no alterations in the volume density of TH-ir fibers in layer I of the prelimbic (PrL) subregion of medial PFC (mPFC), (iii) no alteration in the percentage of TH-ir dopaminergic fibers in layer I of the PrL subregion of mPFC as revealed by TH and/or DBH immunoreactivity. Furthermore, the SHR showed increased locomotor activity than WKY in the open field test.
Conclusions:
The demonstration of no alteration in mesocortical dopaminergic neurons and fiber in SHR raises some concern about the position of SHR as an animal model of the inattentive subtype of ADHD. However, these results strengthen this strain as an animal model of hyperactive/impulsive subtype ADHD for future studies that may elucidate the underlying mechanism mediating hyperactivity and test various treatment strategies.
... Using the diameter (r) value obtained from the mean neuron area data, the average volume was estimated using the 4/3 µr 3 formula. Coefficient of error (CE) and coefficient variation (CV) values for volume and number estimations were calculated according to the formula reported by Gundersen and Jensen (1987). ...
... Very few studies have investigated the total DRG volume after injury, and analyses have mostly been performed using the Cavalieri method (Gundersen and Jensen 1987). Hart et al. reported a 19% volume loss compared to a control group after Fig. 11 a Images from the crush injury group. ...
Peripheral nerve injuries lead to significant changes in the dorsal root ganglia, where the cell bodies of the damaged axons are located. The sensory neurons and the surrounding satellite cells rearrange the composition of the intracellular organelles to enhance their plasticity for adaptation to changing conditions and response to injury. Meanwhile, satellite cells acquire phagocytic properties and work with macrophages to eliminate degenerated neurons. These structural and functional changes are not identical in all injury types. Understanding the cellular response, which varies according to the type of injury involved, is essential in determining the optimal method of treatment. In this research, we investigated the numerical and morphological changes in primary sensory neurons and satellite cells in the dorsal root ganglion 30 days following chronic compression, crush, and transection injuries using stereology, high-resolution light microscopy, immunohistochemistry, and behavioral analysis techniques. Electron microscopic methods were employed to evaluate fine structural alterations in cells. Stereological evaluations revealed no statistically significant difference in terms of mean sensory neuron numbers (p > 0.05), although a significant decrease was observed in sensory neuron volumes in the transection and crush injury groups (p < 0.05). Active caspase-3 immunopositivity increased in the injury groups compared to the sham group (p < 0.05). While crush injury led to desensitization, chronic compression injury caused thermal hyperalgesia. Macrophage infiltrations were observed in all injury types. Electron microscopic results revealed that the chromatolysis response was triggered in the sensory neuron bodies from the transection injury group. An increase in organelle density was observed in the perikaryon of sensory neurons after crush-type injury. This indicates the presence of a more active regeneration process in crush-type injury than in other types. The effect of chronic compression injury is more devastating than that of crush-type injury, and the edema caused by compression significantly inhibits the regeneration process.
... Stereological evaluation of number of NEUN-immunoreactive neurons An optical fractionator unbiased sampling design was used to estimate the number of NEUN-immunoreactive neurons in CA1-CA4, subiculum (SUB), and ERC cortex throughout their full rostrocaudal extent. [12][13][14] The boundaries of ERC were determined according to the location of large, stellate, multipolar neurons in layer II and anatomic landmarks described from a monkey atlas. 15 On the coronal sections, the medial border of ERC was limited rostrally by a group of dense neurons of the amygdala transition area and caudally by two groups of small neurons that belong to parasubiculum and presubiculum. ...
INTRODUCTION
The understanding of the pathological events in Alzheimer's disease (AD) has advanced dramatically, but the successful translation from rodent models into efficient human therapies is still problematic.
METHODS
To examine how tau pathology can develop in the primate brain, we injected 12 macaques with a dual tau mutation (P301L/S320F) into the entorhinal cortex (ERC). An investigation was performed using high‐resolution microscopy, magnetic resonance imaging (MRI), positron emission tomography (PET), and fluid biomarkers to determine the temporal progression of the pathology 3 and 6 months after the injection.
RESULTS
Using quantitative microscopy targeting markers for neurodegeneration and neuroinflammation, as well as fluid and imaging biomarkers, we detailed the progression of misfolded tau spreading and the consequential inflammatory response induced by glial cells.
DISCUSSION
By combining the analysis of several in vivo biomarkers with extensive brain microscopy analysis, we described the initial steps of misfolded tau spreading and neuroinflammation in a monkey model highly translatable to AD patients.
Highlights
Dual tau mutation delivery in the entorhinal cortex induces progressive tau pathology in rhesus macaques.
Exogenous human 4R‐tau coaptates monkey 3R‐tau during transneuronal spread, in a prion‐like manner.
Neuroinflammatory response is coordinated by microglia and astrocytes in response to tau pathology, with microglia targeting early tau pathology, while astrocytes engaged later in the progression, coincident with neuronal death.
Monthly collection of CSF and plasma revealed a profile of changes in several AD core biomarkers, reflective of neurodegeneration and neuroinflammation as early as 1 month after injection.
... The volume density of the main uterine compartments (endometrium, myometrium, and perimetrium) was determined according to the Cavalieri principle [57,58] using the newCAST stereological software package Visiopharm Integrator System (VIS) version 3.2.7.0 (Visiopharm; Denmark). Sampling of 5 µm uterine sections was performed systematically, uniformly at random. ...
Increased fructose consumption and chronic stress, the major characteristics of modern lifestyle, impact human health; however, the consequences of their combination on the uterus remain understudied. In this study, we investigated contractile activity, morphology, and intracellular activity of antioxidant enzymes in uteri from virgin Wistar rats subjected to liquid fructose supplementation and/or unpredictable stress over 9 weeks. Contractile activity and uterine response to oxytocin or adrenaline were examined ex vivo using isolated bath chambers. Fructose supplementation, irrespective of stress, affected uterine morphology by increasing endometrium while decreasing myometrium volume density, attenuated uterine response to increasing doses of oxytocin, and increased glutathione peroxidase activity. Stress, irrespective of fructose, attenuated dose-dependent adrenaline-induced uterine relaxation. Stress, when applied solely, decreased mitochondrial superoxide dismutase activity. In the combined treatment, irregular estrous cycles and both reduced response to oxytocin and to adrenaline (as a consequence of fructose consumption and exposure to stress), along with fructose-related alteration of uterine morphology, were detected. In conclusion, fructose and stress affect uterine contractile activity, irrespective of each other, by inducing completely distinct responses in isolated uteri. In the combined treatment, the effects of both factors were evident, suggesting that the combination exerts more detrimental effects on the uterus than each factor individually.
... The stereologic analysis included the absolute volume of the pituitary gland and the volume occupied by the immunopositive GH cells. Volumes were estimated according to the Cavalieri principle [47]. Sampling of pituitary sections was systematically uniform from the beginning (every 40th section from each tissue block was analyzed). ...
Inflammation, demyelination, and axonal damage to the central nervous system (CNS) are the hallmarks of multiple sclerosis (MS) and its representative animal model, experimental autoimmune encephalomyelitis (EAE). There is scientific evidence for the involvement of growth hormone (GH) in autoimmune regulation. Previous data on the relationship between the GH/insulin like growth factor-1 (IGF-1) axis and MS/EAE are inconclusive; therefore, the aim of our study was to investigate the changes in the GH axis during acute monophasic EAE. The results show that the gene expression of Ghrh and Sst in the hypothalamus does not change, except for Npy and Agrp, while at the pituitary level the Gh, Ghrhr and Ghr genes are upregulated. Interestingly, the cell volume of somatotropic cells in the pituitary gland remains unchanged at the peak of the disease. We found elevated serum GH levels in association with low IGF-1 concentration and downregulated Ghr and Igf1r expression in the liver, indicating a condition resembling GH resistance. This is likely due to inadequate nutrient intake at the peak of the disease when inflammation in the CNS is greatest. Considering that GH secretion is finely regulated by numerous central and peripheral signals, the involvement of the GH/IGF-1 axis in MS/EAE should be thoroughly investigated for possible future therapeutic strategies, especially with a view to improving EAE disease.
... An unbiased counting frame (Gundersen & Jensen, 1987) was superimposed on the optical section stack using the Blend Images plugin in ImageJ. All cells in the lookup section were discarded and marked with a single color. ...
Recent studies have highlighted the potential involvement of reactive oxygen species (ROS) and microglia, a major source of ROS, in the pathophysiology of schizophrenia. In our study, we explored how the second‐generation antipsychotic risperidone (RIS) affects ROS regulation and microglial activation in the hippocampus using a mouse ketamine (KET) model of schizophrenia. KET administration resulted in schizophrenia‐like behaviors in male C57BL/6J mice, such as impaired prepulse inhibition (PPI) of the acoustic startle response and hyper‐locomotion. These behaviors were mitigated by RIS. We found that the gene expression level of an enzyme responsible for ROS production (Nox2), which is primarily associated with activated microglia, was lower in KET/RIS‐treated mice than in KET‐treated mice. Conversely, the levels of antioxidant enzymes (Ho‐1 and Gclc) were higher in KET/RIS‐treated mice. The microglial density in the hippocampus was increased in KET‐treated mice, which was counteracted by RIS. Hierarchical cluster analysis revealed three morphological subtypes of microglia. In control mice, most microglia were resting‐ramified (type I, 89.7%). KET administration shifted the microglial composition to moderately ramified (type II, 44.4%) and hyper‐ramified (type III, 25.0%). In KET/RIS‐treated mice, type II decreased to 32.0%, while type III increased to 34.0%. An in vitro ROS assay showed that KET increased ROS production in dissociated hippocampal microglia, and this effect was mitigated by RIS. Furthermore, we discovered that a NOX2 inhibitor could counteract KET‐induced behavioral deficits. These findings suggest that pharmacological inhibition of ROS production by RIS may play a crucial role in ameliorating schizophrenia‐related symptoms. Moreover, modulating microglial activation to regulate ROS production has emerged as a novel avenue for developing innovative treatments for schizophrenia. image
... (280 μm apart, approximately − 0.7 to -3.5 mm Bregma) were stained with Luxol Fast Blue (LFB) and Cresyl Violet (CV) as per a modified protocol [46]. For analysis, images were captured using a Leica Aperio AT Turbo Brightfield slide scanner, then FIJI/ImageJ (NIH, Bethesda, MD; http://imagej.nih.gov/ij) was used to apply the unbiased Cavalieri method to estimate the volume of the intact dorsal cortex and hippocampus in each hemisphere [47]. Each grid point represented an area of 100 μm², with a sampling frequency of four. ...
Pneumonia is a common comorbidity in patients with severe traumatic brain injury (TBI), and is associated with increased morbidity and mortality. In this study, we established a model of intratracheal Klebsiella pneumoniae administration in young adult male and female mice, at 4 days following an experimental TBI, to investigate how K. pneumoniae infection influences acute post-TBI outcomes. A dose-response curve determined the optimal dose of K. pneumoniae for inoculation (1 x 10^6 colony forming units), and administration at 4 days post-TBI resulted in transient body weight loss and sickness behaviors (hypoactivity and acute dyspnea). K. pneumoniae infection led to an increase in pro-inflammatory cytokines in serum and bronchoalveolar lavage fluid at 24 h post-infection, in both TBI and sham (uninjured) mice. By 7 days, when myeloperoxidase + neutrophil numbers had returned to baseline in all groups, lung histopathology was observed with an increase in airspace size in TBI + K. pneumoniae mice compared to TBI + vehicle mice. In the brain, increased neuroinflammatory gene expression was observed acutely in response to TBI, with an exacerbated increase in Ccl2 and Hmox1 in TBI + K. pneumoniae mice compared to either TBI or K. pneumoniae alone. However, the presence of neuroinflammatory immune cells in the injured brain, and the extent of damage to cortical and hippocampal brain tissue, was comparable between K. pneumoniae and vehicle-treated mice by 7 days. Examination of the fecal microbiome across a time course did not reveal any pronounced effects of either injury or K. pneumoniae on bacterial diversity or abundance. Together, these findings demonstrate that K. pneumoniae lung infection after TBI induces an acute and transient inflammatory response, primarily localized to the lungs with some systemic effects. However, this infection had minimal impact on secondary injury processes in the brain following TBI. Future studies are needed to evaluate the potential longer-term consequences of this dual-hit insult.
Supplementary Information
The online version contains supplementary material available at 10.1186/s12974-024-03093-9.
... AO-stained samples were observed and photographed using an Olympus BX50 fluorescence microscope (Olympus, Hamburg, Germany). The evaluation of the coverage was carried out by the method of quantitative microscopy and with respecting the principles of unbiased sampling [37,38]. Images of the AO-stained surface with bacteria were photographed according to the previously prepared scheme: using an immersion objective (100×, numerical aperture 1.30), every third field of view on a vertical line passing through the center of the sample and every third field of view on a horizontal line passing through the center of the sample were photographed, representing 2 × 5 fields on each sample ( figure 1(b)). ...
Titanium and its alloys are commonly used in modern implantology. Cell viability on the surface of titanium implants depends on the surface topography, roughness, and wettability. Laser treatment is a successful method to control the surface morphology. The aim of this study was to comprehensively investigate the effects of laser ablation on titanium surfaces and their interactions with cells and bacteria. Cell adhesion, proliferation, and bacterial retention on smooth and laser-textured samples of commercially pure and nanostructured titanium of two grades were evaluated. Femtosecond laser treatment effectively enhances the wettability. Titanium grade four exhibits superior adhesion and proliferation rates when compared to titanium grade two. The cytotoxicity of nanostructured titanium is significantly lower, regardless of the surface treatment. Laser treatment resulted in increased short-term cell proliferation on grade two titanium and long-term cell proliferation on nanostructured grade two titanium only. Although the laser ablation has a limited effect on bacterial adhesion, the coverage of less than 1% in most samples indicates that the material itself has an antibacterial effect on the bacterial strain Streptococcus oralis. These findings provide valuable insights into how different material structures and surface treatments can affect cellular response and antibacterial properties for potential use in dental implantology.
... Stereological analysis was conducted to estimate the number of hepatocytes in rats. The paraffin-processed liver samples were serially sectioned as reported by Gundersen and Jensen [18], to provide sections of 10ʯm thickness. 25 tissue sections were selected per group using a systematic uniform random sampling method and stained with H and E. ...
Background
Mercury chloride (HgCl 2 ) damages tissues it comes in contact with in sufficient concentration. This study evaluated the protective effects of n- butanol fraction of Phoenix dactylifera (BFPD) on mercury-triggered liver toxicity in Wistar rats. 25 male rats were divided into 5 groups of 5 rats each. Group I was administered 2 ml/kg of distilled water; group II was administered 5 mg/kg of HgCl 2 ; group III was administered 500 mg/kg of BFPD + 5 mg/kg of HgCl 2 ; group IV was administered 1000 mg/kg of BFPD + 5 mg/kg of HgCl 2 , while group V was administered 100 mg/kg of silymarin + 5 mg/kg of HgCl 2 . orally for 2 weeks. The rats were euthanized and liver tissue blood samples were collected for histological, histochemical, stereological, immunohistochemical, molecular, and biochemical studies.
Results
The results revealed that HgCl 2 induced oxidative stress in the rats evident by histoarchitectural distortions and altered levels of liver enzymes, proteins, and oxidative stress biomarkers when compared to the control. However, BFPD treatment restored these changes. Glutathione peroxidase levels decreased ( p < 0.05) in the HgCl 2− treated group when compared to the control and BFPD-treated groups. HgCl 2 group revealed reduced reactivity with histochemical and immunohistochemical stains (Masson’s Trichrome and B cell Lymphoma 2) when compared to the control, with a significant decrease in quantified liver Bcl-2 stain intensity when compared to the silymarin-treated group. BFPD administration revealed normal staining intensity comparable to the control. HgCl 2 administration revealed a remarked decrease in the number of hepatocytes when compared to the control, BFPD, and silymarin groups. BFPD preserved ( p < 0.05) the stereological features when compared to the HgCl 2 -treated group. GPx activity in the liver decreased ( p < 0.05) with HgCl 2 administration when compared to the control and silymarin-treated groups. BFPD attenuated GPx gene activity to levels similar to the control indicating some level of amelioration against HgCl 2 -induced toxicity.
Conclusions
The ability of BFPD to mitigate HgCl 2 triggered liver alterations could be attributed to the antioxidant property of its flavonoid content. Therefore, BFPD may be a potential candidate for treating and managing liver-induced mercury intoxication.
... Microphotographs at 10x were obtained with a confocal microscope, and then processed using the stitching plugin in FIJI/ImageJ [59] in order to have the whole nucleus on a single microphotograph. All the sections in a 1/6 subseries containing the region of interest were analyzed and the areas estimated using Cavalieri's principle [69]. To obtain the volume of the CA1 region, we multiplied the area by the thickness of the slice (50 µm) and the number of series. ...
Severe psychiatric illnesses, for instance schizophrenia, and affective diseases or autism spectrum disorders, have been associated with cognitive impairment and perturbed excitatory-inhibitory balance in the brain. Effects in juvenile mice can elucidate how erythropoietin (EPO) might aid in rectifying hippocampal transcriptional networks and synaptic structures of pyramidal lineages, conceivably explaining mitigation of neuropsychiatric diseases. An imminent conundrum is how EPO restores synapses by involving interneurons. By analyzing ~12,000 single-nuclei transcriptomic data, we generated a comprehensive molecular atlas of hippocampal interneurons, resolved into 15 interneuron subtypes. Next, we studied molecular alterations upon recombinant human (rh)EPO and saw that gene expression changes relate to synaptic structure, trans-synaptic signaling and intracellular catabolic pathways. Putative ligand-receptor interactions between pyramidal and inhibitory neurons, regulating synaptogenesis, are altered upon rhEPO. An array of in/ex vivo experiments confirms that specific interneuronal populations exhibit reduced dendritic complexity, synaptic connectivity, and changes in plasticity-related molecules. Metabolism and inhibitory potential of interneuron subgroups are compromised, leading to greater excitability of pyramidal neurons. To conclude, improvement by rhEPO of neuropsychiatric phenotypes may partly owe to restrictive control over interneurons, facilitating re-connectivity and synapse development.
... The second equation can be found in Gundersen and Jensen (1987). Finally, using Cochran's formula and Goodman's formula, respectively, and supposing independent estimators of A, B, and E(C), we obtain the following predicted approximations of the squared coefficients of error forF andÊ (Cruz-Orive (2024), Sections A.2.3 and A.2.4): ce 2 (F) = ce 2 ((B) 2 ) + ce 2 (Â). ...
Sickle cell disease causes the deformation of erythrocytes into sickle cells. The study of this process using digital images of peripheral blood smears can help specialists to quantify the number of deformed cells in order to gauge the severity of the illness. A new method for classifying red blood cells into three categories: healthy, sickle cell disease, and other deformations is proposed. This method does not require obtaining the contour of each cell but instead utilizes information obtained from a small number of points, obtained through appropriate geometric sampling and the use of stereological formulas. The parameters utilized for classification are the bending energy times length (E) and the circular shape factor (F). In normal cells, which exhibit an almost circular shape, these parameters typically have values close to (1,1). To assess the effectiveness of classification using the parameters (E,F), a synthetic curve dataset and a dataset of red blood cells are employed, applying various supervised and unsupervised classification methods.
... The same process was repeated a total of 3 times to be calculated at different times. Acer et al, (2007), Roberts et al, (1993), Gundersen and Jensen (1987) were referenced for the estimation of total intracranial volume. Intracranial cavity volumes were estimated using the following equation: ...
Stereology is a powerful scientific method for estimating the real attributes of three-dimensional structures using calculations on two-dimensional photographs. The goal of this study was to determine the cerebral volume of cats using stereological cranial computed tomography (CT) and to uncover dimorphic differences. The study employed 16 adult Van cats (8 females and 8 males). A multislice CT equipment was utilized to scan the craniums of the cats involved in the study. Stereological computations were performed using 12 section images from cranial CT sections with intracranial borders calculated using a systematic random sampling procedure. Linear measurements of the intra-cranial region were also taken. When the findings were analysed, it was discovered that intracranial volume values differed across sexes (P<0.05). It was determined that the measured linear parameters and calculated index values did not show dimorphism between the sexes (P>0.05). As a result, it is thought that the determination of the intracranial volumes of Van cats using stereology method will contribute to clinical sciences in terms of diagnosis, detection and treatment of diseases and will allow the comparison of volume values calculated with different methods.
... Image Analysis -Quanti cation of PAS Reactivity PAS-stained micrographs were quanti ed for PAS reactivity to glycogen moiety as described by Amber et al. 24 which involved measuring staining intensity using a computer running image analysis software (ImageJ® NIH, US) according to the manufacturer's speci cations. The ImageJ® region of interest (ROI) manager tool for analysis of speci c areas of the micrographs was employed 25 . The mean gray values for three ROIs were obtained and means were computed and analyzed. ...
Mercury is a highly toxic substance that poses a serious threat to living organisms. This work evaluated the protective effects of n- butanol fraction of Phoenix dactylifera Linn (BFPD) on mercury-induced kidney toxicity in Wistar rats. 25 rats were divided into 5 groups containing 5 rats each. Group I was administered 2 ml/kg of distilled water; group II was administered 5 mg/kg of mercury chloride (HgCl 2 ); groups III and IV received 500 mg/kg and 1000 mg/kg of BFPD followed by 5 mg/kg of HgCl 2 respectively. Group V was treated with 100 mg/kg of silymarin followed by 5 mg/kg of HgCl 2 . All administrations were oral and lasted for 2 weeks after which the rats were euthanized and blood and kidney samples were collected for biochemical, histological, and histochemical studies respectively. HgCl 2 induced oxidative stress resulting in nephrotoxicity in the rats noticeable by altered levels of Na ²⁺ , Ca ²⁺ , K ⁺ , Cl ⁻ and HCO 3 ⁻ , and activities of SOD and catalase when compared to the control. However, BFPD treatment ameliorated these alterations. The group treated with HgCl 2 showed histological variations in the kidney such as dilated Bowman’s capsule and glomerular shrinkage while histochemical analysis revealed reduced reactivity to glycogen moiety when compared to the control. Treatment with BFPD protected the histoarchitectural properties of the kidney comparable to the control. In conclusion, BFPD protected the kidney against HgCl 2 -induced nephrotoxicity in rats due to its antioxidant (flavonoid) properties. Therefore, BFPD may be considered a noble candidate for treating and managing HgCl2-related nephrotoxicity.
... PAS-stained micrographs were quantified for PAS reactivity to glycogen moiety as described by Amber et al. 24 which involved measuring staining intensity using a computer running image analysis software (ImageJ® NIH, US) according to the manufacturer's specifications. The ImageJ® region of interest (ROI) manager tool for analysis of specific areas of the micrographs was employed 25 . The mean gray values for three ROIs were obtained and means were computed and analyzed. ...
Mercury is a highly toxic substance that poses a serious threat to living organisms. This work evaluated the protective effects of n-butanol fraction of Phoenix dactylifera Linn (BFPD) on mercury-induced kidney toxicity in Wistar rats. 25 rats were divided into 5 groups containing 5 rats each. Group I was administered 2 ml/kg of distilled water; group II was administered 5 mg/kg of mercury chloride (HgCl2); groups III and IV received 500 mg/kg and 1000 mg/kg of BFPD followed by 5 mg/kg of HgCl2 respectively. Group V was treated with 100 mg/kg of silymarin followed by 5 mg/kg of HgCl2. All administrations were oral and lasted for 2 weeks after which the rats were euthanized and blood and kidney samples were collected for biochemical, histological, and histochemical studies respectively. HgCl2 induced oxidative stress resulting in nephrotoxicity in the rats noticeable by altered levels of Na 2+ , Ca 2+ , K + , Cl-and HCO3-, and activities of SOD and catalase when compared to the control. However, BFPD treatment ameliorated these alterations. The group treated with HgCl2 showed histological variations in the kidney such as dilated Bowman's capsule and glomerular shrinkage while histochemical analysis revealed reduced reactivity to glycogen moiety when compared to the control. Treatment with BFPD protected the histoarchitectural properties of the kidney comparable to the control. In conclusion, BFPD protected the kidney against HgCl2-induced nephrotoxicity in rats due to its antioxidant (flavonoid) properties. Therefore, BFPD may be considered a noble candidate for treating and managing HgCl2-related nephrotoxicity.
... The obtained sections were collected in phosphate-buffered saline (PBS), and, after that, transferred into de Olmos cryoprotectant solution where they were stored at −20 • C until further processing. From the entire set of sections collected from each block, 4 series were formed by using a systematic, random sampling method [69,70]. Accordingly, the first section was randomly selected from the first group of 4 collected sections, and the remaining were sampled at regular intervals of 160 µm (i.e., 1 out of 4 sections). ...
The basolateral amygdala (BLA) contains interneurons that express neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP), both of which are involved in the regulation of functions and behaviors that undergo deterioration with aging. There is considerable evidence that, in some brain areas, the expression of NPY and VIP might be modulated by acetylcholine. Importantly, the BLA is one of the brain regions that has one of the densest cholinergic innervations, which arise mainly from the basal forebrain cholinergic neurons. These cholinergic neurons depend on nerve growth factor (NGF) for their survival, connectivity, and function. Thus, in this study, we sought to determine if aging alters the densities of NPY- and VIP-positive neurons and cholinergic varicosities in the BLA and, in the affirmative, if those changes might rely on insufficient trophic support provided by NGF. The number of NPY-positive neurons was significantly reduced in aged rats, whereas the number of VIP-immunoreactive neurons was unaltered. The decreased NPY expression was fully reversed by the infusion of NGF in the lateral ventricle. The density of cholinergic varicosities was similar in adult and old rats. On the other hand, the density of cholinergic varicosities is significantly higher in old rats treated with NGF than in adult and old rats. Our results indicate a dissimilar resistance of different populations of BLA interneurons to aging. Furthermore, the present data also show that the BLA cholinergic innervation is particularly resistant to aging effects. Finally, our results also show that the reduced NPY expression in the BLA of aged rats can be related to changes in the NGF neurotrophic support.
... Stereological analysis was conducted to estimate the global volume of the kidney in the rats. The processed kidney samples were serially sectioned as reported by Gundersen and Jensen [22] to provide sections of 10ʯm thickness. Tissue sections were selected using a systematic uniform random sampling method and stained with H and E. ...
... We calculated the demyelination areas by manually tracing each section obtained per lesion using Image J and summing the areas across sections. We used the following formula 36,37 : ...
Background
Inflammatory demyelinating diseases of the central nervous system, such as multiple sclerosis, are significant sources of morbidity in young adults despite therapeutic advances. Current murine models of remyelination have limited applicability due to the low white matter content of their brains, which restricts the spatial resolution of diagnostic imaging. Large animal models might be more suitable but pose significant technological, ethical and logistical challenges.
Methods
We induced targeted cerebral demyelinating lesions by serially repeated injections of lysophosphatidylcholine in the minipig brain. Lesions were amenable to follow-up using the same clinical imaging modalities (3T magnetic resonance imaging, ¹¹C-PIB positron emission tomography) and standard histopathology protocols as for human diagnostics (myelin, glia and neuronal cell markers), as well as electron microscopy (EM), to compare against biopsy data from two patients.
Findings
We demonstrate controlled, clinically unapparent, reversible and multimodally trackable brain white matter demyelination in a large animal model. De-/remyelination dynamics were slower than reported for rodent models and paralleled by a degree of secondary axonal pathology. Regression modelling of ultrastructural parameters (g-ratio, axon thickness) predicted EM features of cerebral de- and remyelination in human data.
Interpretation
We validated our minipig model of demyelinating brain diseases by employing human diagnostic tools and comparing it with biopsy data from patients with cerebral demyelination.
Funding
This work was supported by the 10.13039/501100001659DFG under Germany's Excellence Strategy within the framework of the Munich Cluster for Systems Neurology (EXC 2145 SyNergy, ID 390857198) and TRR 274/1 2020, 408885537 (projects B03 and Z01).
... Cavalieri's principle originates from the method developed by Johannes Kepler to calculate the volumes of wine barrels (Pitlyk, 2006). Bonaventura Cavalieri developed a method to calculate the volumes of tissues and organs that interact with the surrounding structures and this method is frequently preferred by researchers in stereological methods (Gundersen & Jensen, 1987). Computed tomography helps physicians in the diagnosis, diagnostics, treatment, follow-up, and management of diseases since the day it was produced (Kalender, 2006). ...
The intracranial cavity contains vital organs such as the brain, cerebellum and meninges. The veterinary field employment in this region is limited. For this reason, obtaining information about the intracranial cavity, the operation to be performed on this region and information about the world will benefit our unit. It aimed to examine the width between gender differences and expectations by calculating the intracranial volume stereological and 3D models using computed tomography sections. In the study, 7 male and 6 female adult Hamdani sheep were used. The intracranial cavity was modelled in 3D using computed tomography sections. Intracranial volume was estimated using CT slices according to the Cavalieri principle. Morphometric measurements were obtained from the intracranial cavity. In the study, intracranial volume was calculated as 120.50±7.59 cm3 in females and 137.29±9.79 cm3 in males in measurements made using Computed Tomography. Intracranial volume calculation made using the Cavalieri's principle was calculated as 125.33±5.20cm3 in females and 136.89±9.13 cm3in males. In the configured CT models, a target difference was detected between the male and female volume values calculated on it (p < 0.05). There was a difference between males and females in stereologically viable intracranial volume (p < 0.05). A high level of density was observed between the results of both methods. In the mean of the data, there was no difference between the objectives (p > 0.05). Morphometric measurements determined that the maximum cranial cavity width parameter was between females and males (p < 0.005). In index 1 and index 2 calculated with the obtained partitions, a circulating difference between genders was observed (p < 0.05). In the resulting state, the 3-dimensional intracranial cavity models and the intracranial volume fraction estimated by the Cavalieri principle are conserved. There are high limits between the methods. Feeding future intracranial volume and clinical care.
... Images were used to count paracellular junctions and characterised according to VE-cadherin staining pattern at cell-cell borders: continuous or disrupted. A grid was used by ImageJ software so that all the junctional regions had the same chance of being counted, and junctions from every other square which did not cross the 'forbidden line' were analysed [23,24]. ...
In vitro studies have shown that Wharton’s jelly mesenchymal stem cells (WJ-MSCs) can cross umbilical and uterine endothelial barriers and up-regulate endothelial junctional integrity from sub-endothelial niches. This pericytic behaviour may be lost in pregnancies complicated by gestational diabetes (GDM), where increased vascular permeability and junctional disruption are reported. The aim of the present study was to investigate whether WJ-MSCs isolated from GDM pregnancies displayed any changes in morphology, proliferation, VEGF-A secretion, and their ability to influence paracellular junctional composition and permeability. WJ-MSCs were isolated from human umbilical cords from normal pregnancies (nWJ-MSCs, n=13) and those complicated by GDM (gWJ-MSCs), either diet-controlled (d-GDM, n=13) or metformin-treated (m-GDM, n=9). We recorded that 4-fold more WJ-MSCs migrated from m-GDM, and 2.5-fold from d-GDM cord samples compared with the normal pregnancy. gWJ-MSCs showed a less predominance of spindle-shaped morphology and secreted 3.8-fold more VEGF-A compared with nWJ-MSCs. The number of cells expressing CD105 (Endoglin) was higher in gWJ-MSCs compared with nWJ-MSCs (17%) at P-2. The tracer leakage after 24 h across the HUVEC + gWJ-MSCs bilayer was 22.13% and 11.2% higher in the m-GDM and d-GDM, respectively, HUVEC + nWJ-MSCs. Transfection studies with siRNAs that target Endoglin were performed in n-WJ-MSCs; transfected cells were co-cultured with HUVEC followed by permeability studies and VE-cadherin analyses. Loss of Endoglin also led to increased VEGF-A secretion, increased permeability and affected endothelial stabilization. These results reinforce the pericytic role of nWJ-MSCs to promote vascular repair and the deficient ability of gWJ-MSCs to maintain endothelial barrier integrity.
... Image Analysis -Quanti cation of PAS Reactivity PAS-stained micrographs were quanti ed for PAS reactivity to glycogen moiety as described by Amber et al. 24 which involved measuring staining intensity using a computer running image analysis software (ImageJ® NIH, US) according to the manufacturer's speci cations. The ImageJ® region of interest (ROI) manager tool for analysis of speci c areas of the micrographs was employed 25 . The mean gray values for three ROIs were obtained and means were computed and analyzed. ...
Introduction:
Mercury is a highly toxic metal that exerts adverse effects on humans and animals. Date palm (Phoenix dactylifera) is used in folk medicine to treat fever, and inflammation, among others. The present study aimed to evaluate the protective effects of aqueous and ethanol extracts of Phoenix dactylifera on mercury-induced kidney damage in Wistar rats.
Design:
45 male rats (80–125g) were divided into nine groups (I-XI). Group 1 received 1mg/kg of distilled water, group II received mercury chloride (HgCl2) at 5 mg/kg; group III was pretreated with100 mg/kg of silymarin followed by 5 mg/kg of HgCl2; groups IV and V were pretreated with 500 mg/kg and 1000 mg/kg of aqueous extract of Phoenix dactylifera (AFPD) respectively, followed by 5 mg/kg of HgCl2; groups VI and VII were pretreated with 500 mg/kg and 1000 mg/kg of ethanol extract of Phoenix dactylifera L. (EFPD) respectively, followed by 5 mg/kg of HgCl2 while group VIII and IX received 1000 mg/kg of AFPD and EFPD respectively. After two weeks of oral administration, the animals were sacrificed and the blood and kidney tissue were collected for analyses.
Results
The present study showed that MDA significantly increased in HgCl2 group when compared to control while histological and histochemical changes in the kidney revealed severe degenerative changes. AFPD and EFPD extracts restored serum enzymes, electrolytes and antioxidant enzymes activity with significant increase in SOD activity in the AFPD (high dose) when compared to the HgCl2 treated group. This indicates some level of protection against HgCl2-induced changes in the kidneys of Wistar rats. The protective activity of the AFPD and EFPD may be attributed to the antioxidant properties of the phytochemicals, such as flavonoids, tannins etc.
Conclusion
The aqueous and ethanol extracts of Phoenix dactylifera are potential candidates for the management and treatment of ROS-induced kidney diseases.
... The areas covered by matrix polysaccharides, eDNA and microbial cells were determined by threshold-based segmentation of the red, green and blue channel images, respectively. For each z-stack, biovolumes were estimated according to the Cavalieri principle by multiplying the areas covered by matrix polysaccharides, eDNA and microbial cells with the interslice distance [36]. The effect of enzymatic treatment on biofilm biovolumes (determined by CV staining) and cell indices (determined by RCTA) was analyzed by one-way ANOVA with Dunnett's correction for multiple comparisons. ...
Introduction:
The dental biofilm matrix is an important determinant of virulence for caries development and comprises a variety of extracellular polymeric substances that contribute to biofilm stability. Enzymes that break down matrix components may be a promising approach to caries control, and in light of the compositional complexity of the dental biofilm matrix, treatment with multiple enzymes may enhance the reduction of biofilm formation compared to single enzyme therapy. The present study investigated the effect of the three matrix-degrading enzymes mutanase, beta-glucanase, and DNase, applied separately or in combinations, on biofilm prevention and removal in a saliva-derived in vitro-grown model.
Methods:
Biofilms were treated during growth to assess biofilm prevention or after 24 h of growth to assess biofilm removal by the enzymes. Biofilms were quantified by crystal violet staining and impedance-based real-time cell analysis, and the biofilm structure was visualized by confocal microscopy and staining of extracellular DNA (eDNA) and polysaccharides.
Results:
The in vitro model was dominated by Streptococcus spp., as determined by 16S rRNA gene amplicon sequencing. All tested enzymes and combinations had a significant effect on biofilm prevention, with reductions of >90% for mutanase and all combinations including mutanase. Combined application of DNase and beta-glucanase resulted in an additive effect (81.0% ± 1.3% SD vs. 36.9% ± 21.9% SD and 48.2% ± 14.9% SD). For biofilm removal, significant reductions of up to 73.2% ± 5.5% SD were achieved for combinations including mutanase, whereas treatment with DNase had no effect. Glucans, but not eDNA decreased in abundance upon treatment with all three enzymes.
Conclusion:
Multi-enzyme treatment is a promising approach to dental biofilm control that needs to be validated in more diverse biofilms.
... The area of the GCL was calculated at each distance from Bregma. To obtain the volume, the sum of the area measured was multiplied by the section sampling fraction (8) and the section thickness (30 µm) (Gundersen and Jensen, 1987;Gundersen et al., 1988;Latchney et al., 2014Latchney et al., , 2015. The Gunderson coefficient of variance for each mouse was always <10% (Latchney et al., 2014(Latchney et al., , 2015. ...
Adult neurogenesis is a persistent phenomenon in mammals that occurs in select brain structures in both healthy and diseased brains. The tumor suppressor gene, phosphatase and tensin homolog deleted on chromosome 10 ( Pten ) has previously been found to restrict the proliferation of neural stem/progenitor cells (NSPCs) in vivo . In this study, we aimed to provide a comprehensive picture of how conditional deletion of Pten may regulate the genesis of adult NSPCs in the dentate gyrus of the hippocampus and the subventricular zone bordering the lateral ventricles. Using conventional markers and stereology, we quantified multiple stages of neurogenesis, including proliferating cells, immature neurons (neuroblasts), and apoptotic cells in several regions of the dentate gyrus, including the subgranular zone (SGZ), outer granule cell layer (oGCL), molecular layer, and hilus at 4 and 10 weeks of age. Our data demonstrate that conditional deletion of Pten in mice produces successive increases in dentate gyrus proliferating cells and immature neuroblasts, which confirms the known negative roles Pten has on cell proliferation and maturation. Specifically, we observe a significant increase in Ki67+ proliferating cells in the neurogenic SGZ at 4 weeks of age, but not 10 weeks of age. We also observe a delayed increase in neuroblasts at 10 weeks of age. However, our study expands on previous work by providing temporal, subregional, and neurogenesis-stage resolution. Specifically, we found that Pten deletion initially increases cell proliferation in the neurogenic SGZ, but this increase spreads to non-neurogenic dentate gyrus areas, including the hilus, oGCL, and molecular layer, as mice age. We also observed region-specific increases in apoptotic cells in the dentate gyrus hilar region that paralleled the regional increases in Ki67+ cells. Our work is accordant with the literature showing that Pten serves as a negative regulator of dentate gyrus neurogenesis but adds temporal and spatial components to the existing knowledge.
... We also measured telencephalon volume (minus medial and dorsal cortical volumes) to assess if population of origin and stress treatment effects were specific to the cortices or representative of global changes within the brain [63][64][65]. The left and right hemispheres of the medial cortex, dorsal cortex and the remainder of the telencephalon were contoured in their entirety at 5×. Cortical volume estimations were generated with the Cavalieri procedure [66] using a 200 µm grid. Finally, we estimated medial cortical neuron soma volume with the optical fractionator nucleator procedure using four rays and a 250 µm grid, at 100×, measuring three randomly selected cells per counting frame. ...
Stress experienced during ontogeny can have profound effects on the adult phenotype. However, stress can also be experienced intergenerationally, where an offspring's phenotype can be moulded by stress experienced by the parents. Although early-life and intergenerational stress can alter anatomy, physiology, and behaviour, nothing is known about how these stress contexts interact to affect the neural phenotype. Here, we examined how early-life and intergenerational stress affect the brain in eastern fence lizards (Sceloporus undulatus). Some lizard populations co-occur with predatory fire ants, and stress from fire ant attacks exerts intergenerational physiological and behavioural changes in lizards. However, it is unclear if intergenerational stress, or the interaction between intergenerational and early-life stress, modulates the brain. To test this, we captured gravid females from fire ant invaded and uninvaded populations, and subjected offspring to three early-life stress treatments: (1) fire ant attack, (2) corticosterone, or (3) a control. Corticosterone and fire ant attack decreased some aspects of the neural phenotype while population of origin and the interaction of early-life stress and population had no effects on the brain. These results suggest that early-life stressors may better predict adult brain variation than intergenerational stress in this species.
... We quantified numbers of microglia in the entire striatum and TH+ or NeuN+ neurons in the SNpc using stereology with the optical fractionator method (Table S2) and Stereo Investigator 7 software (MBF Bioscience). Coefficient of error attributable to the sampling was calculated according to Gundersen and Jensen (Gundersen & Jensen, 1987). Errors ≤0.10 were acceptable. ...
L‐Dopa, while treating motor symptoms of Parkinson's disease, can lead to debilitating L‐Dopa‐induced dyskinesias, limiting its use. To investigate the causative relationship between neuro‐inflammation and dyskinesias, we assessed if striatal M1 and M2 microglia numbers correlated with dyskinesia severity and whether the anti‐inflammatories, minocycline and indomethacin, reverse these numbers and mitigate against dyskinesia. In 6‐OHDA lesioned mice, we used stereology to assess numbers of striatal M1 and M2 microglia populations in non‐lesioned (naïve) and lesioned mice that either received no L‐Dopa (PD), remained non‐dyskinetic even after L‐Dopa (non‐LID) or became dyskinetic after L‐Dopa treatment (LID). We also assessed the effect of minocycline/indomethacin treatment on striatal M1 and M2 microglia and its anti‐dyskinetic potential via AIMs scoring. We report that L‐Dopa treatment leading to LIDs exacerbates activated microglia numbers beyond that associated with the PD state; the severity of LIDs is strongly correlated to the ratio of the striatal M1 to M2 microglial numbers; in non‐dyskinetic mice, there is no M1/M2 microglia ratio increase above that seen in PD mice; and reducing M1/M2 microglia ratio using anti‐inflammatories is anti‐dyskinetic. Parkinson's disease is associated with increased inflammation, but this is insufficient to underpin dyskinesia. Given that L‐Dopa‐treated non‐LID mice show the same ratio of M1/M2 microglia as PD mice that received no L‐Dopa, and, given minocycline/indomethacin reduces both the ratio of M1/M2 microglia and dyskinesia severity, our data suggest the increased microglial M1/M2 ratio that occurs following L‐Dopa treatment is a contributing cause of dyskinesias.
... The area (A) of the cross-section can be calculated by multiplying the total number of points (Σp) and the area represented by a point [a(p)]. 16 A ¼ ...
The use of Methylphenidate (MP) can have adverse effects on bone growth and mineralization. This study aimed to investigate the underlying pathophysiology of MP-induced skeletal deficits in growing rats using stereological and immunohistochemical methods. Male rats, aged 4 weeks, were orally treated with MP through an 8-h/day water drinking protocol. The rats ( n=30) were randomly divided into three groups: MP-High Dose (30/60 mg/kg/day MP), MP-Low Dose (4/10 mg/kg/day MP), and control (water only). After 13 weeks, the femoral bones were assessed using calliper measurements, dual-energy X-ray absorptiometry, and biomechanical evaluation. The total femur volume, cartilage volume, growth zone volume, and volume fractions were determined using the Cavalieri method. Immunohistochemical analyses were conducted using alkaline phosphatase and anti-calpain antibody staining. Rats exposed to MP exhibited significant reductions in weight gain, femoral growth, bone mineralization, and biomechanical integrity compared to the control group. The total femoral volume of MP-treated rats was significantly lower than that of the control group. The MP-High Dose group showed significantly higher ratios of total cartilage volume/total femoral volume and total growth zone volume/total femoral volume than the other groups. Immunohistochemical evaluation of the growth plate revealed reduced osteoblastic activity and decreased intracellular calcium deposition with chronic MP exposure. The possible mechanism of MP-induced skeletal growth retardation may involve the inhibition of intracellular calcium deposition in chondrocytes of the hypertrophic zone in the growth plate. In this way, MP may hinder the differentiation of cartilage tissue from bone tissue, resulting in reduced bone growth and mineralization.
... For all sections, the parameters asf and ssf were set to 30% and 33.3%, respectively, while tsf was measured individually in each section]. The accuracy of the method was evaluated using Gundersen's m1 coefficient of error (CE) (Gundersen & Jensen, 1987), which amounted to 0.009 (indicative of very high accuracy). ...
The dendritic arbour of striatal projection neurons (SPNs) is the primary anatomical site where dopamine and glutamate inputs to the basal ganglia functionally interact to control movement. These dendritic arbourisations undergo atrophic changes in Parkinson's disease. A reduction in the dendritic complexity of SPNs is found also in animal models with severe striatal dopamine denervation. Using 6‐hydroxydopamine (6‐OHDA) lesions of the medial forebrain bundle as a model, we set out to compare morphological and electrophysiological properties of SPNs at an early versus a chronic stage of dopaminergic degeneration. Ex vivo recordings were performed in transgenic mice where SPNs forming the direct pathway (dSPNs) express a fluorescent reporter protein. At both the time points studied (5 and 28 days following 6‐OHDA lesion), there was a complete loss of dopaminergic fibres through the dorsolateral striatum. A reduction in dSPN dendritic complexity and spine density was manifest at 28, but not 5 days post‐lesion. At the late time point, dSPN also exhibited a marked increase in intrinsic excitability (reduced rheobase current, increased input resistance, more evoked action potentials in response to depolarising currents), which was not present at 5 days. The increase in neuronal excitability was accompanied by a marked reduction in inward‐rectifying potassium (Kir) currents (which dampen the SPN response to depolarising stimuli). Our results show that dSPNs undergo delayed coordinate changes in dendritic morphology, intrinsic excitability and Kir conductance following dopamine denervation. These changes are predicted to interfere with the dSPN capacity to produce a normal movement‐related output.
... Microphotographs at 10x were obtained with a confocal microscope, and then processed using the stitching plugin in FIJI/ImageJ 99 in order to have the whole nucleus on a single microphotograph. All the sections in a 1/6 subseries containing the region of interest were analyzed and the areas estimated using Cavalieri's principle 109 . To obtain the volume of the CA1 region, we multiplied the area by the thickness of the slice (50-µm) and the number of series. ...
Erythropoietin (EPO) aids in rectifying hippocampal transcriptional networks and synaptic structures of pyramidal lineages, thereby mitigating mood and cognition-associated disorders. An imminent conundrum is how EPO restores synapses by involving interneurons. By analyzing ∼ 12,000 single-nuclei transcriptomic data, we generated a comprehensive molecular atlas of hippocampal interneurons, resolved into 15 interneuron subtypes. Next, we studied molecular alterations upon recombinant human (rh)EPO and saw that gene expression changes relate to synaptic structure, trans-synaptic signaling and intracellular catabolic pathways. Putative ligand-receptor interactions between pyramidal and inhibitory neurons, regulating synaptogenesis, are altered upon rhEPO. An array of in/ex vivo experiments confirms that specific interneuronal populations exhibit reduced dendritic complexity, synaptic connectivity, and changes in plasticity-related molecules. Metabolism and inhibitory potential of interneuron subgroups are compromised, leading to greater excitability of pyramidal neurons. To conclude, improvement by rhEPO of neuropsychiatric phenotypes may partly owe to restrictive control over interneurons, facilitating re-connectivity and synapse development.
... For NeuN-stained brain sections, slides were scanned with an Aperio Scanscope AT Turbo microscope (Leica Biosystems) at ×20 magnification under the resolution of 0.50 μm/pixel, and analysed using Aperio Image Scope software. Estimation of volume was done using Cavalieri estimator based on the area of LGN in equally spaced sections throughout the anteroposterior extent of this nucleus (Atapour et al., 2017;Gundersen & Jensen, 1987). For neuronal density, counting frames (150×100μm 2 ) were placed systematically on each LGN section covering both degenerated and undegenerated sectors. ...
Lesions of the primary visual cortex (V1) cause retrograde neuronal degeneration, volume loss and neurochemical changes in the lateral geniculate nucleus (LGN). Here we characterise the timing of these processes, by comparing the LGN of adult marmoset monkeys following various recovery times after unilateral V1 lesions. Observations in NeuN-stained sections obtained from animals with very short (3 days) recoveries showed that the volume and neuronal density in the LGN ipsilateral to the lesions were similar to those in the contralateral hemispheres. However, neuronal density in the LGN lesion projection zones (LPZ) dropped rapidly thereafter, with a loss of ~50% of the population occurring within a month of the lesions. This level of neuronal loss was then sustained for the remainder of the range of recovery times, up to >3 years post-lesion. In comparison, shrinkage of the LGN progressed more gradually, not reaching a stable value until 6 months post lesion. We also determined the time course of the expression of the calcium-binding protein calbindin (CB) in magnocellular (M) and parvocellular (P) layer neurons, a recently described form of neurochemical plasticity triggered by V1 lesions. We found that CB expression could be detected in surviving M and P neurons as early as 1 month after a lesion, with the percentage of neurons showing this neurochemical phenotype showing subtle changes over 6 months. Our study shows that there is a limited time window for any possible interventions aimed at reducing secondary neuronal loss in the visual afferent pathways following damage to V1.
... We carried out stereology using StereoInvestigator software (MBF Bioscience, Williston, VT, USA) and estimated the numbers of OSNs in the olfactory epithelium and mitral cells in the olfactory bulb using the optical fractionator technique (Mouton, 2011;West et al., 1991). After determining the first and last sections that contained the structure of interest, we selected eight-to-12 intervening sections in a systematic-random fashion (i.e., random starting section with evenly spaced sections that spanned the entire bulb thereafter) for stereological analysis (Gundersen et al., 1999;Gundersen & Jensen, 1987;Mouton, 2011). We traced the outline of the olfactory epithelium or mitral cell layer in each slice at 40Â magnification using a Wacom computer screen that showed a live feed from the video camera. ...
Comparative studies are a common way to address large‐scale questions in sensory biology. For studies that investigate olfactory abilities, the most commonly used metric is olfactory bulb size. However, recent work has called into question the broad‐scale use of olfactory bulb size. In this paper, we use three neuroanatomical measures with a more mechanistic link to olfactory function (number of olfactory sensory neurons (OSNs), number of mitral cells (MCs), and number of glomeruli) to ask how species with different diets may differ with respect to olfactory ability. We use phyllostomid bats as our study system because behavioral and physiological work has shown that fruit‐ and nectar‐feeding phyllostomids rely on odors for detecting, localizing, and assessing potential foods, while insect‐eating species do not. Therefore, we predicted that fruit‐ and nectar‐feeding bats would have larger numbers of these three neuroanatomical measures than insect‐eating species. In general, our results supported the predictions. We found that fruit‐eaters had greater numbers of OSNs and glomeruli than insect‐eaters, but we found no difference between groups in number of MCs. We also examined the allometric relationship between the three neuroanatomical variables and olfactory bulb volume, and we found isometry in all cases. These findings lend support to the notion that neuroanatomical measures can offer valuable insights into comparative olfactory abilities, and suggest that the size of the olfactory bulb may be an informative parameter to use at the whole‐organism level.
... These point densities were determined according to a pilot study. The appropriate coefficient of error (≤0.05) and coefficient of variation (≤0.2) values were estimated using a formula [Gundersen and Jensen, 1987]. The volume estimation of the components and the whole of the structure was performed by using point-counting grids placed on the cross-sectional images on the PC (shown in Fig. 1). ...
Momordica charantia (MC) is a traditional plant widely used since ancient times for wound healing. This study evaluated its potential effects on tendon healing. Adult Male Wistar albino rats (n=32, 8 rats in each group) were anesthetized, and their Achilles tendons were prepared for surgical procedures. Group 1 (Cont= control group) were not subjected to any surgery and were used as a control group for baseline values. Group 2 (PR= primary repair group) underwent primary repair (PR) with a monofilament suture after a full-thickness incision of the Achilles tendon. A full-thickness incision was also made to the Achilles tendon of Group 3 (CT=collagen tube administered group), followed by PR and collagen tube insertion. In Group 4 (MC= Momordica charantia-administered group), 1 ml of MC extract was applied locally on the collagen tube in addition to the surgical procedure applied to Group 3. The Achilles tendons were excised on the postoperative 40th day and examined stereologically, histologically, and bioinformatically. Data showed that the total volume of the collagen fibers was higher in MC and CT groups than in the PR group. The total volume of the tendon was decreased in MC and CT groups than in the Cont group. The ratios between the volumes of the collagen fibers and total tendon in the MC and CT groups were significantly different from PR; but not different from the Cont group. Additionally, MC improved tenoblastic activity, collagen production, and neovascularization. Bioinformatic interactions showed that the proteases of MC could trigger the signals playing a role on vasculogenesis, reducing inflammation, and contributing to tenoblast activation and collagen remodeling. MC extract ameliorates the healing of injured tendon and can provide satisfactory tendon repair. Further works are recommended to explore the healing capacity of MC.
Precise and unbiased quantitative estimations are often necessary in biomedical sciences. Stereological procedures, which were first applied in the 1970s, are widely used nowadays. Our study assessed a number of quantitative estimators to characterize a well-defined human brain structure, the caudate nucleus (CN), using magnetic resonance (MR) and histological images, i.e., CN volume and number and volume of neurons in the CN. The study emphasizes the importance of precise sampling of the human CN, for which well-known stereological tools such as the Cavalieri, disector, nucleator, and point-sampled intercept methods were used. The article offers a detailed description of the process followed to obtain simple, exact, and punctual data—a CN volume of 3.9 cm3, a total of 234 · 106 neurons, and a neuronal volume of roughly 1200 μm3. It also provides guidelines and describes conditions and limitations for applying these stereological methods.
Background: Design-based (unbiased) stereology is considered the best practice for quantifying morphometric parameters of stained biological structures in tissue sections. These methods use systematic random sampling and unbiased geometric probes to ensure that estimates of stereology parameters converge on the expected (true) values as tissue sampling proceeds. A critical caveat is that the practical applications of these theoretically unbiased methods to biological tissue can confound the accuracy of stereology estimates.
New Method: Here, we show that calculations based on block advance, i.e., the instrument setting, on sectioning partially processed tissue can introduce substantial nonrandom error into stereology estimates of total region volume (\( \hat{V_{\mathrm{Ref}}} \)) by Cavalieri point-counting and total cell number (\( \hat{N_{\mathrm{Cell}}} \)) by disector-based methods.
Comparison with Existing Method: We provide proof that the use of block advance produces a similar error for both \( \hat{N_{\mathrm{Cell}}} \) calculated by the volume-based two-stage (NV · \( \hat{V_{\mathrm{Ref}}} \)) approach and fraction-based methods. Finally, a formula is given for removing this error from \( \hat{V_{\mathrm{Ref}}} \) and \( \hat{N_{\mathrm{Cell}}} \)using the final post-processing section thickness.
Volume estimations are crucial for many neuroscience studies, allowing the evaluation of changes in the size of brain areas that may have relevant functional consequences. Classical histological methods and modern human brain imaging techniques rely on obtaining physical or digital sections, with a known thickness, of the organ to be analyzed. This “slicing” strategy is associated with an ineludible loss of information about the three-dimensional organization of the analyzed structures, especially affecting the precision of volumetric measurements. To overcome this problem, several methods have been developed. One of the most commonly used approaches for volume estimation is the classical Cavalieri’s method. Within this book chapter, we provide first an overview of Cavalieri’s method and propose a new one, named the truncated cone shape (TCS) method, for the estimation of volumes from tissue sections. Second, we compare the accuracy of both methods using computer-generated objects of different shapes and sizes. We conclude that, more frequently, the TCS method provides a better estimate of real volumes than Cavalieri’s method. And third, we describe a protocol to estimate volumes using a self-developed and freely available tool for ImageJ: VolumestJ (https://github.com/Jorvalgl/VolumestJ). This new tool helps to implement both Cavalieri’s and TCS methods using digital images of tissue sections. We consider that VolumestJ will facilitate the labor of researchers interested in volume estimations.
The isotropic virtual planes (IVP) method is a global spatial sampling approach for length estimation. Thanks to computer-assisted microscopy, IVP can be applied to thick sections oriented in any particular direction, as the stereological probe is randomly rotated inside the section and, therefore, avoids the possible bias that might occur when structures with a given preferred orientation are analyzed. We have applied this stereological tool to estimate axonal length in two different experimental/methodological approaches: (1) axons from populations of projection neurons and (2) axons from single projection neurons, visualized in mice or human tissues. Our results demonstrate that the IVP is an accurate stereological tool that can be used to estimate the axonal length of single or groups of neurons. In the case of single neurons, the IVP allows collecting these quantitative data efficiently, preventing the need to reconstruct the entire cell’s structure manually. The fact that this method is valid to analyze groups of neurons, whose axons provide terminal arborizations that can be either scattered in broad brain regions or restricted to specific brain areas, makes the IVP a suitable tool to be applied in connectomic experiments performed in either small brains, as in rodents, or primates, where the reconstruction of the entire axon is a tedious and time-consuming task. The analysis of the axonal length is of great importance since it is proportional to the number of synapses the axon establishes with its postsynaptic targets, and it is affected by some pathological conditions.
FiberLean microfibrillated cellulose (MFC) was assessed against the recent definition of a nanomaterial published by the European Union. FiberLean MFC is made by mechanical grinding with the aim of fibrillating traditional wood pulp fibres to generate a highly networked high surface area structure capable of reinforcing a matrix as a filler or modifying the rheology of liquids and gels. The networked structure is complex; consisting of a broad range of fibre sizes which are challenging for any one characterisation technique to accurately represent. Scanning electron microscopy allows investigators to identify the boundaries of discrete complex fibre structures; the challenge is to represent the whole fibre size range. In this study a method is presented for the unbiased quantification of fibre width size distribution from systematic uniform random sampled high-resolution areas (164 µm \(\times\) 164 µm) using automated scanning electron microscope software. From such images the boundaries of both large- and small-scale fibres could be identified and measured, thus represent the complete size range. The resulting size distribution of FiberLean MFC generated using this method does not meet the criteria specified in the EU definition of a nanomaterial.
This study explores the multifaceted influence of litter size, maternal care, exercise, and aging on rats' neurobehavioral plasticity and dentate gyrus microglia dynamics. Body weight evolution revealed a progressive increase until maturity, followed by a decline during aging, with larger litters exhibiting lower weights initially. Notably, exercised rats from smaller litters displayed higher body weights during the mature and aged stages. The dentate gyrus volumes showed no significant differences among groups, except for aged sedentary rats from smaller litters, which exhibited a reduction. Maternal care varied significantly based on litter size, with large litter dams showing lower frequencies of caregiving behaviors. Behavioral assays highlighted the detrimental impact of a sedentary lifestyle and reduced maternal care/large litters on spatial memory, mitigated by exercise in aged rats from smaller litters. The microglial dynamics in the layers of dentate gyrus revealed age-related changes modulated by litter size and exercise. Exercise interventions mitigated microgliosis associated with aging, particularly in aged rats. These findings underscore the complex interplay between early-life experiences, exercise, microglial dynamics, and neurobehavioral outcomes during aging.
Diabetes mellitus is a group of chronic metabolic conditions, all of which are characterized by elevated blood glucose levels resulting from the body's inability to produce insulin or resistance to insulin action, or both. Insulin is the main hormone that regulates glucose production and use by body tissues and organs. Diabetes affects all systems in the body, including the liver. Streptozotocin is an antibiotic that performs pancreatic β cell destruction and is widely used to induce experimental diabetes. In this study; It was aimed to calculate the volume and cell numbers of the morphometric changes caused by streptozotocin-induced diabetes in the rat liver using stereological methods. Twenty adult male rats were used in the study. Four groups were created randomly. Blood glucose values of all groups were measured. Diabetes was induced by administering different doses of streptozotocin (55 mg/kg and 65 mg/kg). After 48 hours, blood glucose values were measured again. Four weeks later, liver tissues of all rats from the control and experimental groups were taken under anesthesia and fixed in 10% formaldehyde. The fixed liver tissues were divided into equal parts by physical fractionation. It was fixed in paraffin after passing through dehydration stages. Histological sections were prepared. It was stained with Hematoxylin-Eosin, examined under a light microscope and photographed. The total volume of the liver was calculated using the Cavalieri Principle and the number of hepatocytes was calculated with the unbiased counting frame of the dissector method. In the diabetes-related groups, the increase in liver volume and cell number was significant compared to the control group (p<0.05). As a result, streptozotocin injection and the resulting diabetes lead to an increase in liver volume and hepatocyte numbers.
Purpose
A lesion to primary visual cortex (V1) in primates can produce retrograde transneuronal degeneration in the dorsal lateral geniculate nucleus (LGN) and retina. We investigated the effect of age at time of lesion on LGN volume and retinal ganglion cell (RGC) density in marmoset monkeys.
Methods
Retinas and LGNs were obtained about 2 years after a unilateral left-sided V1 lesion as infants (n = 7) or young adult (n = 1). Antibodies against RBPMS were used to label all RGCs, and antibodies against CaMKII or GABAA receptors were used to label nonmidget RGCs. Cell densities were compared in the left and right hemiretina of each eye. The LGNs were stained with the nuclear marker NeuN or for Nissl substance.
Results
In three animals lesioned within the first 2 postnatal weeks, the proportion of RGCs lost within 5 mm of the fovea was ∼twofold higher than after lesions at 4 or 6 weeks. There was negligible loss in the animal lesioned at 2 years of age. A positive correlation between RGC loss and LGN volume reduction was evident. No loss of CaMKII-positive or GABAA receptor-positive RGCs was apparent within 2 mm of the fovea in any of the retinas investigated.
Conclusions
Susceptibility of marmoset RGCs to transneuronal degeneration is high at birth and declines over the first 6 postnatal weeks. High survival rates of CaMKII and GABAA receptor-positive RGCs implies that widefield and parasol cells are less affected by neonatal cortical lesions than are midget-pathway cells.
Bacterial biofilms have a complex and heterogeneous three-dimensional architecture that is characterized by chemically and structurally distinct microenvironments. Confocal microscopy-based pH ratiometry and fluorescence lectin-binding analysis (FLBA) are well-established methods to characterize pH developments and the carbohydrate matrix architecture of biofilms at the microscale. Here, we developed a combined analysis, pH-FLBA, to concomitantly map biofilm pH and the distribution of matrix carbohydrates in bacterial biofilms while preserving the biofilm microarchitecture. As a proof of principle, the relationship between pH and the presence of galactose- and fucose-containing matrix components was investigated in dental biofilms grown with and without sucrose. The pH response to a sucrose challenge was monitored in different areas at the biofilm base using the ratiometric pH-sensitive dye C-SNARF-4. Thereafter, the fucose- and galactose-specific fluorescently labeled lectins Aleuria aurantia lectin (AAL) and Morus nigra agglutinin G (MNA-G) were used to visualize carbohydrate matrix components in the same biofilm areas and their immediate surroundings. Sucrose during growth significantly decreased biofilm pH (P < 0.05) and increased the amounts of both MNA-G- and AAL-targeted matrix carbohydrates (P < 0.05). Moreover, it modulated the biofilm composition towards a less diverse community dominated by streptococci, as determined by 16S rRNA gene sequencing. Altogether, these results suggest that the production of galactose- and fucose-containing matrix carbohydrates is related to streptococcal metabolism and, thereby, pH profiles in dental biofilms. In conclusion, pH-FLBA using lectins with different carbohydrate specificities is a useful method to investigate the association between biofilm pH and the complex carbohydrate architecture of bacterial biofilms.
IMPORTANCE
Biofilm pH is a key regulating factor in several biological and biochemical processes in environmental, industrial, and medical biofilms. At the microscale, microbial biofilms are characterized by steep pH gradients and an extracellular matrix rich in carbohydrate components with diffusion-modifying properties that contribute to bacterial acid–base metabolism. Here, we propose a combined analysis of pH ratiometry and fluorescence lectin-binding analysis, pH-FLBA, to concomitantly investigate the matrix architecture and pH developments in microbial biofilms, using complex saliva-derived biofilms as an example. Spatiotemporal changes in biofilm pH are monitored non-invasively over time by pH ratiometry, while FLBA with lectins of different carbohydrate specificities allows mapping the distribution of multiple relevant matrix components in the same biofilm areas. As the biofilm structure is preserved, pH-FLBA can be used to investigate the in situ relationship between the biofilm matrix architecture and biofilm pH in complex multispecies biofilms.
In vitro studies have shown that Wharton's jelly mesenchymal stem cells (WJ-MSCs) can cross umbilical and uterine endothelial barriers and upregulate endothelial junctional integrity from sub-endothelial niches. This pericytic behaviour may be lost in pregnancies complicated by gestational diabetes (GDM), where increased vascular permeability and junctional disruption are reported. The aim of this study was to investigate whether WJ-MSCs isolated from GDM pregnancies displayed any changes in morphology, proliferation, VEGF-A secretion, and their ability to influence paracellular junctional composition and permeability. WJ-MSCs were isolated from human umbilical cords from normal pregnancies (nWJ-MSCs; n=13) and those complicated by GDM (gWJ-MSCs); either diet-controlled (d-GDM; n=13) or metformin-treated (m-GDM; n=9). We recorded that 4-fold more WJ-MSCs migrated from m-GDM, and 2.5-fold from d-GDM cord samples compared to the normal pregnancy. gWJ-MSCs showed a less predominance of spindle-shaped morphology and secreted 3.8-fold more VEGF-A compared to nWJ-MSCs. The number of cells expressing CD105 (Endoglin) was higher in gWJ-MSCs compared to nWJ-MSCs (17%) at P-2. The tracer leakage after 24h across the HUVEC + gWJ-MSCs bilayer was 22.13% and 11.2% higher in the m-GDM and d-GDM, respectively HUVEC + nWJ-MSCs. Transfection studies with siRNAs that target Endoglin were performed in n-WJ-MSCs; transfected cells were co-cultured with HUVEC followed by permeability studies and VE-cadherin analyses. Loss of Endoglin also led to increased VEGF-A secretion, increased permeability and affected endothelial stabilization. These results reinforce the pericytic role of nWJ-MSCs to promote vascular repair and the deficient ability of gWJ-MSCs to maintain endothelial barrier integrity.
Amaç: Alkol toksik bir ajan olup, gün geçtikçe tüketimi artmaktadır. Tüm doku ve organlar üzerinde zararlı etkileri mevcut olan alkolün, çalışmamızda dalak üzerinde oluşturduğu etkileri stereolojik ve immunohistokimyasal yöntemlerle incelemeyi amaçladık. Materyal ve Metot: Çalışma için 13 adet Wistar albino cinsi erkek sıçan alındı. Rastgele Kontrol (6), Etanol (7) olarak 2 gruba ayrıldı. Etanol grubuna oral yolla su yerine %6,4 (v/v) dozunda etanol verildi. Kontrol grubuna herhangi bir uygulama yapılmadı. Uygulamalara 18 gün devam edildi. Deney sonunda anestezi altında dalak dokusu çıkarıldı. Rutin ışık mikroskopik histolojik takip prosedürü ve immunohistokimyasal boyama için doku hazırlık aşamaları uygulandı, parafine gömüldü. Akabinde 5 µm kalınlığında ilki rastgele olmak üzere her 40. kesit alınarak, ortalama 10 kesit alındı. Alınan kesitler Hematoksilen & Eosinle boyandı. Stereolojik ölçümde Cavalieri prensibinin modifiye metodu kullanıldı. Shtereom 1.5 version paket programında verilen noktalı alan cetveliyle total doku volüm oranları ölçüldü. Doku kesitlerine Kollajen Tip IV ve Fibronektin immunohistokimyasal boyama uygulandı. İmmunohistokimyasal ve stereolojik değerlendirmeler yapıldı. Sonuçlar istatistiki olarak karşılaştırıldı. Bulgular: Stereolojik ölçümlerde dalak total volümü ve kırmızı pulpa volümünün etanol grubunda kontrol grubuna göre artmış olduğu ve istatistiki olarak anlamlı olduğu tespit edildi (p0.05). Kollajen Tip IV ve Fibronektin’in immunuhistokimyasal değerlendirilmesinde kapsül ve trabeküller alanlarda kontrol grubuyla karşılaştırıldığında etanol grubunda tutulumunun ciddi oranda azalmış olduğu tespit edildi. Sonuç: Çalışmamızın stereolojik ve immunohistokimyasal bulgularıyla alkolün immun ve hematopoetik sistemin kavşak noktasında yer alan dalak üzerindeki olumsuz etkileri ortaya konuldu.
There is a significant need for additional therapy to improve outcomes for newborns with acute Hypoxic-ischemic (HI) encephalopathy (HIE). New evidence suggests that insulin could be neuroprotective. This study aimed to investigate whether intranasal insulin attenuates HI-induced brain damage and neurobehavioral dysfunction in neonatal rats. Postnatal day 10 (P10), Sprague-Dawley rat pups were randomly divided into Sham + Vehicle, Sham + Insulin, HI + Vehicle, and HI + Insulin groups with equal male-to-female ratios. Pups either had HI by permanent ligation of the right common carotid artery followed by 90 min of hypoxia (8% O2) or sham surgery followed by room air exposure. Immediately after HI or Sham, pups were given fluorescence-tagged insulin (Alex-546-insulin)/vehicle, human insulin (25 μg), or vehicle in each nare under anesthesia. Shortly after administration, widespread Alex-546-insulin-binding cells were detected in the brain, primarily co-localized with neuronal nuclei-positive neurons on double-immunostaining. In the hippocampus, phospho-Akt was activated in a subset of Alex-546-insulin double-labeled cells, suggesting activation of the Akt/PI3K pathway in these neurons. Intranasal insulin (InInsulin) reduced HI-induced sensorimotor behavioral disturbances at P11. InInsulin prevented HI-induced increased Fluoro-Jade C+ degenerated neurons, cleaved caspase 3+ neurons, and volume loss in the ipsilateral brain at P11. There was no sex-specific response to HI or insulin. The findings confirm that intranasal insulin provides neuroprotection against HI brain injury in P10 rats associated with activation of intracellular cell survival signaling. If further pre-clinical research shows long-term benefits, intranasal insulin has the potential to be a promising non-invasive therapy to improve outcomes for newborns with HIE.
The IntelliCage is an automated home-cage system that allows researchers to investigate the spontaneous behavior and learning abilities of group-housed mice. The IntelliCage enables us to increase the standardization and reproducibility of behavioral outcomes by the omission of experimenter–mouse interactions. Although the IntelliCage provides a less stressful environment for animals, standard IntelliCage protocols use controlled water access as the motivational driver for learning. To overcome possible water restrictions in slow learners, we developed a series of novel protocols based on appetitive learning, in which mice had permanent access to plain water but were additionally rewarded with sweetened water upon solving the task. C57BL/6NCrl female mice were used to assess the efficacy of these sweet reward-based protocols in a series of learning tasks. Compared to control mice tested with standard protocols, mice motivated with a sweet reward did equal to or better in operant performance and place learning tasks. Learning of temporal rules was slower than that in controls. When faced with a combined temporal x spatial working memory task, sweet-rewarded mice learned little and chose plain water. In a second set of experiments, the impact of environmental enrichment on appetitive learning was tested. Mice kept under enriched environment (EE) or standard housing (SH) conditions prior to the IntelliCage experiments performed similarly in the sweet-rewarded place learning task. EE mice performed better in the hippocampus-dependent spatial working memory task. The improved performance of EE mice in the hippocampus-dependent spatial working memory task might be explained by the observed larger volume of their mossy fibers. Our results confirm that environmental enrichment increases complex spatial learning abilities and leads to long-lasting morphological changes in the hippocampus. Furthermore, simple standard IntelliCage protocols could easily be adapted to sweet rewards, which improve animal welfare by removing the possibility of water restriction. However, complex behavioral tasks motivated by sweet reward-based learning need further adjustments to reach the same efficacy as standard protocols.
Testis is considered the main organ of the male reproductive system. Dogs are used as a suitable experimental model of testicular diseases in humans. From the veterinary aspect, several disorders have been reported to affect the testis in dogs. Thus, the objective of the present study was to investigate the morphometrical features of the dog testis using design‐based stereology. The testes of six male dogs were used. Isotropic, uniform random sections were obtained and processed for light microscopy. Testicular total volume and the fractional volume of the seminiferous tubules, interstitial tissue and germinal epithelium were measured using the Cavalieri's estimator and the point counting system. Germinal epithelial surface area was estimated using test lines, and total length of seminiferous tubules was analysed using the counting frames. The total volume of testis was calculated 13.64 ± 1.94 cm ³ . The relative volume fractions of the seminiferous tubules, interstitial tissue and germinal layer expressed as a percentage of total testicular volume were found to be 75.87 ± 6.11%, 23.68 ± 5.15% and 64.15 ± 4.82%, respectively. The surface area of the germinal layer was 915.25 ± 150.48 cm ² . The thickness of germinal layer was estimated to be 96.18 ± 10.72 μm. The total length of seminiferous tubules measured 290.8 ± 35.86 m. No statistical difference in investigated parameters was found between the left and right testes ( p > 0.05). Our data might contribute to the male reproductive knowledge, help develop experimental studies in this field and possibly lead to advancement in the diagnosis and treatment of testicular diseases in the dog.
Introduction
Neurogenesis within the dentate gyrus is thought to play an important role in cognitive processes such as reversal learning and pattern separation. The α7 nicotinic acetylcholine receptor (α7 nAChR) is expressed early in newly formed granule cells of the dentate gyrus, though its role in neurogenesis and related cognitive function is not fully understood.
Methods
To better characterize relevant function of α7 nAChRs, we performed unbiased stereology to quantify hippocampal granule cells, pyramidal cells, and total volume and used a touchscreen operant spatial discrimination/reversal task to test pattern separation in a global α7 nAChR knockout mouse line.
Results
The knockout resulted in an ≈22% reduction in granule cells and a ≈ 20% reduction in pyramidal cells in both sexes, with no change in total hippocampal volume. However, the knockout impaired performance in the touchscreen task for males only. The sex-dependent difference in behavioral, but not stereological, results suggest a divergence in the structure–function relationship in males versus females. Detailed analyses revealed males were more biased by the initial reversal contingency relative to females indicating a potential source of the sex-specific interaction with the loss of α7 nAChRs.
Discussion
These findings argue that the α7 nAChR plays a critical role in hippocampal development, not just granule cell neurogenesis, and plays a sex-dependent role in cognitive function.
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