Optical Fractionator Method Research Articles

Ontogenetic changes in the tyrosine hydroxylase immunoreactive preoptic area in the small-spotted catshark Scyliorhinus canicula (L., 1758) females: catecholaminergic involvement in sexual maturation.

The catecholaminergic component of the brain-pituitary-gonadal axis, which mediates the influence of external and internal stimuli on the central nervous system and gonad development in vertebrates, is largely unexplored in Chondrichthyes. We considered Scyliorhinus canicula (L., 1758) females as a model for this vertebrate's class, to assess the involvement of the catecholaminergic system of the brain in its reproduction. Along the S. canicula reproductive cycle, we characterized and evaluated differences in somata morphometry and the number of putative catecholaminergic neurons in two brain nuclei: the periventricular preoptic nucleus, hypothesized to be a positive control for ovarian development, and the suprachiasmatic nucleus, examined as a negative control. 16 S. canicula wild females were sampled and grouped in maturity stages (immature, maturing, mature, and mature egg-laying). The ovary was histologically processed for the qualitative description of maturity stages. Anti-tyrosine hydroxylase immunofluorescence was performed on the diencephalic brain sections. The immunoreactive somata were investigated for morphometry and counted using the optical fractionator method, throughout the confocal microscopy. Qualitative and quantitative research confirmed two separate populations of immunoreactive neurons. The modifications detected in the preoptic nucleus revealed that somata were more numerous, significantly smaller in size, and more excitable during the maturing phase but decreased, becoming slightly bigger and less excitable in the egg-laying stage. This may indicate that the catecholaminergic preoptic nucleus is involved in the control of reproduction, regulating both the onset of puberty and the imminent spawning. In contrast, somata in the suprachiasmatic nucleus grew in size and underwent turnover in morphometry, increasing the total number from the immature-virgin to maturing stage, with similar values in the more advanced maturity stages. These changes were not linked to a reproductive role. These findings provide new valuable information on Chondrichthyes, suggesting the existence of an additional brain system implicated in the integration of internal and environmental cues for reproduction.

COMPARISON OF STEREOLOGY METHODS FOR ASSESSING AGE-RELATED EFFECTS ON IMMUNOSTAINED BRAIN CELLS

Abstract The primary benefit of stereology methods is quantification of well-stained biological objects in tissue sections with the ability to adjust sampling intensity to achieve desired levels of precision. The advent of hand-crafted algorithms and artificial intelligence-based deep learning (DL) provides an opportunity for more standardized collection of stereology data with enhanced efficiency and higher reproducibility compared to state-of-the-art manual stereology. We contrasted and compared the performance of four manual, semi-automatic, and fully automatic approaches for generating data for total number of Neu-N immunostained neurons in neocortex (NCTX) in the mouse brain. The gold standard for these studies was manual counts using the state-of-the-art optical fractionator method on 3-D reconstructed serial z-axis image stacks through a known tissue volume (disector stacks). To allow for direct methodological comparisons on the same images, disector stacks were automatically converted into extended depth of field (EDF) images in which all neurons in the disector stack were imaged at each cell’s maximal plane of focus. Total number of Neu-N neurons on the same EDF images were counted by a fully automatic hand-crafted method [automatic segmentation algorithm (ASA)] and a semi-automatic method [ASA counts manually corrected for false positives and negatives]. All comparison counts were done using unbiased frames and counting rules with total counts of NeuN-immunostained neurons by the optical fractionator method. The results were comparable across methods with wide variations in throughput efficiency and inter-rater agreement. These results are discussed with respect to applications to experimental studies of brain aging, neuroinflammation and neurodegenerative disease.

A disector-based framework for the automatic optical fractionator

Stereology-based methods provide the current state-of-the-art approaches for accurate quantification of numbers and other morphometric parameters of biological objects in stained tissue sections. The advent of artificial intelligence (AI)-based deep learning (DL) offers the possibility of improving throughput by automating the collection of stereology data. We have recently shown that DL can effectively achieve comparable accuracy to manual stereology but with higher repeatability, improved throughput, and less variation due to human factors by quantifying the total number of immunostained cells at their maximal profile of focus in extended depth of field (EDF) images. In the first of two novel contributions in this work, we propose a semi-automatic approach using a handcrafted Adaptive Segmentation Algorithm (ASA) to automatically generate ground truth on EDF images for training our deep learning (DL) models to automatically count cells using unbiased stereology methods. This update increases the amount of training data, thereby improving the accuracy and efficiency of automatic cell counting methods, without a requirement for extra expert time. The second contribution of this work is a Multi-channel Input and Multi-channel Output (MIMO) method using a U-Net deep learning architecture for automatic cell counting in a stack of z-axis images (also known as disector stacks). This DL-based digital automation of the ordinary optical fractionator ensures accurate counts through spatial separation of stained cells in the z-plane, thereby avoiding false negatives from overlapping cells in EDF images without the shortcomings of 3D and recurrent DL models. The contribution overcomes the issue of under-counting errors with EDF images due to overlapping cells in the z-plane (masking). We demonstrate the practical applications of these advances with automatic disector-based estimates of the total number of NeuN-immunostained neurons in a mouse neocortex. In summary, this work provides the first demonstration of automatic estimation of a total cell number in tissue sections using a combination of deep learning and the disector-based optical fractionator method.

The effects of different herbals on the rat hippocampus exposed to electromagnetic field for one hour during the prenatal period

The purpose of this study was to highlight the possible effects on the hippocampus of the electromagnetic field (EMF) emitted by mobile phones, and to investigate whether these potential effects can be reduced using various antioxidant substances. Twenty-seven female Wistar albino rats were divided into nine equal groups, each containing three pregnant rats aged 8–10 weeks and weighing 200–250 gr. The EMF groups were exposed to 900 Megahertz (MHz) EMF for 1 h (hr) a day for 21 days. No EMF exposure was applied to the Cont and also the groups given only Garcinia kola (GK), Momordica charantia (MC), and thymoquinone (TQ). The Sham group was kept in the polycarbonate EMF exposure system, but was not exposed to EMF. Four weeks after birth, rat pups were subjected to behavioural tests. Brain tissue samples were evaluated using histological, stereological, functional, and immunohistochemical methods. The numbers of pyramidal neurons in the rat cornu ammonis (CA) were determined using the optical fractionator method. Superoxide dismutase (SOD) and catalase (CAT) enzyme activities in the blood samples were also evaluated. The analysis data indicated that total pyramidal neuron numbers were decreased significantly in the CA of the EMF (1 hr) group (p < 0.01). Our results also showed that the protective effect of MC was more potent than that of the other antioxidant substances (p < 0.01). A 900 MHz EMF can cause deleterious changes in the brain. It can also be suggested that GK, MC and TQ are capable of reducing these adverse effects.

Stereological analysis of hippocampus in rat treated with chemotherapeutic agent oxaliplatin.

Oxaliplatin (OX) has been widely used for treatment of colorectal and other cancers. Adverse effect of OX and other anticancer agents on cognition have been reported, but studies on the effects of chemotherapy on brain structure are scarce. This study describes the morphometrical features of the hippocampus structures in rat following OX treatment using design-based stereological methods. Ten male Wistar rats were randomised into two groups. The rats from OX group received 2.4 mg/kg OX in vehicle for 5 consecutive days every week for 2 weeks intraperitoneally. Controls received vehicle only. Cavalieri's method and the optical fractionator method were used for volume and neuron estimation, respectively. Cavalieri's method was used to estimate volume and showed that the volume of the hippocampus was significantly decreased in OX group (31.84 ± ± 1.24 mm3) compared with the vehicle control group (36.95 ± 3.48 mm3). The optical fractionator method was used to estimate neuron number and showed that the number of neurons in dentate gyrus, cornu ammonis 1 and 3 in OX group (8.147 ± 2.84 × 105, 4.257 ± 0.59 × 105 and 2.133 ± 0.22 × 105, respectively) did not differ from those of vehicle control group (7.36 ± 1.42 × 105, 3.521 ± ± 0.54 × 105 and 1.989 ± 0.46 × 105, respectively). These findings suggested that OX treatment induces loss of hippocampal volume without neuronal loss which might help to clarify the mechanism by which OX affects cognition and to improve preventive treatment strategies.

Stereological Analysis of Early Gene Expression Using Egr-1 Immunolabeling After Spreading Depression in the Rat Somatosensory Cortex.

Early growth response-1 (Egr-1), defined as a zinc finger transcription factor, is an upstream master switch of the inflammatory response, and its expression can be used to investigate the spatial and temporal extent of inflammatory changes in the brain. Cortical spreading depression (CSD) is characterized as a slowly propagating (2–5 mm/min) depolarization wave through neurons and astrocytes in humans that contributes to migraines and possibly to other brain pathologies. In rodents, CSD can be induced experimentally, which involves unilateral depolarization that is associated with microglial and astrocyte responses. The impact of CSD on structures beyond the affected hemisphere has not been explored. Here, we used an optical fractionator method to investigate potential correlations between the number of and period of the eletrophysiologic record of CSD phenomena and Egr-1 expression in ipsilateral and contralateral hemispheres. CSD was elicited by the restricted application of a 2% KCl solution over the left premotor cortex. Electrophysiological events were recorded using a pair of Ag/AgCl agar-Ringer electrodes for 2 or 6 h. An optical fractionator was applied to count the Egr-1 positive cells. We found that CSD increased Egr-1 expression in a time- and event-dependent manner in the ipsilateral/left hemisphere. Although CSD did not cross the midline, multiple CSD inductions were associated with an increased number of Egr-1 positive cells in the contralateral/right hemisphere. Thus, repeated CSD waves may have far reaching effects that are more global than previously considered possible. The mechanism of contralateral expression is unknown, but we speculate that callosal projections from the depolarized hemisphere may be related to this phenomenon.

Dysmaturation of Somatostatin Interneurons Following Umbilical Cord Occlusion in Preterm Fetal Sheep.

IntroductionCerebral white matter injury is the most common neuropathology observed in preterm infants. However, there is increasing evidence that gray matter development also contributes to neurodevelopmental abnormalities. Fetal cerebral ischemia can lead to both neuronal and non-neuronal structural-functional abnormalities, but less is known about the specific effects on interneurons.ObjectiveIn this study we used a well-established animal model of fetal asphyxia in preterm fetal sheep to study neuropathological outcome. We used comprehensive stereological methods to investigate the total number of oligodendrocytes, neurons and somatostatin (STT) positive interneurons as well as 3D morphological analysis of STT cells 14 days following umbilical cord occlusion (UCO) in fetal sheep.Materials and MethodsInduction of asphyxia was performed by 25 min of complete UCO in five preterm fetal sheep (98–100 days gestational age). Seven, non-occluded twins served as controls. Quantification of the number of neurons (NeuN), STT interneurons and oligodendrocytes (Olig2, CNPase) was performed on fetal brain regions by applying optical fractionator method. A 3D morphological analysis of STT interneurons was performed using IMARIS software.ResultsThe number of Olig2, NeuN, and STT positive cells were reduced in IGWM, caudate and putamen in UCO animals compared to controls. There were also fewer STT interneurons in the ventral part of the hippocampus, the subiculum and the entorhinal cortex in UCO group, while other parts of cortex were virtually unaffected (p > 0.05). Morphologically, STT positive interneurons showed a markedly immature structure, with shorter dendritic length and fewer dendritic branches in cortex, caudate, putamen, and subiculum in the UCO group compared with control group (p < 0.05).ConclusionThe significant reduction in the total number of neurons and oligodendrocytes in several brain regions confirm previous studies showing susceptibility of both neuronal and non-neuronal cells following fetal asphyxia. However, in the cerebral cortex significant dysmaturation of STT positive neurons occurred in the absence of cell loss. This suggests an abnormal maturation pattern of GABAergic interneurons in the cerebral cortex, which might contribute to neurodevelopmental impairment in preterm infants and could implicate a novel target for neuroprotective therapies.

Automatic ground truth for deep learning stereology of immunostained neurons and microglia in mouse neocortex

Collection of unbiased stereology data currently relies on relatively simple, low throughput technology developed in the mid-1990s. In an effort to improve the accuracy and efficiency of these integrated hardware-software-digital microscopy systems, we have developed an automatic segmentation algorithm (ASA) for automatic stereology counts using the unbiased optical fractionator method. Here we report on a series of validation experiments in which immunostained neurons (NeuN) and microglia (Iba1) were automatically counted in tissue sections through a mouse neocortex. In the first step, a minimum of 100 systematic-random z-axis image stacks (disector stacks) containing NeuN- and Iba1-immunostained cells were automatically collected using a software-controlled 3 axes (XYZ) stage motor. In the second step, each disector stack was converted to an extended depth of field (EDF) image in which each cell is shown at its optimal plane of focus. Third, individual neurons and microglia were segmented and the regional minimas were extracted and used as seed regions for cells in a watershed segmentation algorithm. Finally, the unbiased disector frame and counting rules were used to make unbiased parameter estimates for neurons and microglia cells. The results for both NeuN neurons and Iba1 microglia were compared to manual counts made by a moderately experienced data collector from the same disector stacks. The final results show lower error rates for counts of Iba1-immunostained microglia cells than for quantifying NeuN-immunostained neurons, most likely due to less three-dimensional overlapping of Iba1 cells. We report that the throughput efficiency of using ASA to automatically annotate images of Iba1 microglia is more than five times greater than that of manual stereology counts of the same sections. Moreover, we show that ASA is significantly more accurate in counting microglia cells than a moderately experienced data collector (about 10% higher overall accuracy) when both were compared to counts by an expert neurohistologist. Thus, the ASA method applied to EDF images from disector stacks can be extremely useful to automate and increase the accuracy of cell counts, which could be especially helpful and cost-effective when expert help is not available. Another potential use of our ASA approach is to generate unsupervised ground truth as an efficient alternative to manual annotation for training deep learning models, as shown in our ongoing work.

Neuroprotective Effect of Echinacoside in Subacute Mouse Model of Parkinson's Disease.

Objective To study the protective effect of Echinacoside for 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induced dopaminergic (DA) neurons injury in the subacute mouse model of Parkinson's disease (PD) and to explore its mechanism of action. Methods We chose 10 weeks of healthy wild type C57BL/6 male mice, hypodermic MPTP 30 mg/kg/day, five days, to prepare PD subacute mouse model. Behavior indexes of open field test and pole test were applied to examine the function of ECH to PD subacute mice model of PD sample action. The effects of ECH on dopaminergic neurons and astrocyte were examined using Immunohistochemistry including tyrosine hydroxylase (TH) and glial fibrillary acidic protein (GFAP) expression. The total numbers of TH-positive neurons and GFAP-positive cells in the substantia nigra pars compacts (SNpc) and ventral tegmental area (VTA) were obtained stereologically using the optical fractionator method. Enzyme-linked immunosorbent assay (ELISA) method was used to detect the inflammatory cytokines in the serum, including TNF-α (Ttumor necrosis factor alpha) and IFN-γ (interferon gamma). Protein expressions of ionized calcium binding adaptor molecule 1 (IBA-1), TNF-α, Cleaved caspase-3, glial derived neurotrophic factor (GDNF), and phosphorylated and total extracellular signal-regulated kinase (p-ERK and ERK) in the anatomical region of substantia nigra (SN) were tested by protein immunoblot method (i.e., Western blotting). Results ECH reversed the reduction of total distance in open field test in MPTP-induced PD model mice (P < 0.01), shortened the return time and total time of PD subacute model mice in pole test (P < 0.01, P < 0.05), significantly reversed the reduction of TH positive neurons induced by MPTP (P < 0.05), and reduced the activation of astrocytes (P < 0.05). Meanwhile, ECH significantly inhibited the expression of IBA-1, Cleaved caspase-3, and TNF-α in midbrain of MPTP model mice (P < 0.05, P < 0.05, and P < 0.05) and upregulated the expression of GDNF (P < 0.05). And ECH lowered the level of TNF-α and IFN-γ in serum (P < 0.05, P < 0.05). Conclusion ECH has protective effects on the MPTP subacute model mice, its mechanism may be through inhibiting activation of microglia and astrocytes, reducing inflammatory reaction and promoting the secretion of neurotrophic factors, and eventually resulting in the reduction of the DA neurons apoptosis.

Automated Cell Counts on Tissue Sections by Deep Learning and Unbiased Stereology

In recent decades stereology-based studies have played a significant role in understanding brain aging and developing novel drug discovery strategies for treatment of neurological disease and mental illness. A major obstacle to further progress in a wide range of neuroscience sub-disciplines remains the lack of high-throughput technology for stereology analyses. Though founded on methodologically unbiased principles, commercially available stereology systems still rely on well-trained humans to manually count hundreds of cells within each region of interest (ROI). Even for a simple study with 10 controls and 10 treated animals, cell counts typically require over a month of tedious labor and high costs. Furthermore, these studies are prone to errors and poor reproducibility due to human factors such as subjectivity, variable training, recognition bias, and fatigue. Here we propose a deep neural network-stereology combination to automatically segment and estimate the total number of immunostained neurons on tissue sections. Our three-step approach consists of (1) creating extended-depth-of-field (EDF) images from z-stacks of images (disector stacks); (2) applying an adaptive segmentation algorithm (ASA) to label stained cells in the EDF images (i.e., create masks) for training a convolutional neural network (CNN); and (3) use the trained CNN model to automatically segment and count the total number of cells in test disector stacks using the optical fractionator method. The automated stereology approach shows less than 2% error and over 5× greater efficiency compared to counts by a trained human, without the subjectivity, tedium, and poor precision associated with conventional stereology.

The Effect of Agomelatine Treatment on Diabetes-Induced Cognitive Impairments in Rats: Concomitant Alterations in the Hippocampal Neuron Numbers.

Researches that are related to the central nervous system complications of diabetes have indicated higher incidence of cognitive disorders in patients. Since the variety of nootropic drugs used in clinics is limited and none of them consistently improves the outcomes, new and effective drug alternatives are needed for the treatment of diabetes-induced cognitive disorders. Based on the nootropic potential of agomelatine, the promising efficacy of this drug on cognitive impairments of diabetic rats was investigated in the current study. Experimental diabetes model was induced by streptozotocin. After development of diabetes-related cognitive impairments in rats, agomelatine (40 and 80 mg/kg) was administrated orally for two weeks. Cognitive performance was assessed by Morris water-maze and passive avoidance tests. Then, the total numbers of neurons in both dentate gyrus and Cornu Ammonis (CA) 1–3 subfields of the hippocampus were estimated by the optical fractionator method. Agomelatine treatment induced notable enhancement in the learning and memory performance of diabetic rats. Moreover, it reversed the neuronal loss in the hippocampal subregions of diabetic animals. Obtained results suggest that agomelatine has a significant potential for the treatment of diabetes-induced cognitive impairments. However, therapeutic efficacy of this drug in diabetic patients suffering from cognitive dysfunctions needs to be confirmed by further clinical trials.

Neuroprotective effect of rapamycin against Parkinson's disease in mice

To investigate the effect of rapamycin on Parkinson's disease (PD) and its underlying mechanism in mice. Sixty SPF adult male C57BL/6 mice were randomly divided into control group, model group and treatment group. 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine(MPTP) was used to induce Parkinson's disease in model group and treatment group. All mice were trained to cross the runway and were subjected to computer-assisted CatWalk. The numbers of tyrosine hydroxylase positive (TH+) neurons in the substantia nigra (SN) were assessed by unbiased stereology using the optical fractionator method; protein expression was detected by Western blot analysis; and glutathione peroxidase (GSH-Px), malondialdehyde (MDA) and superoxide dismutase (SOD) were detected by spectrophotometry. In the model group, a decrease in stride rate and an increase in variation of stance and swing were noted by CatWalk system (P<0.05 or P<0.01); the numbers of TH+ neurons decreased (P<0.01); expression of p-Akt, p-S6K, p-S6 and p-ULK increased (all P<0.01); LC3-Ⅱ/Ⅰ ratio decreased (P<0.01); MDA level was elevated while the levels of SOD and GSH-PX were reduced (all P<0.01). Compared with the model group, after treated with rapamycin, the abnormal behavior including the stride length, variation of stance and swing and step patterns induced by MPTP were all improved (P<0.05 or P<0.01); the numbers of TH+ neurons increased (P<0.05); the expression of p-Akt, p-S6K, p-S6 and p-ULK was suppressed (all P<0.01); the LC3-Ⅱ/Ⅰ ratio was upregulated (P<0.05); MDA level decreased while the levels of GSH-Px and SOD increased (all P<0.01). Rapamycin inhibits the activation of mTOR pathway, which contributes to protect against the loss of dopaminergic neurons and provide behavioral improvements in mice with Parkinson's disease. These results are partially related to the ability of rapamycin in inducing autophagy and reducing oxidative stress.

EARLY gestational exposure to isoflurane causes persistent cell loss in the dentate gyrus of adult male rats

BackgroundOur previous research showed that 4 h of maternal anesthesia with isoflurane during early gestation in pregnant rats leads to a deficit in spatial memory of adult male offspring. Because spatial memory is predominantly a hippocampally-mediated task, we asked the question if early gestational exposure to isoflurane affects development of the hippocampus in the offspring.FindingsPreviously behaviorally characterized adult male rats that were exposed to isoflurane during second trimester were sacrificed at 4 months of age (N = 10 and 13, control and isoflurane groups, respectively) for quantitative histology of hippocampal subregions. Sections were stained with cresyl violet and the total number of cells in the granular layer of the dentate gyrus and the pyramidal cell layer in the CA1 region were determined by a blinded observer using unbiased stereological principles and the optical fractionator method. Data were analyzed using Student’s t test; P < 0.05 was accorded statistical significance. Stereological examination revealed 9% fewer cells in the granular layer of the dentate gyrus of isoflurane-exposed adult rats compared to controls (1,002,122 ± 84,870 vs. 1,091,829 ± 65,791, respectively; Mean ± S.D, *P = 0.01). In contrast, there were no changes in the cell number in the CA1 region, nor were there changes in the volumes of both regions.ConclusionsOur results show that maternal isoflurane anesthesia in rodents causes region-specific cell loss in the hippocampus of adult male offspring. These changes may, in part, account for the behavioral deficits reported in adult rats exposed to isoflurane in utero.

Single dose ketamine injection affects activation of cells in the nucleus accumbens of prenatally stressed rats

Objectives: The nucleus accumbens (NAc) has recently been implicated in the pathophysiology of depression. In animals displaying depressive-like behavior following chronic stress exposure, glutamatergic transmission increases in the NAc. NMDA receptor antagonist ketamine act as an antidepressant, especially in treatment-resistant cases. In this study, we aimed to investigate the effects of single-dose ketamine application in the activation of cells in the NAc of prenatally stressed rats. Methods: Sprague-Dawley dams were exposed to immobilization stress during the last week of pregnancy for 3 hours. Male offspring were divided into four groups at postnatal day 40. Prenatal stress and control groups received either a single dose of ketamine (10 mg/kg, i.p.) or same doses of saline injections. Immediate gene activation was stimulated by forced swim for 6 minutes and assessed by c-Fos immunohistochemistry. The total number of activated cells in the core and shell subregions was estimated by optical fractionator method. Results: The total number of activated cells in the shell subregion significantly decreased in prenatally stressed rats. Ketamine administration reversed their activation level similar to those of control group. Although activation of cells did not change in the core region following prenatal stress, ketamine treatment enhanced the activation of cells in this region both control and prenatally stressed animals. Conclusion: These results suggest that prenatal stress influences the activation of NAc in a subdivision specific manner, but ketamine treatment could act on both core and shell regions by affecting glutamatergic limbic information that flows from shell to core subregion of the NAc.

Hemispheric differences in the number of parvalbumin-positive neurons in subdivisions of the rat basolateral amygdala complex

The amygdala is a bilateral temporal lobe brain region which plays an important role in emotional processing. Past studies on the amygdala have shown hemispheric differences in amygdalar processes and responses associated with specific pain and fear behaviors. Despite the functional differences in the amygdala, few studies have been performed to characterize whether anatomical differences exist between the left and right amygdala. Parvalbumin (PV) is a phenotypic marker for an inhibitory interneuronal population in cortical brain structures such as the basolateral amygdala complex (BLC). This study examined the number of PV-positive neurons in the left and right BLC of adult, male Long-Evans rats using unbiased stereology. Coronal sections through the rostral-caudal extent of the BLC were immunohistochemically-stained for PV and the optical fractionator method was used to obtain an unbiased estimate of the number of PV-positive neurons in subdivisions through the BLC. The lateral and basolateral amygdala divisions of the BLC were analyzed, were subdivided into the dorsolateral, ventrolateral and ventromedial and the posterior, anterior and ventral subdivisions, respectively. The results indicate that there are significantly more PV-positive neurons in the left basolateral amygdala compared to the right, with a significant difference specifically in the posterior subdivision. This difference in PV neuronal number could help explain the distinct hemispheric roles of the BLC in the behavioral processing following exposure to painful and fearful stimuli.

Effects of prenatal PPAR-γ agonist rosiglitazone exposure on rat hippocampus development in a time-dependent manner: A stereological and histopathological study.

Rosiglitazone is in the thiazolidinedione class of drugs used in the treatment of type 2 diabetes mellitus. It works as an insulin sensitizer by binding to the peroxisome proliferator-activated receptor gamma. We investigated the effects of prenatally administered rosiglitazone on pyramidal cell numbers and morphologies in the hippocampus at postnatal period using histochemical and stereological techniques, congenital morphological properties and the number of offspring in rats. Eighteen female rats were grouped into control (C), low-dose rosiglitazone (LDR) and high-dose rosiglitazone (HDR). LDR pregnant rats received 2 mg/kg/day of rosiglitazone via oral gavage during the first 16 days of the pregnancy. HDR rats received 5 mg/kg/day. The infants were grouped into newborn (NB), 4 week (4 W) and 12 week (12 W). A side from histopathologic and congenital assessments, stereological analyses were performed using the optical fractionator method. Congenital anomaly was not detected in any of the rosiglitazone treatment groups, and their number of offspring was similar to that of the C group. Stereological counts revealed a significant reduction in the number of hippocampal pyramidal cells in the C and LDR groups but not in the HDR group until birth to 12th week. When NB groups were compared, the number of pyramidal cells in the HDRNB group was less than those in the LDRNB and CNB groups. HDR affected apoptosis or the proliferation and maturation of progenitor cells to the pyramidal neuron during neurodevelopment in the hippocampus, whereas LDR did not adversely affect neuronal development and did not cause congenital anomalies.

Stereological Estimation of Dopaminergic Neuron Number in the Mouse Substantia Nigra Using the Optical Fractionator and Standard Microscopy Equipment.

In pre-clinical Parkinson's disease research, analysis of the nigrostriatal tract, including quantification of dopaminergic neuron loss within the substantia nigra, is essential. To estimate the total dopaminergic neuron number, unbiased stereology using the optical fractionator method is currently considered the gold standard. Because the theory behind the optical fractionator method is complex and because stereology is difficult to achieve without specialized equipment, several commercially available complete stereology systems that include the necessary software do exist, purely for cell counting reasons. Since purchasing a specialized stereology setup is not always feasible, for many reasons, this report describes a method for the stereological estimation of dopaminergic neuronal cell counts using standard microscopy equipment, including a light microscope, a motorized object table (x, y, z plane) with imaging software, and a computer for analysis. A step-by-step explanation is given on how to perform stereological quantification using the optical fractionator method, and pre-programmed files for the calculation of estimated cell counts are provided. To assess the accuracy of this method, a comparison to data obtained from a commercially available stereology apparatus was performed. Comparable cell numbers were found using this protocol and the stereology device, thus demonstrating the precision of this protocol for unbiased stereology.

Prolonged sleep deprivation decreases cell proliferation and immature newborn neurons in both dorsal and ventral hippocampus of male rats

Previous studies have indicated that sleep deprivation negatively affects hippocampal neurogenesis, which may explain the reason for the relation between sleep loss and depression. Increasing evidence indicates that the hippocampus is anatomically and functionally segregated along a dorsolateral (cognitive function)/ventromedial (control for mood and stress response) axis. Thus, the present study was conducted to elucidate regional differences in the adverse effects of sleep deprivation on hippocampal neurogenesis. Male Sprague-Dawley rats were subjected to sleep deprivation using the “platform on the water” method for 24- or 72-h. Quantification of hippocampal cell proliferation and immature newborn neurons was stereologically estimated using immunostaining with Ki-67 and doublecortin (DCX), respectively, by optical fractionator method. A consecutive three days of sleep deprivation significantly reduced the density of Ki-67- and DCX-immunopositive cells both in the dorsal and ventral hippocampal subgranular zone and the decrease in DCX-labeled cells was more pronounced in the ventral hippocampus than in dorsal region. Our results indicate that prolonged sleep deprivation decreases hippocampal cell proliferation and neurogenesis in both the dorsal and ventral dentate gyrus. Future studies will be needed to clarify the impact of sleep deprivation-induced decreases in hippocampal neurogenesis on the development of depression.

Neurovascular plasticity of the hippocampus one week after a single dose of ketamine in genetic rat model of depression.

Glutamatergic system and the structural plasticity hypothesis are principal components for rapid and sustained antidepressant effects of novel antidepressant therapeutics. This study represents the first investigation of the structural plasticity of the hippocampus as one of the main contributed mechanisms to the sustained anti-depressive effect of ketamine. Flinders Sensitive Line (FSL) and Flinders Resistant Line (FRL) rats were given a single intraperitoneal injection of ketamine (15 mg/kg) or saline 7 days before perfusion-fixed. The optical fractionator method was used to estimate the total number of neurons in the granular cell layer. Microvessel length in the molecular layer of DG was evaluated with global spatial sampling method. By use of the physical disector method, the number of synapses was estimated. The volume of the hippocampus was larger in the FRL-vehicle rats compared with FSL-vehicle group and in FSL-ketamine versus FSL-vehicle rats (P < 0.05). The number of non-perforated synapses was significantly higher in the FSL-ketamine versus FSL-vehicle group, (P = 0.01). A significant effect of ketamine on enhancement of the number of neurons in DG in FSL rats was observed (P = 0.01). The total length of the microvessels 1 week after ketamine treatment in the FSL rats significantly increased (P < 0.05). Our results indicate that neurovascular changes of hippocampus could be one of the possible mechanisms underlying the sustained antidepressant effect of ketamine by reversing alteration of the number of the excitatory synapses, neuronal number and length of the microvessels in the hippocampus. © 2016 Wiley Periodicals, Inc.

Apoptotic cell death and temporal expression of apoptotic proteins Bcl-2 and Bax in the hippocampus, following binge ethanol in the neonatal rat model.

Binge-like ethanol (EtOH) exposure during the early rat neonatal period results in acute cell loss in specific brain regions, but such acute cell death has not been well established in the hippocampus. Binge alcohol exposure can also result in protein expression changes in the cerebellum that could alter cell fate, but this has not been reported for the hippocampal subregions. This study investigates acute apoptotic cell death in hippocampal regions CA1, CA3, and dentate gyrus (DG) following a binge EtOH exposure on postnatal day (PN) 6, PN8, or PN6+8 and the alteration in pro- and anti-apoptotic proteins following a single EtOH binge on PN6. Apoptotic cell death was quantified 12hours after EtOH binge exposure using the optical fractionator method. Western blot analysis determined expression of pro-apoptotic Bax and anti-apoptotic Bcl-2, 12, 24, and 48hours after binge EtOH exposure on PN6. The Bcl-2:Bax ratio was used as a measure of vulnerability to apoptosis. Acute apoptosis increased significantly 12hours following PN6 or 8 EtOH exposure in CA1, CA3, and DG, but the magnitude of apoptotic cell death was significantly greater in CA1 than in CA3 and DG, which did not differ. Significant cell death was not detected when a PN8 EtOH exposure was preceded by exposure on PN6. Binge EtOH exposure on PN6 resulted in a significant increase in expression of Bcl-2 and the Bcl-2:Bax ratio in the CA1/DG region at 24hours after EtOH exposure on PN6. The Bcl-2:Bax ratio in the CA3 region was not altered. This study shows that repeated binge exposure does not have a cumulative effect on the magnitude of acute apoptotic cell death. This finding may be explained in part by changes in the Bcl-2:Bax ratio after a single binge EtOH exposure.