Implementation of Unbiased Stereology Method for Organ Volume Estimation using Image Processing

Stereology had been widely used around the globe for estimating the value of the biological structure. It interlinks the connection between the dimensions using a sample that is 2D for a quantification in 3D structure. Volume estimation using stereology method is the most common and basic quantification that shows an example of how stereology works. Stereology method has a drawback when it comes to a lot of samples, so it is common for a researcher to use the computer to help to quantify and to do repetitive work, which is known as computer-assisted stereology quantification. The purpose of this research is to develop software that can assist researchers using stereology method for estimating the volume of a sample object from its 2D digital images with point counting method. The software was built based on the manual procedure of volume estimation using stereology method performed by experienced researchers. The volume estimation using the software were then compared with the results of the manual procedure by the experts on the same samples, i.e., the kidney and trigeminal ganglion of a rat. The experimental results showed that the relative difference of the volume estimations between manual and computer assisted calculations were 0.93% for rat kidney sample and 10.81%. TA1 trigeminal ganglion sample. Based on the results, the software estimation can be used for further analysis

Pengaruh Ekstrak Etanol Pegagan (Centella asiatica ) Terhadap Volume Hippocampus Tikus (Rattus norvegicus) Pasca Stres Kronik

Background. Chronic stress caused by restraint stress induces an increase in corticosterone (glucocorticoid) that result decreased hippocampal vollume. Centella asiatica has long been used for various neurological disturbances in Southeast Asian countries. Aims. The research aims to investigate the effects of ethanol extracts of Centella Asiatica to prevent the decrease of hippocampal vollume after chronic stress. Methods. Thirty male adult rats (Sprague Dawley) with body weight of 250-350 gram was randomly subdivided into six groups of treatments: Nonstress control group was given 2% Pulvis Gummi Arabicum (PGA), Stress control groups( PGA 2 %), groups treated with fluoxetine 10mg/kgbw/day , and Ca150,  Ca300, Ca600 containing ethanol extracts of Centella asiatica with doses 150 mg/kgbw/day, 300 mg/kgbw/day, 600 mg/kgbw/day a, respectively followed by restraint stress.  After 21 days of stress, rats underwent Morris Water Maze test for 6 days, perfused, and hippocampus was collected for histological processing. Toluidine blue staining is used to asses the estimation of hippocampal volume. Results. The estimated hippocampal volume were 3.1512 ± 1.01 (nonstress), 2.4736 ± 0.10 (stress), 2.7018 ± 1.06 (Ca150), 2.7405 ± 1.19 (Ca300), 2.2678 ± 0.82  (Ca600) and 2.5818 ± 0.70 (Fluoxetine). Statistic test showed that there were no significant diferrence between stress control group and groups treated. Conclusion. There is no the significant effects of Ethanol extracts of Centella Asiatica to prevent the decrease of hippocampal vollume after chronic stress

Prefrontal cortex cell proliferation of adult rats after chronic stress treated with ethanolic extract of Centella asiatica (L) Urban.

The decrease of proliferation level of cells in several regions of adult brain is found afterstress exposure. One of such area is prefrontal cortex. Herbal medicine as antistresshas been used widely. Centella asiatica (L) Urban extract was reported to have potentcompounds to increase brain function. The objective of this study is to investigate theeffects of ethanolic extract C. asiatica on cell proliferation of the adult prefrontal cortexin rats (PFC) after chronic stress. Adult male Sprague-Dawley rats, weighing 260-390g were randomly assigned into six experimental groups, with five rats per group, i.e.Group 1 as nomal control without chronic stress, Group 2 as stress control, Group 3 aspositive control given fluoxetine, Group 4-6 as treatment groups given 150; 300 and 600of ethanolic extrac C. asiatica, respectively. Extract were administered orally to the ratsfollowing a period of restraint duration of 6 hours/day for 21 days. Bromodeoxyuridine(BrdU) immunohistochemistry was used to label the proliferated cells. Physical fractionatormethod was used to estimate the total number of proliferated cells. One-way analysis ofvariance (ANOVA) followed by Tukey post hoc test was used to evaluate the differencebetween groups. BrdU-labeled cells on medial prefrontal cortex were as follows:1715.3±1345.1 (Group 1), 2659.2±2250.6 (Group 2), 4077.4±2415.3 (Group 3),1784.1±908.3 (Group 4), 3056.6±4263.3 (Group 5), and 2153.4±2259.4 (Group 6).No significance difference between groups was observed (p>0.05). In conclusion, theadministration of ethanolic extract of C. asiatica does not influence cell proliferation onprefrontal cortex of rats after chronic stress

Moderate-intensity interval exercise but not high-intensity interval exercise improves the spatial memory of ovariectomized rats

Physical exercise exerts beneficial effects on the spatial learning and memory. High-intensity interval exercise (HIIE) has been proposed as a time-efficient physical exercise regimen. On the other hand, there were evidences that HIIE increased oxidative stress biomarkers and reduced antioxidant capacity, which resulted in oxidative damage. The present study aimed to investigate the effects of high-intensity interval exercise and moderate-intensity interval exercise on oxidative stress biomarkers and oxidative enzymes activity in the hippocampus and the spatial memory of ovariectomized rats. A total of 16 female Sprague Dawley rats aged 12 weeks were randomly assigned into 4 groups, i.e. the sham-operated (SO), ovariectomized without exercise (O), ovariectomized with high-intensity interval exercise (HIIE), and ovariectomized with moderate-intensity interval exercise (MIIE) groups. Rats of the exercise groups (HIIE & MIIE groups) performed 6 sessions of interval exercise per week for 6 weeks. The spatial memory of rats was measured using the Morris water maze procedure. The malondialdehyde (MDA) levels and activity of catalase (Cat) as well as glutathione peroxidase (GPx) in hippocampus were determined using spectrophotometry method. The spatial learning and memory retention of the moderate-intensity interval exercise group was significantly better than that of the high-intensity interval exercise group. The GPx activity of MIIE group was higher than any other groups. The SO group had the lowest hippocampal MDA level and highest Cat activity among groups. Moderate-intensity interval exercise reduces the ovariectomy induced-oxidative stress in the hippocampus and improves spatial learning and memory retention of ovariectomized rats

THE EFFECTS OF EARLY LIFE UNDERNUTRITION ON LONGEVITY AND ASPECTS OF AGEING OF THE RODENT BRAIN

Diet restriction has been widely acknowledged among gerontologists as a method to delay or retard ageing, and extend the lifespan of many organisms. A considerable and growing number of studies have been dedicated to reveal the effects of diet restriction on factors that may be involved in the ageing of a given organism. Despite this, a comprehensive explanation of the exact mechanisms that may be involved remain largely uncertain. The hypothesis that has gained the most support is the reduced oxidative damage hypothesis although even this hypothesis needs further research to confirm or deny its validity. The present project aimed to examine (1) the effects of relatively short periods of undernutrition at differing stages of early life (i.e. pre- and post-weaning) on the lifespan of mice, (2) the effects of these periods of undernutrition on the oxidative stress status of the brain of rodents, and (3) to investigate whether any alterations in this status correlate with changes at the cellular and behavioural levels in the animals. The oxidative stress parameters investigated included the levels of mRNA and activities of major anti-oxidant enzymes (i.e. manganese superoxide dismutase [MnSOD], copper/ zinc superoxide dismutase [Cu/ZnSOD], catalase, and glutathione peroxidase 1 [GPx 1]), as well as measures of reduced glutathione (GSH), and lipid peroxidation. Further studies estimated the effects of undernutrition during different stages of early life on the cell numbers in the hippocampal formation and on spatial and motor coordination behaviours. The present study found that there were no differences in the maximum, mean, and median lifespan between mice undernourished during either the pre- or postweaning periods and well-fed controls. However, some long-term changes in body weight and the reduced incidence of pathological disorders were observed in some of the undernourished groups of animals. There were some long-term alterations in the levels of mRNA of Cu/ZnSOD in both the forebrain and cerebellum of the mice and rats that had been undernourished during the pre-weaning period. In addition, there were some transient increases in the levels of mRNA of MnSOD in the cerebellum of the mice and catalase in the forebrain of the undernourished rats. These alterations in gene expression, however, did not correlate with the activity levels of the enzymes in most instances. This finding suggests that pre-weaning undernutrition may regulate the anti-oxidant enzymes at the transcriptional level differently from the posttranscriptional, translational, or post-translational levels. Another observation in this set of experiments was the long-lasting increase in the levels of GSH in the forebrain of mice following pre-weaning undernutrition. The exact significance of this change on the ageing process of an animal remains uncertain at present. Pre-weaning undernutrition did not affect the total number of CA2-CA3 pyramidal neurons of the hippocampus of the rats. However, such undernutrition did cause significant long-term alterations in the behavioural performance of the mice in the learning tasks used in this study. These effects on behaviour were not observed in mice undernourished during the post-weaning period. The significance of these results to the various hypotheses on the effects of diet restriction on an animals ageing is discussed in detail

Hasil Review "Turmeric (Curcuma longa L.) Extract may Prevent the Deterioration of Spatial Memory and the Deficit of Estimated Total Number of Hippocampal Pyramidal Cells of Trimethyltin-Exposed Rats."

Context: Protection of neurons from degeneration is an important preventive strategy for dementia. Much of the dementia pathology implicates oxidative stress pathways. Turmeric (Curcuma longa L.) contains curcuminoids which has anti-oxidative and neuro-protective effects. These effects are considered to be similar to those of citicoline which has been regularly used as one of standard medications for dementia. Objective: This study aimed at investigating the effects of turmeric rhizome extract on the hippocampus of trimethyltin (TMT)-treated Sprague-Dawley rats. Materials and methods: The rats were divided randomly into six groups, i.e., a normal control group (N); Sn group, which was given TMT chloride; Sn-Cit group, which was treated with citicoline and TMT chloride; and three Sn-TE groups, which were treated with three different dosages of turmeric rhizome extract and TMT chloride. Morris water maze test was carried out to examine the spatial memory. The estimated total number of CA1 and CA2–CA3 pyramidal cells was calculated using a stereological method. Results: The administration of turmeric extract at a dose of 200 mg/kg bw has been shown to prevent the deficits in the spatial memory performance and partially inhibit the reduction of the number of CA2–CA3 regions pyramidal neurons. Discussion: TMT-induced neurotoxic damage seemed to be mediated by the generation of reactive oxygen species and reactive nitrogen species. Turmeric extract might act as anti inflammatory as well as anti-oxidant agent. Conclusions: The effects of turmeric extract at a dose of 200 mg/kg bw seem to be comparable to those of citicoline. Keywords: Curcuma longa L.; trimethyltin; Morris water maze; pyramidal cell; stereolog

The number, size, and type of axons in rat subcortical white matter on left and right sides: A stereological, ultrastructural study

Abundant evidence indicates important functional differences between the two cerebral hemispheres of humans, although the cellular basis of these differences is unknown. A recent hypothesis proposes that these functional differences depend on differences between sides in the "repertoire" of axonal conduction delays for cortico-cortical axons. In morphological terms this corresponds to differences in caliber, or proportion, of myelinated versus unmyelinated axons. Several behavioural studies have indicated that cerebral asymmetry occurs in rodents, in which rigorous morphological analysis is possible. The hypothesis was therefore tested for the first time in adult male Wistar rats, using transmission electron microscopy and stereological methods. Subcortical white matter was compared between left and right sides in three regions (frontal, parietal, and occipital). The average caliber and numerical density of unmyelinated and myelinated axons was compared between sides and between regions. All data were corrected for shrinkage. No significant differences between sides were found in the average caliber of either type of axon in any region. The numerical density of either type of axon also yielded no significant differences between sides in any region. Significant differences were evident between regions in both caliber and numerical density of the two axonal types, and these quantitative data are reported. The proportion of unmyelinated axons in the lateral white matter was also higher than in previous studies of hemispheric white matter that studied the corpus callosum. The present study provides no evidence supporting the hypothesis that functional hemispheric specialization is due to differences in axonal number, caliber or type

Spatial Memory Disturbance Following Transient Brain Ischemia is Associated with Vascular Remodeling in Hippocampus

A number of studies have investigated the effects of ischemic injury on functional and cellular characteristics of hippocampus. There is only a limited study on vascular remodeling of it. The present study aimed at examining vascular remodeling in hippocampus and spatial memory disturbances after transient brain ischemia. Male Wistar rats were randomly divided into four groups, i.e. sham operated (SHAM), transient brain ischemia with 1 day reperfusion (IR1), 3 day reperfusion (IR3), and 10 days reperfusion (IR10) groups. Transient brain ischemia was induced by bilateral common carotid artery occlusion (BCCAO). The spatial memory test was performed using the Morris water maze (MWM) in SHAM and IR10 groups. The rats were euthanized at day 1, 3 or 10 after BCCAO depending on the groups. The mRNA expressions of SOD2, Bcl-2, NeuN, eNOS, endothelin-1 (ET-1), CD31, VE-cadherin and vascular remodeling of the hippocampus were examined. There were deteriorations of spatial learning ability in IR10 group. The percentages of SOD2 and Bcl-2, the expression of NeuN, decreased and the vascular remodeling was observed in the ischemic groups. The eNOS and CD31 expressions were less in IR10, the VE-cadherin expression was less in all ischemic groups than in SHAM group, while ET-1 expression in IR1 group was higher than any other groups. The spatial memory deterioration after BCCAO is associated with vascular remodeling in hippocampus, characterized by lumen narrowing and smooth muscle thickening of microvessels

Effects of treadmill exercise training on cerebellar estrogen and estrogen receptors, serum estrogen, and motor coordination performance of ovariectomized rats

Objective(s): The present study aims at examining the motor coordination performance, serum and cerebellar estrogen, as well as ERβ levels, of ovariectomized rats (as menopausal model) following regular exercise. Materials and Methods:Ten female Sprague Dawley ratsaged 12 weeks old were randomly divided into two groups; all of which underwent ovariectomy. The first group was treated with regular exercise of moderate intensity, in which the rats were trained to run on a treadmill for 60 min per day for 12 weeks. The second group served as control. Rotarod test was carried out before and after exercise treatment. All rats were euthanized thereafter, and blood and cerebellums of the rats were collected. The serum and cerebellar estrogen as well as cerebellar ERβ levels were measured using ELISA assays. Results: The number of falls in the rotarod task of the exercise group was significantly lower than that of control group. The cerebellar estrogen level of the exercise group was significantly higher than that of control group. Accordingly, there was a significantly negative correlation between the number of falls and cerebellar estrogen level in the exercise group. Conclusion:The present study shows that a lengthy period of regular exercise improves the cerebellar estrogen level and motor coordination performance in ovariectomized rats