Hüseyin Aslan

Caffeic acid phenethyl ester protects rabbit brains against permanent focal ischemia by antioxidant action: A biochemical and planimetric study

The present study was conducted to investigate whether caffeic acid phenethyl ester (CAPE), an active component of propolis extract, has a protective effect on brain injury after focal permanent cerebral ischemia, and to determine the possible antioxidant mechanisms. Cerebral infarction in adult male New Zealand rabbits was induced by microsurgical procedures producing right focal permanent middle cerebral artery occlusion (pMCAO). CAPE was administered to the treatment group after pMCAO at a dose of 10 micromol kg(-1) once a day intraperitoneally for 7 days. Neurological deficits were evaluated, using a modified six-point scale. Spectrophotometric assay was used to determine the contents of malondialdehyde (MDA), glutathione (GSH), catalase (CAT), nitric oxide (NO) and xanthine oxidase (XO). In the ipsilateral hemisphere, the infarct volume of the brain was assessed in brain slices stained with heamatoxylen and eosin. The results showed that treatment with CAPE significantly reduced the percentage of infarction in the ipsilateral hemisphere compared with the ischemia group. CAPE treatment significantly attenuated the elevation of plasma MDA, CAT and XO content (p<0.05), whereas it significantly increased the levels of plasma GSH and NO (p<0.05). Therefore, subacute CAPE administration plays a protective role in focal pMCAO due to attenuation of lipid peroxidation and its antioxidant activity. All of these findings suggest that CAPE provides neuroprotection against cerebral ischemia injury through its antioxidant action.

Determination of Oestrous Cycle of the Rats by Direct Examination: How Reliable?

For determination of the oestrous cycle in rats classical Papanicolaou technique has long been used successfully. Instead of using many stains in Papanicolaou, staining the vaginal secretions with only methylene blue has also been defined. Recently a new technique in which vaginal samples are directly examined under light microscope has been introduced. The aim of this study was to assess the reliability of this new technique by comparing it with the classical staining techniques. From 20 Wistar rats 60 vaginal samples were collected with a micropipette, three from each. Briefly, the vagina was flushed two to three times then the fluid was placed onto a glass slide. The fluid was equally distributed onto three glass slides. The glass slides were coded. Two samples were stained with Papanicoloau and methylene blue while the other one was examined directly. Determination of the phases of the oestrous cycle was made by the same histologist who was blinded to the groups and coding system. After determination of the oestrous phase in all samples, the results were compared and it was found that the results were matching. In conclusion, the same results can be obtained with the direct examination technique and this technique is reliable, so there is no need to use relatively time‐consuming, less practical and more expensive techniques such as Papanicolaou or methylene blue.

Effects of formaldehyde exposure on granule cell number and volume of dentate gyrus: a histopathological and stereological study.

The hippocampal formation is a complex region of the brain related to memory and learning. The purpose of the present study was to determine whether exposure of neonatal rats to formaldehyde (FA) had either early or delayed effects on the numbers of granule cells in the dentate gyrus (DG). After birth, the neonatal male Wistar rats were exposed throughout a 30-day period to various concentrations of FA: 0 (control group), 6 ppm (low concentration group) and 12 ppm (high concentration group). This was done by placing them for 6 h/day and 5 days per week in a glass chamber containing FA vapor. Then, five animals from each group were anesthetized and decapitated on postnatal day (PND) 30, and the remaining five animals were sacrificed on PND 90 by intracardiac perfusion using 10% neutral buffered FA solution. The Cavalieri principle of stereological approaches was used to determine the volume of the DG in these sections. The optical fractionator counting method was used to estimate the total number of granule cells in the DG. The appearance of granule cells was normal under light microscopy in all PND 30 and PND 90 groups. There were significant age-related reductions in the volume of the DG at PND 90 irrespective of which group was examined. Significant age-related neuron loss was also determined at PND 90 compared to that at PND 30. Rats treated with a high concentration FA were found to have fewer granule cells than either the animals treated with a low concentration FA or the control group (p<0.01 and p<0.01, respectively) at PND 90 but not at PND 30. These findings clearly indicate that granule cells in the DG may be vulnerable to stress and the concentration of FA to which they are exposed during early postnatal life, and also that a neurotoxic effect of high dose FA on cell number is only seen after a long time period. These results may explain why some disorders do not appear until later life.

Effect of prenatal exposure to an anti-inflammatory drug on neuron number in cornu ammonis and dentate gyrus of the rat hippocampus: A stereological study

Prenatal exposed to an anti-inflammatory drug is a major problem for the developing central nervous system. It is not well known the effect of prenatal exposed to a non-steroidal anti-inflammatory drug on the hippocampus. Total neuron number in one side of the cornu ammonis (CA) and gyrus dentatus (GD) of the hippocampal formation in control and drug-treated (diclofenac sodium, DS) groups of male rats was estimated using the optical fractionator technique. Each main group has also two subgroups that are 4 weeks old (4W-old) and 20 weeks old (20W-old). In CA, no significant difference between 4W-old DS-treated and their control was found, but a significant difference was observed between 20W-old DS-treated and their controls. A decreasing of neuron number was 12% for 20W-old DS-treated group. In GD, a decreasing of the granule cell number in 4W-old of DS-treated group was seen but an increasing of granule cell number was found in the 20W-old drug-treated rats in comparison to its control group, 7% and 9%, respectively. Although an increasing of neuron number in CA at the control group was seen with age, from 4th week to 20th week (10%), age-dependent substantial granule cell decline (17%) was observed in GD. No age effect on the total cell numbers of CA and GD of the drug-treated groups was seen in comparison to 4W-old week and 20W-old. A pronounced neuron loss observed in the drug-treated group may be attributed to the neurotoxicity of diclofenac sodium (DS) on the developing hippocampal formation. Age-dependent neuron increase in the CA of 20W-old and neuron decline in GD of 20W-old control groups may be a result of a dual effect of saline injection during the fetal life, since these animals were exposed to a stress of 15-day-period of saline injection, prenatal stress. The reason of no age effect on CA and GD cell number in the drug-treated groups may be attributed to the depletion of the progenitor cells due to neurotoxicity of DS in the fetal life of these animals.

A simple technique for localizing consecutive fields for disector pairs in light microscopy: Application to neuron counting in rabbit spinal cord following spinal cord injury

Locating the same microscopic fields in consecutive sections is important in stereological analysis. The tools for achieving this requirement have limited number in practice. This paper presents a simple and inexpensive technique for localizing the same fields on disector pairs in conventional light microscopes equipped with widely available dial indicators. It is partly a modification of equipment previously described. The presented procedure requires two light microscopes equipped with dial indicators and modified slide clips. An application of the present system was shown in a model of spinal cord injury (SCI). A midthoracic laminectomy was performed leaving the dura intact. A contusion was done at the level of midthoracic spinal cord segments (T7-T8) by dropping a 10-g mass from a height of 30 cm. The subjects were randomly divided into three groups (10 animals in each): hypothermia group, methylprednisolone group, and traumatic spinal cord injury alone group. Present results show that treatment with hypothermia after spinal cord trauma has a neuroprotective effect on cell damage but not in the methylprednisolone treatment group.

Morphological, stereological and radiological changes in pinealectomized chicken cervical vertebrae.

Abstract:  Considerable evidence supports the hypothesis that melatonin plays an important role in osteogenesis. We carried out an experiment to investigate histological and radiological changes in the cervical vertebrae of pinealectomized chickens. Thirty new‐hatched chicks were divided into two groups: pinealectomized group (n = 15) and nonpinealectomized control (n = 15). Surgical pinealectomies were performed in Hybro broiler chickens at the age of 3 days. At 8 wk, one animal from each group was examined using computed tomography scanner to obtain density histograms of four consecutive vertebrae between the 4th and 7th cervical vertebral segments of chickens. At the end of the study, all animals were killed for subsequent measurement of bone mineral density (BMD) and macroscopic, radiographic and histopathological evaluation of specimens. We found that the profile of the density histogram in nonpinealectomized control group showed a spike profile for the vertebral body, indicating an increase in the amount of higher density tissues in this region, while a plateau‐shaped profile was obtained for the vertebral body in pinealectomy group, indicating the presence of heterogenous bone tissue. Accordingly, the mean value of BMD in pinealectomy group was significantly lower at the vertebral body in chickens compared with control subjects (P < 0.001). At the end of the experiment, almost all of the chickens in pinealectomy group developed a scoliotic curvature and the mean weight and length of the cervical vertebral bodies of the pinealectomized chickens were significantly lower than those of control group (P < 0.001). Although the numerical density of osteocytes and osteoblasts in pinealectomy group was significantly higher than that from the control group, total number of osteocytes but not osteoblasts in cervical vetrebrae from pinealectomized animals was significantly lower than that from nonpinealectomized control animals (P < 0.05). In conclusion, the results of the current study demonstrated for the first time pinealectomy‐induced histomorphometrical changes in chicken vertebral column using stereological methods, suggesting that pineal gland/melatonin may have an osteoinductive effect on bone formation, but further studies are needed to elucidate the relationship of our findings with some disorders such as postmenopausal and/or senile osteoporosis.

Effect of prenatal exposure to diclofenac sodium on Purkinje cell numbers in rat cerebellum: a stereological study.

Diclofenac sodium (DS) is commonly used as a non-steroidal anti-inflammatory drug. Although several adverse effects are clearly established, it is still unknown whether prenatal exposure to DS has an effect on the development of the cerebellum. In this study, we investigated the total number of Purkinje cells of the cerebellum in a control group and in a DS-treated group of male rats using a stereological method. The DS in a dose of 1 mg/kg daily was intraperitoneally injected to the drug-treated group of pregnant rats beginning from the 5th day after mating for a period of 15 days during pregnancy. Physiological serum at 1 ml dose was intraperitoneally injected to the control group of pregnant rats at the same period. After delivery, male offspring were obtained and each main group was divided into two subgroups that were 4-week-old (4W-old) and 20-week-old (20W-old). Our results showed that the total number of Purkinje cells in offspring of drug-treated rats was significantly lower than in the offspring of control animals. These results suggest that the Purkinje cells of a developing cerebellum may be affected by administration of DS during the prenatal period.

Effect of a High Fat Diet on Quantitative Features of Adipocytes in the Omentum: An Experimental, Stereological and Ultrastructural Study

Background: Omental adipose tissue specimens of female rats that were fed a high fat (HF) diet were evaluated stereologically and histopathologically. To our knowledge, there is no stereological study on numerical density, nuclear height and volume of adipocytes in omental adipose tissue in the female rat fed a HF diet in the literature. Method: 20 female Spraque Dawley rats were used in the study. 10 of the animals were fed HF diet consisting of 30% of calories from fat for 3 months. The remaining 10 rats, the control group, were fed a normal diet. After the experimental procedure, all animals were anesthetized and omental adipose tissues in the same area were dissected and fixed for the histochemical process using a mixture of 3% glutaraldehyde and 1% osmium tetraoxide in 0.1 M phosphate buffer. After embedding of tissues in araldite CY 212, semi-thin and thin sections were cut. The semi-thin sections were stained with toluidine blue. The physical dissector counting method was used for estimation of numerical density and nuclear height of adipocytes. Cavalieri principle was used for the estimation of adipocyte volume; volume fraction approach was applied to find the volume fraction of adipose tissue components. Results: The mean numerical density of adipocytes in the HF diet group was significantly higher than the control. The mean nuclear height of adipocytes was also very high in the HF diet group. The volume fraction of adipose mass was increased whereas the extracellular matrix volume fraction was reduced in the HF diet group compared to the controls. The mean volume of adipocytes in the HF diet group was also significantly higher than in the control group. At the light microscopy level, it was found that adipocytes were enlarged and gaining irregular shape in the HF diet group. Thicker basal lamina and electron dense lipid content were also found in this group at the electron microscopy level. Conclusion: Lipid content and number of adipocytes in the adipose tissue of HF diet rats were higher than in the controls. Thus, HF diet induces increase in body weight via both hypertrophy and hyperplasia of adipocytes.

Effects of postnatal formaldehyde exposure on pyramidal cell number, volume of cell layer in hippocampus and hemisphere in the rat : A stereological study

The purpose of the present study was to determine whether exposure of neonatal rats to formaldehyde (FA) had either early or delayed effects on the numbers of pyramidal cells in the cornu ammonis (CA) of the hippocampus. Neonatal Wistar rats were exposed to 0 ppm (control group), 6 ppm and 12 ppm (high concentration group) of FA concentrations throughout the 30-day period following the birth by placing them for 6 h/day in a glass chamber containing FA vapor. Then, some of the animals from each FA-treated group were anesthetized and decapitated at the day 30, and the remaining ones were killed at the day 90. The brains were removed immediately and fixed in 10% neutral-buffered FA solution. The Cavalieri principle was used to determine the volumes of the CA and the entire cerebral hemisphere. The optical fractionator counting method was used to estimate the total number of pyramidal cells in the CA. The appearance of pyramidal cells was normal under light microscopy at both postnatal day (PND) 30 and PND 90 in all groups. There were concentration-related volume changes of CA at PND 30 and PND 90; low concentration of FA significantly increased, whereas high concentration decreased the volume of CA in comparison of the control at PND 30. Importantly, high concentration of FA at PND 90 increased the volume of CA in comparison of the low concentration but not with the control. Furthermore, low and high concentrations of FA decreased the volume of hemisphere at PND 30, whereas a reverse effect of these concentrations was observed at the hemisphere of PND 90 in comparison of the control. In both CA and cerebral hemisphere, an age-related volume decrease in both control and low/high concentration groups were found. On the other hand, there were significant age-related reductions in the total number of pyramidal cells at 90 days of age irrespective of the groups examined. Rats treated with high concentration FA were seen to have significantly fewer pyramidal cell neurons than either the animals treated with low concentration FA or control groups (p<0.01). These observations indicate that pyramidal cells in the hippocampus may be vulnerable to FA exposure during the early period of life.

Inhibitory effect of pinealectomy on the development of cerebellar granule cells in the chick: A stereological study

Melatonin has some effects upon morphological features of various structures in small animals and human being. However, there has been no investigation concerning its physiological role on development of cerebellar granule cells. In this study, the changes induced by pinealectomy procedure on cerebellar development and their granule cell numbers in the chick were investigated using quantitative stereological methods. A total of 15 Hybro Broiler newly hatched chicks were randomly divided into three equal groups: pinealectomy group (n=5) and non-pinealectomized control group (n=5) and sham-operated group (n=5). Pinealectomy procedure and sham operation were done in 3-day-old chicks and all animals were sacrificed for histopathological evaluation and subsequent stereological analysis in the 8th week. Here, it was observed that pinealectomy significantly reduces the granular cell number in cerebellar cortex of the chicks (P<0.001). The present study is the first stereological study to evaluate the histomorphological effects of pinealectomy on the cerebellar granule cells of the chick. We suggest that the granule cell loss in the cerebellar cortex is due to developmental retardation in early postnatal period, although its exact mechanism is not clear. Based on our findings, we intimate that pineal gland/melatonin might play an important role in the development of cerebellar granule cells in the chick.