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.

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.

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.

Neonatal pinealectomy induces Purkinje cell loss in the cerebellum of the chick: a stereological study.

Melatonin plays an important role in certain physiological functions and morphological features of various structures. In the current study, the effects of pinealectomy on Purkinje cell number and morphological features of developing cerebellum in the chick were investigated using stereological methods. Fifteen Hybro Broiler newly hatched chicks were divided into three groups: a pinealectomized group (n = 5), sham-operated group (n = 5) and a non-pinealectomized control group (n = 5). Surgical pinealectomy was performed in 3-day-old chicks. In the 8th week, all animals were sacrificed for histopathological evaluation and subsequent stereological analysis. Each layer volume of molecular (+Purkinje cell), granular and white matter in the cerebellum was estimated in all animals. It was found that there was no significant difference for the volume of whole cerebellum and also molecular (+Purkinje cell) layer in these groups (P > 0.05). Nevertheless, the values of granular layer and white matter of sham-operated group were significantly different from those of control and pinealectomized animals (P < 0.01). It was also observed that pinealectomy significantly reduces the Purkinje cell number in cerebellar cortex (P < 0.01). The present study is the first stereological study to demonstrate the histomorphological effects of pinealectomy on the cerebellum in the chick. Our results suggest that pineal gland/melatonin might play an important role in morphological features of the developing cerebellum in the chick.

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.

Pinealectomy exaggerates and melatonin treatment suppresses neuroma formation of transected sciatic nerve in rats: gross morphological, histological and stereological analysis.

At present, an intensive effort for prevention of neuroma formation following peripheral nerve section continues. It has been recently suggested that surgical pinealectomy (Px) induces elevation of the collagen content in the granulation tissue of a wound, while melatonin application after Px suppresses elevation of the collagen accumulation in the tissue. The aim of the present study was to assess whether melatonin had the ability to suppress collagen production and neuroma formation following peripheral nerve transection. A total of 40 male rats (four groups of 10) were left intact (intact controls) or sham operated (sham group), were Px, or were Px and given melatonin (Px + melatonin group). All animals underwent a surgical intervention consisting of right sciatic nerve neurectomy. After 4 wk, the animals were killed following intracardiac perfusion. Gross morphology of neuroma formation in the proximal nerve segment was examined and proximal neuroma evaluated. Macroscopic and microscopic findings revealed that Px caused a proliferation of connective tissue and large neuroma formation at the proximal ends of transected nerves. Stereological analysis showed that there was a statistically significant reduction in connective tissue content of the same region in Px animals treated with melatonin (P < 0.005). The results achieved in a rodent model of sciatic nerve neuroma formation showed that there was a positive correlation between macroscopic and microscopic observations, and that melatonin enhanced axonal regeneration presumably due to its inhibitory effect on neuroma formation.