Süleyman Kaplan

Unbiased estimation of the liver volume by the Cavalieri principle using magnetic resonance images

OBJECTIVE It is often useful to know the exact volume of the liver, such as in monitoring the effects of a disease, treatment, dieting regime, training program or surgical application. Some non-invasive methodologies have been previously described which estimate the volume of the liver. However, these preliminary techniques need special software or skilled performers and they are not ideal for daily use in clinical practice. Here, we describe a simple, accurate and practical technique for estimating liver volume without changing the routine magnetic resonance imaging scanning procedure. MATERIALS AND METHODS In this study, five normal livers, obtained from cadavers, were scanned by 0.5 T MR machine, in horizontal and sagittal planes. The consecutive sections, in 10 mm thickness, were used to estimate the whole volume of the liver by means of the Cavalieri principle. The volume estimations were done by three different performers to evaluate the reproducibility. RESULTS There are no statistical differences between the performers and real liver volumes (P > 0.05). There is also high correlation between the estimates of performers and the real liver volume (r = 0.993). CONCLUSION We conclude that the combination of MR imaging with the Cavalieri principle is a non-invasive, direct and unbiased technique that can be safely applied to estimate liver volume with a very moderate workload per individual.

Protection of nigral cell death by bilateral subthalamic nucleus stimulation

In Parkinson disease (PD), the subthalamic nucleus (STN) becomes hyperactive (disinhibited), which is reported to cause excitotoxic damage to midbrain dopaminergic neurons. Here, we examined whether silencing of the hyperactive STN by chronic bilateral deep brain stimulation (DBS) increased the survival of midbrain dopaminergic neurons in a rat model of PD. High-precision design-based stereologic examination of the total number of neurons and tyrosine tydroxylase (TH) immunoreactive neurons in the substantia nigra pars compacta revealed that STN DBS resulted in a significant survival of these neurons. These data provide the first evidence in vivo that bilateral STN DBS is useful for protecting midbrain dopaminergic neurons from cell death in PD.

Effects of prenatal exposure to a 900 MHz electromagnetic field on the dentate gyrus of rats: a stereological and histopathological study.

Electromagnetic fields (EMFs) inhibit the formation and differentiation of neural stem cells during embryonic development. In this study, the effects of prenatal exposure to EMF on the number of granule cells in the dentate gyrus of 4-week-old rats were investigated. This experiment used a control (Cont) group and an EMF exposed (EMF) group (three pregnant rats each group). The EMF group consisted of six offspring (n=6) of pregnant rats that were exposed to an EMF of up to 900 megahertz (MHz) for 60 min/day between the first and last days of gestation. The control group consisted of five offspring (n=5) of pregnant rats that were not treated at all. The offspring were sacrificed when they were 4 weeks old. The numbers of granule cells in the dentate gyrus were analyzed using the optical fractionator technique. The results showed that prenatal EMF exposure caused a decrease in the number of granule cells in the dentate gyrus of the rats (P<0.01). This suggests that prenatal exposure to a 900 MHz EMF affects the development of the dentate gyrus granule cells in the rat hippocampus. Cell loss might be caused by an inhibition of granule cell neurogenesis in the dentate gyrus.

High frequency stimulation of the subthalamic nucleus improves speed of locomotion but impairs forelimb movement in parkinsonian rats

The subthalamic nucleus (STN) plays an important role in motor and non-motor behavior in Parkinsons disease, but its involvement in gait functions is largely unknown. In this study, we investigated the role of the STN on gait in a rat model of PD using the CatWalk method. Parkinsonian rats received bilateral high frequency stimulation (HFS) with different stimulation amplitudes of the STN. Rats were rendered parkinsonian by bilateral injections of 6-hydroxydopamine (6-OHDA) into the striatum. One group of 6-OHDA animals was implanted bilaterally with stimulation electrodes at the level of the STN. Stimulations were performed at 130 Hz (frequency), 60 micros (pulse width) and varying amplitudes of 0, 3, 30 and 150 microA. Rats were evaluated in an automated quantitative gait analysis method (CatWalk method). After behavioral evaluations, rats were killed and the brains processed for histological stainings to determine the impact of the dopaminergic lesion (tyrosine hydroxylase immunohistochemistry) and the localization of the electrode tip (hematoxylin-eosin histochemistry). Results show that bilateral 6-OHDA infusion significantly decreased (70%) the number of dopaminergic cells in the substantia nigra pars compacta (SNc). Due to 6-OHDA treatment, the gait parameters changed considerably. There was a general slowness. The most pronounced effects were seen at the level of the hind paws. Due to implantation of STN electrodes the step pattern changed. STN electrical stimulation improved the general slowness but induced slowing of the forelimb movement. Furthermore, we found that HFS with a medium amplitude significantly changed speed, the so-called dynamic aspect of gait. The static features of gait were only significantly influenced with low amplitude. Remarkably, STN stimulation affected predominantly the forepaws/limbs.

A simple technique to measure the movements of the microscope stage along the x and y axes for stereological methods

Measurements of microscope stage movements in the x and y directions are of importance for some stereological methods such as the optical disector and optical fractionator. The length of stage movements can be measured with great precision and accuracy using a suitable motorized stage, which is generally a computer‐assisted instrument. This type of equipment is generally too expensive for and not readily available in many laboratories. This paper describes a simple method to measure the movements of the microscope stage along the x and y directions, which can be used for purposes such as systematic uniform random sampling. It needs a microscope attachment consisting of two dial indicators; one of them is used to measure the amount of stage movement along the x‐axis and the other measures the amount of movement along the y‐axis. Movements of the stage on the micrometre‐scale can be measured easily using this device.

Enhancement of nerve regeneration and orientation across a gap with a nerve graft within a vein conduit graft: a functional, stereological, and electrophysiological study.

In this study, the right sciatic nerves of 40 rats were used to determine whether a nerve graft within a vein graft might accelerate and facilitate axonal regeneration, compared with a nerve graft alone. The animals were separated into four groups, as follows: group 1, sham control; group 2 (control), segmental nerve resection and no repair; group 3, segmental nerve resection and nerve grafting; group 4, segmental nerve resection and reconstruction with a nerve graft within a vein conduit graft. For all groups, sciatic functional indices were calculated before the operation and on postoperative days 7 and 90. On postoperative day 90, the sciatic nerves were reexposed and nerve conduction velocities were recorded. The sciatic nerves were harvested from all groups for counting of the myelinated axons with a stereological method. No statistically significant differences with respect to return of gait function, axon count, or nerve conduction were noted between groups 3 and 4 (p > 0.05). However, functional recovery in group 4 on postoperative day 90 was significant, compared with group 2 (p < 0.05); the recovery difference between groups 2 and 3 was not significant (p > 0.05). This study was not able to demonstrate any functional benefits with the use of a nerve graft within a vein graft, compared with standard nerve grafting.

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.

Calibration of the stereological estimation of the number of myelinated axons in the rat sciatic nerve: A multicenter study

Several sources of variability can affect stereological estimates. Here we measured the impact of potential sources of variability on numerical stereological estimates of myelinated axons in the adult rat sciatic nerve. Besides biological variation, parameters tested included two variations of stereological methods (unbiased counting frame versus 2D-disector), two sampling schemes (few large versus frequent small sampling boxes), and workstations with varying degrees of sophistication. All estimates were validated against exhaustive counts of the same nerve cross sections to obtain calibrated true numbers of myelinated axons (gold standard). In addition, we quantified errors in particle identification by comparing light microscopic and electron microscopic images of selected consecutive sections. Biological variation was 15.6%. There was no significant difference between the two stereological approaches or workstations used, but sampling schemes with few large samples yielded larger differences (20.7+/-3.7% SEM) of estimates from true values, while frequent small samples showed significantly smaller differences (12.7+/-1.9% SEM). Particle identification was accurate in 94% of cases (range: 89-98%). The most common identification error was due to profiles of Schwann cell nuclei mimicking profiles of small myelinated nerve fibers. We recommend sampling frequent small rather than few large areas, and conclude that workstations with basic stereological equipment are sufficient to obtain accurate estimates. Electron microscopic verification showed that particle misidentification had a surprisingly variable and large impact of up to 11%, corresponding to 2/3 of the biological variation (15.6%). Thus, errors in particle identification require further attention, and we provide a simple nerve fiber recognition test to assist investigators with self-testing and training.

Platelet gel does not improve peripheral nerve regeneration: an electrophysiological, stereological, and electron microscopic study.

Although use of platelet gel (PG) for promoting tissue regeneration is a popular approach because of its capacity to accelerate tissue regeneration, to our knowledge, its effects on peripheral nerve have still not been elucidated. Therefore, the aim of this study was to investigate effects of PG on sciatic nerve regeneration using electrophysiology, stereology, and electron microscopy. The study was performed using five groups of rats: sham operated (Sham), collagen tube conduit (CT), collagen tube conduit plus platelet gel (CT + PG), autogenous nerve graft (ANG), and primary repair (PR) groups. Gap length for CT and CT + PG groups is 1 cm. Electrophysiology showed that nerve conduction velocity was not different among experimental groups; the amplitude of compound action potential of PR group was significantly higher than other groups. Examination of the nerves showed that Sham group not only had a larger axon diameter but also a thicker myelin sheath. A higher number of myelinated axon was found in both ANG and PR groups in comparison to Sham, CT, and CT+PG groups. There is no significant difference between morphological quantities of CT+PG and CT group. It was expected that regeneration degree of the nerve fibers of CT+PG group would be better than CT group, which was the control group permitting to disclose the presence of a positive effect of PG on nerve regeneration, but this was not the case. Therefore, our results suggest that PG does not improve axon regeneration after microsurgical reconstruction of a nerve gap by collagen tubes.