Sinan Canan

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.

Hippocampal Volume in Schizophrenia and Its Relationship with Risperidone Treatment: A Stereological Study

The purpose of this study was to assess the significance of the hippocampal volume differences and its relation with risperidone treatment in schizophrenia. In schizophrenic patients who were on risperidone treatment (n = 11) and in healthy volunteers (n = 11), volumes of the hippocampi were estimated using magnetic resonance images (MRIs). A detailed systematic series of coronal MRIs of the entire brain (3 mm thickness, T1-weighted, TR/TE 400/10 ms) was obtained for each subject. All estimations were done according to Cavalieri’s method by a modified point-counting grid placed on surface areas of hippocampal slices. The mean right and left hippocampal volumes in schizophrenics and control subjects were 1,059.4 and 1,003.2 mm3, and 1,780.1 and 1,589.1 mm3, respectively. The corresponding coefficients of errors were 0.05 and 0.068, and 0.059 and 0.081, respectively. The volumes of left and right hemispheres were not significantly different in both schizophrenic patients and controls (p > 0.05). However, a statistically significant difference (p < 0.05) was found between hippocampal volumes of the schizophrenic patients and controls. In conclusion, the hippocampal volume of the schizophrenic patients is significantly smaller than of the healthy controls. The patients who responded well to risperidone treatment had significantly greater hippocampal volumes than the patients who did not respond properly. Thus, hippocampal volume may be a predictor of the treatment response of schizophrenics to risperidone.

Prenatal exposure to a non-steroidal anti-inflammatory drug or saline solution impairs sciatic nerve morphology: a stereological and histological study

The toxic effect of non‐steroidal anti‐inflammatory drugs (NSAIDs) during development has been widely investigated. While it has been shown that these drugs impair central nervous development and compromise the neural activity, the effects of these substances on the development of peripheral nerves are still not clarified. In the present study, sciatic nerves withdrawn from three experimental groups of 4‐week‐old rats, prenatally exposed to either saline solution, or diclofenac sodium, and controls not exposed to any substance, were evaluated in terms of axon number, cross‐sectional area of axon and myelin sheet thickness as well as of the ultrastructure of nerve fibers. Comparisons of stereological estimations among these three groups showed that axon number and mean axon cross‐sectional area, but not average myelin sheet thickness, were significantly decreased in rats that were exposed to both diclofenac sodium and also to the saline solution, in comparison of the control group. Electron microscope analysis revealed, in both treated groups, deterioration of myelin sheaths that was more pronounced in rats that were exposed to diclofenac sodium. Altogether, these findings show that the prenatal administration of both diclofenac sodium and saline solution impairs peripheral nervous system development, thus suggesting that this potential teratogenic effect should be also taken into consideration in the clinical use of these substances in pregnant patients.

Estimation of numerical density and mean synaptic height in chick hippocampus 24 and 48 hours after passive avoidance training.

The effects of passive avoidance learning on synaptic morphology and number in the dorsolateral hippocampus of chick were investigated at 24 and 48 h after training. Chicks of both sexes were used. The numerical density of synapses and mean synaptic height were determined using design-based quantitative electron microscopic techniques. Our results suggest that after training there is a significant increase in synaptic density in the dorsolateral hippocampus of chicks at both 24 and 48 h, and also that the mean synaptic height was significantly different between trained and control groups. The increase in synaptic density was due to shaft (type II) synapses. It is known that during synaptogenesis, shaft synapses are formed first and are then converted to spine synapses. The only hemispheric asymmetry was found in the 24 h water-trained (W-trained) males where the numerical density of spine synapses was significantly higher in the left hippocampus. No significant differences due to gender in either numerical synaptic density or synapse height were observed at either 24 and 48 h. Comparison of the 24 h with 48 h groups showed an increase in shaft synaptic density over time in the W-trained groups, and an increased density of both shaft and spine synapses with time in methylanthranilate-trained (MeA-trained) chicks. These results demonstrate that the dorsolateral hippocampus of the chick shows synaptic changes at both 24 and 48 h after training and implicates this region in the long-term memory process.

An efficient stereological sampling approach for quantitative assessment of nerve regeneration

Aims: The aim of the present study was to find the most efficient sampling strategy for stereological analysis of peripheral nerve, including the number of myelinated nerve fibres, axon cross‐sectional area and myelin sheet thicknesses of nerve fibres. Methods: Two groups of rats underwent experimental resection of the tibial and peroneal nerves. The first group received tibial‐peroneal end to end autograft repair (n = 6). Tibial and peroneal nerves were isolated, transected, and separated 1 cm distal to the trifurcation, where they lay adjacent to each other by a 1‐cm gap, then repaired with an autologous nerve graft taken from the tibial nerve. The proximal stump of the tibial nerve and distal stump of the peroneal nerve were connected to each other by means of the nerve graft. The second group received tibial‐peroneal repair with a flexible collagen tube (n = 6). After 90 days of recovery, animals were sacrificed and nerve segments were removed and sectioned for microscopy. Three different sampling strategies, that is, small, medium and large step sizes were applied to obtain each quantitative parameter. Results: There are no significant differences between these sampling strategies with respect to total number of myelinated nerve fibres, axon cross‐sectional area and myelin sheet thicknesses of nerve fibres. Conclusion: Findings show that one can achieve the desired estimate precisely with a rather large and less time‐consuming sampling approach. In addition, it was observed that the size discrepancy of nerve regeneration can be improved by collagen tube conduit even with a 1‐cm gap.

Detailed spectral profile analysis of penicillin-induced epileptiform activity in anesthetized rats

Penicillin model is a widely used experimental model for epilepsy research. In the present study we aimed to portray a detailed spectral analysis of penicillin-induced epileptiform activity in comparison with basal brain activity in anesthetized Wistar rats. Male Wistar rats were anesthetized with i.p. urethane and connected to an electrocorticogram setup. After a short period of basal activity recording, epileptic focus was induced by injecting 400IU/2 microl penicillin-G potassium into the left lateral ventricle while the cortical activity was continuously recorded. Basal activity, latent period and the penicillin-induced epileptiform activity periods were then analyzed using both conventional methods and spectral analysis. Spectral analyses were conducted by dividing the whole spectrum into different frequency bands including delta, theta (slow and fast), alpha-sigma, beta (1 and 2) and gamma (1 and 2) bands. Our results show that the most affected frequency bands were delta, theta, beta-2 and gamma-2 bands during the epileptiform activity and there were marked differences in terms of spectral densities between three investigated episodes (basal activity, latent period and epileptiform activity). Our results may help to analyze novel data obtained using similar experimental models and the simple analysis method described here can be used in similar studies to investigate the basic neuronal mechanism of this or other types of experimental epilepsies.

A calcium channel blocker flunarizine attenuates the neurotoxic effects of iron

Iron is a metal highly concentrated in liver and brain tissue, and known to induce neuronal hyperactivity and oxidative stress. It has been established that iron levels rise in the brain in some neurodegenerative diseases such as Parkinsons and Alzheimers diseases (AD). A body of evidence indicates a link between neuronal death and intracellular excessive calcium accumulation. The aim of the present study was to investigate the effects of a calcium antagonist, flunarizine, on neurotoxicity induced by intracerebroventricular (i.c.v.) iron injection. For this reason rats were divided into three groups as control, iron and iron+flunarizine groups. Animals in iron and iron+flunarizine groups received i.c.v. FeCl3 injection (200 mM, 2.5 μl), while control rats received the same amount of saline into the cerebral ventricles. Rats in iron+flunarizine group also received i.c.v. flunarizine (1 μM, 2 μl) following FeCl3 injection. All animals were kept alive for ten days following the operation and animals in iron+flunarizine group received intraperitoneal (i.p.) flunarizine injections once a day (10 mg/kg/day) during this period. After ten days, rats were sacrificed. The total numbers of neurons in hippocampus of all rats were estimated with the latest, unbiased stereological techniques. Findings of the present study suggest that flunarizine may attenuate the neurotoxic effects of iron injection by inhibiting the cellular influx of excessive calcium ions.

Examination of Age‐dependent effects of fetal ethanol exposure on behavior, hippocampal cell counts, and doublecortin immunoreactivity in rats

Ethanol is known as a potent teratogen having adverse effects on brain and behavior. However, some of the behavioral deficits caused by fetal alcohol exposure and well expressed in juveniles ameliorate with maturation may suggest some kind of functional recovery occurring during postnatal development. The aim of this study was to reexamine age‐dependent behavioral impairments in fetal‐alcohol rats and to investigate the changes in neurogenesis and gross morphology of the hippocampus during a protracted postnatal period searching for developmental deficits and/or delays that would correlate with behavioral impairments in juveniles and for potential compensatory processes responsible for their amelioration in adults. Ethanol was delivered to the pregnant dams by intragastric intubation throughout 7–21 gestation days at daily dose of 6 g/kg. Isocaloric intubation and intact control groups were included. Locomotor activity, anxiety, and spatial learning tasks were applied to juvenile and young‐adult rats from all groups. Unbiased stereological estimates of hippocampal volumes, the total number of pyramidal and granular cells, and double cortin expressing neurons were carried out for postnatal days (PDs) PD1, PD10, PD30, and PD60. Alcohol insult during second trimester equivalent caused significant deficits in the spatial learning in juvenile rats; however, its effect on hippocampal morphology was limited to a marginally lower number of granular cells in dentate gyrus (DG) on PD30. Thus, initial behavioral deficits and the following functional recovery in fetal‐alcohol subjects may be due to more subtle plastic changes within the hippocampal formation but also in other structures of the extended hippocampal circuit. Further investigation is required.

The effect of autogenous vein grafts on nerve repair with size discrepancy in rats: An electrophysiological and stereological analysis

Aside from anatomical repairs, the reestablishment of sensory and motor innervations for proper functional recovery is one of the fundamental objectives of reconstructive surgery. The heterotopic transfer of autologous tissues is likely to result in a size discrepancy between the donor and recipient nerves, which will have a negative influence on regeneration. Twenty Wistar albino female rats were used in a study that was divided into two main groups: tibial-peroneal (TP) and peroneal-tibial repair (PT). Both types of nerves were exposed on the hind legs with the nerves cut on the right side, while the proximal stump of the tibial nerve and distal stump of the peroneal nerve were sutured to each other. These groups are also called end-to-end neurorrhaphy groups (EtoE). On the left side, the tibial and peroneal nerves were cut on the same level as on the right side. After the end-to-end epineural suturing of the nerve, the vein graft was slid over to the repair zone under irrigation. These are called the vein graft group (VG). All processes mentioned above were also done for the PT group. On the 90th postoperative day, anesthetized animals were fixed prone on a board, with the nerves carefully dissected for electrophysiological recording. Stereological methods for an estimation of the total number of myelinated fiber, a mean axonal cross-section area and the thickness of the myelin sheet were used. In TP and PT groups, nerve conduction velocities were found to be higher within the VG group. Nevertheless; the difference was only significant in the PT group. In both TP and PT groups, the increase in the axon number, axon area and myelin thickness were statistically different in favor of the vein graft sides. An appearance of vacuoles and degenerated pertinacious material within the myelin sheath of EtoE sides was seen. A histomorphological examination of the sections proximal to, from, and distal to the repair zone over three months revealed less epineural scarring, a thinner epineurium, more regenerated axons and fewer inflammatory cells in groups where vein grafting was used, because the vein graft provided additional mechanical and chemical support in the size discrepancy of the nerve regeneration.