Animals
Twenty-one adult male Wistar albino rats (220- 250 g) were divided into three groups as control (n=7), iron (n=7) and iron+ nicardipine (n=7). All animals were obtained from Medical and Surgical Research Faculty of Ondokuz Mayis University. All animals were kept in constant laboratory conditions and supplied with food and water ad-libidum.
Operation
Animals were kept away from food for 12 hours prior to surgery and all animals were weighted just before the surgical operation. Anesthesia was induced by i.p. injection of ketamine hydrochloride (100 mg/kg)+10 mg/kg xylazine. Animals were fixed to a stereotaxic apparatus and details of the surgical procedure can be seen in Bostanci et al.13. Rats in the control group received 2.5 µl saline while rats in iron group received 200 mM (2.5 µl) FeCl3 (FeCl36H2O, Sigma, St. Louis, USA)3. Rats in iron+nicardipine group received the same amount of FeCl3 and i.c.v. nicardipine (Sigma, St. Louis, USA) (1 µM, 2 µl). Then, incisions were sutured and incision area was cleaned using 10% povidon iodide (Aklar Chemistry, Ankara, Turkey) just prior the placement of the animals to their cages. All animals were survived for ten days following the surgery. Only the rats belonging to iron+nicardipine group received additional i.p. nicardipine treatment as 10 mg/kg/day for ten days12.
After ten days, all animals were perfused intracardially under deep urethane (Sigma, St. Louis, USA) anesthesia (1.25 g/kg, i.p.) with 10% formaldehyde (Aklar Chemistry,Ankara, Turkey) and saline, buffered for pH=7.6. After the completion of the perfusion process all animals were decapitated, brains
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were removed immediately and placed in the same fixative for postfixation. After the cerebra and cerebelli were separated physically, cerebelli were processed using the standard histological techniques and embedded in paraplast (Sigma, St. Louis, USA) embedding media. Serial tissue sections obtained using a rotary microtome (Leica RM 2135) in horizontal plane with a section thickness of 40 µm. The slides were stored overnight in the oven (60 °C) and stained with Cresyl violet (Sigma, St. Louis, USA) staining. Approval of Ethical Committee of Ondokuz Mayis University has been obtained prior to experiments and all animal work was performed according to the Experimental Animal Care Rules of European Community Council.
Section sampling and the determination of total Purkinje number
The cytoarchitectonic characteristics of the Purkinje cell layer were identified using the criteria of Gundersen14. According to the pilot study, 14-18 sections were sampled in a systematic random fashion (ssf: 1/7) out of a total of 120 horizontal sections per individual cerebellum. First sections were chosen randomly from the first set of 7 sections containing the cerebellum and then the consecutive samples were selected with a fixed interval of 7 sections.
The counting and analysis of Purkinje cells were performed with a stereology workstation using the optical fractionator counting method (CAST-GRID-Computer Assisted Stereological Toolbox-Olympus, Denmark)15. Cell counts were done using a sampling scheme optimized for a total of approximately 500 cell counts per individual. Determined Purkinje sectional areas were scanned automatically using consecutive steps with 200x200 µm x-y size. Every step in this scanning was individually analyzed with optical dissector probes using 100x oil-objectives. During optical dissector application, an unbiased counting frame comprising the 20% of the total step area was used for particle sampling and counting. Thus, the area sampling fraction (asf) is determined as (587/40.000) µm2.
According to previous pilot studies, a fixed dissector height of 10 µm was predetermined and used throughout the study. It was left a 5 µm for upper guard zone, applied particle counting through a 10 µm dissector height and measured the section thickness. All such measurements were done using a digital microcator (Hidenhein, Germany), incorporated in the stereological analysis system. Thus, the final sampling stage, generally called the thickness sampling fraction (tsf) was calculated by [Dissector Height]/[Mean Section Thickness]. Average section thickness was estimated for each section by measuring the thickness of every 10th field of counting with a random start and by averaging the measured thickness values for each section. The total average of section thickness was 29±2 µm among all animals. After completing a throughout sampling for all sampled sections, properly sampled Purkinje cells were counted as dissector particles (Q-). Total number of cerebellar Purkinje cells (N) was then calculated using the following formulation: N = [1/ssf] x [1/asf] x [1/tsf] x ΣQ-
Statistical analysis
Statistical analysis was performed using SPSS statistical software package (version: 12.0). Differences between groups, the estimated number of the Purkinje cell, were analysed with one way ANOVA and Post Hoc Tukey tests. The P-level was set at P < 0.05.