Experiments were performed in SHR and WKY, born and raised in the animal care facility of the Department of Neuroscience and Behavioral Neurosciences, School of Medicine of Ribeirão Preto, in a controlled environment (room temperature between 21-23°C, air humidity between 40 and 70% and dark/light cycle of 12 h), housed in plastic cages (3-4 animals to a cage) with free access to tap water and rat chow throughout the experiments. All experimental procedures adhered to The Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences and published by the National Institutes of Health (Copyright © 1996 by the National Academy of Sciences), and were approved by the Institutional Ethics Committee for Animal Research (CETEA--Comitê de Ética em Experimentação Animal, protocol number 184/2005). Effort was made to minimize the number of animals used.
Male (N = 6) and female (N = 6) SHR and WKY, brothers and sisters, 20 weeks old were anesthetized with sodium thiopental (Thionembutal, 40 mg kg, i.p.) and a catheter was inserted into the femoral artery for measurement of arterial pressure (AP). Recordings of the systolic (SAP), diastolic (DAP), mean arterial pressure (MAP) and heart rate (HR) were performed as described elsewhere [11, 12]. After the recordings, rats were perfused through the left ventricle first with a 0.05 M phosphate-buffered saline solution, pH 7.4 and then with a 2.5% glutaraldehyde solution in 0.1 M cacodylate buffer, pH 7.2. Both right and left sural nerves, from their origin in the hip (5-7 mm distal to the greater trochanter) through their distal branching at the lateral malleolus level, were carefully dissected without stretching, removed in one piece and placed in the fixative solution for an additional 12 hour. They were washed in cacodylate buffer, pH 7.2, and proximal (close to the origin) and distal (close to terminal branching) segments (of approximately 3 mm each) were cut and processed for epoxy resin embedding (PolyBed 812®, Polysciences Inc., Warrington, PA, USA) as described elsewhere [13–15]. Samples of all four experimental groups were histologically processed at once so that they were submitted to absolutely the same experimental conditions throughout the experiments.
Semithin (0.2-0.3 μm thick) transverse sections of the fascicles were stained with 1% toluidine blue and examined with the aid of an Axiophot II photomicroscope (Carl Zeiss, Jena, Germany). The images were sent via a digital camera (TK- 1270, JVC, Victor Company of Japan Ltd, Tokyo, Japan) to an IBM/PC where they were digitized. For the study of myelinated fibers, the endoneural space was observed with an optical set including an oil immersion lens (100 ×), optovar (1.6 ×), camera (0.5 ×) and an 8 × computerized magnification, which provided images with good resolution for morphometry.
The endoneural space was fully scanned without overlap of the microscopic fields, using an automatic motorized stage (Carl Zeiss, Jena, Germany). Scannings generated 9 to 17 microscopic fields of 640 × 470 pixels, which were used to count and automatically measure the myelinated fibers and their respective axons. Fibers at the upper and left edges of the microscopic fields were counted whereas those at the lower and right edges were not counted ("forbidden line") in order to avoid counting the same fiber twice. All myelinated fibers present in the endoneural space were counted. Morphometric parameters of the fascicles and myelinated fibers of sural nerve segments were obtained as described previously for other nerves [7, 14–16]. Briefly, the total number of myelinated fibers and the total number of Schwann cell nuclei present in each fascicle transverse section were counted. The area and lesser diameter of each fascicle (excluding the perineurium) as well as each myelinated fiber (defined by the axon and its respective myelin sheath excluding the Schwann cell nucleus when present) and respective axon (fiber excluding the myelin sheath) were measured with image analysis software (KS 400, Kontron 2.0, Eching Bei München, Germany). The lesser diameter better represents the diameter of a non-circular fascicle and fiber [13, 17, 18]. The area values are directly calculated by the software ("filled area" function), not calculated by from perimeter value. The percentage of the total cross-sectional area of the endoneural space occupied by the myelinated fibers was calculated, and hereafter referred as the percentage of occupancy of the myelinated fibers [6, 7, 13–15]. The myelinated fibers and Schwann cell nuclei densities were calculated. For myelinated fibers, both axonal diameter and total fiber diameter were automatically measured. The ratio between the two diameters, the g ratio (which indicates the degree of myelination), was obtained [19, 20]. Myelin sheath area was calculated for each myelinated fiber measured. For diameter measuring, fiber and axon forms are taken into account by "circularity index" calculation for each fiber. When fibers present index higher than 0.45, they are measured. Thus, fibers that are too oblong or irregular are automatically discharged. Histograms of population distribution of myelinated fibers and axons, separated into class intervals increasing by 1.0 μm were constructed. Histograms of the g ratio distribution separated into class intervals increasing by 0.1 were also prepared. The investigators were blind to group identities throughout the experiments.
Morphometric data were tested for normal distribution by the Kolmogorov-Smirnov normality test followed by the Levene test for variance equivalence. If data presented a normal distribution and equivalent variance, comparisons were made between proximal and distal segments in the same group by paired Student's t-test. Otherwise, comparisons were made by Wilcoxon's non-parametric test for paired samples. For comparisons between right and left segments in the same group, normally distributed data were tested using the unpaired Student's t-test. Alternatively, comparisons were made by the Mann-Whitney non-parametric test. Comparisons between groups were made by one-way analysis of variance (ANOVA) followed by Holm-Sidak post hoc test. Comparisons between histograms were made by one-way analysis of variance (ANOVA) on Ranks provided that the distributions did not pass the normality test. For all applied statistical tests, differences were considered significant when p < 0.05. Data are presented as mean ± standard error of the mean (SEM).