Effect of melatonin on ram adrenal gland 1175


Immunofluorescence on Fixed Sections Immunofluorescence has been performed in fixed tissue sections as described earlier (Zhang et al., 2007). Sections were deparaffinized with Rot-histol and rehydrated with a series of descending grades of ethanol then washed with PBS 3×5 min each. After that antigen retrieval was carried out in 0.1Msodiumcitrate buffer solution (pH=6) for 5 min using a microwave (600 Watt). Sections were washed three times for 5 min each with 1× PBS, followed by incubating sections in with blocking solution formed of PBS containing 10% normal donkey serum and 0.2% triton X-100 for 2 h at room temperature to minimize nonspecific labeling and increase the permeability to adrenal tissue. Subsequently, sections were incubated with a mouse monoclonal anti-TH antibody diluted at 1:500 and anti-synaptophysin1 diluted at 1:400 in blocking solution overnight at 4°C. On the 2nd day, the sectionswere washed in PBT (3 ×10 min each), then sections were incubated with Alexa Fluor® secondary antibody Dar 568 and Dam 488 for 2 h at room temperature in the dark to preserve fluorescence. Finally, the sections washed (3 ×5 min each) in 1× PBS and mounted with Flouromount-G. Slides were viewed with a Axioplan 2 epifluorescence microscope (Zeiss, Göttingen, Germany).


Semithin Sections and Electron Microscopic Studies


Adrenal glands were preserved by immersion in a mixture of 3% paraformaldehyde–glutaraldehyde fixative and left overnight (Karnovsky, 1965). After fixation, the samples were washed in 0.1 mol/L phosphate buffer and osmicated 1% osmium tetroxide in 0.1 mol/L Na-phosphate buffer at pH 7.3. After that, the samples were dehydrated in a graded series of ethanol followed by propylene oxide and embedded in Araldite. Semithin sections were cut at 1-µm thickness with a Reichert Ultracut (Leica, Germany) and stained with toluidine blue for light microscopy. Ultrathin sections were done with Ultrotom VRV (LKB


Bromma, Germany). The sections (70nm) were stained with uranyl acetate and lead citrate (Reynolds, 1963) and exami- ned in a JEOL 100CX II transmission electron microscope (TEM) (JEOL, Tokyo, Japan) at the Electron Microscopy Unit of Assiut University.


Digitally Colorization of TEM Images


To increase the visual contrast between several structures on the same electron micrograph, we have digitally colored-


specific elements [e.g., telocytes (TCs), ganglion cells, nerve fibers, Schwann cells, etc.] to make them more visible for the readers. All the elements were carefully hand colored in Adobe Photoshop software version 6.


Morphometrical and Statistical Analysis


The morphometric studies were performed on the semithin sections and TEM images of adrenal cortex and medulla of both control and treated animals using Leica Q 500MCimage analyzer (Leica, Germany). They were performed by two


RESULTS


Light Microscopy The Adrenal Cortex The adrenal cortex was arranged into three distinct zones: zona glomerulosa, zona fasciculate, and zona reticularis.


In the control group. The zona glomerulosa was composed of clusters of polygonal cells (10.6±1.3 µm in diameter) contained vesicular rounded nuclei with 1–2 nucleoli and their cytoplasm was provided by lipid droplets of variable size. These cell clusters were surrounded by abundant connective tissues containing rare TCs (Fig. 1a). The fasciculate zone was consisted of large polyhedral cells (15.6±0.95µm in diameter), arranged radially with central rounded nuclei and clearly visible nucleoli with large lipid droplets in the cytoplasm (Fig. 1c). In the reticularis zone, their cells (10.1±0.7µmin diameter) were arranged in a network manner surrounding narrow blood sinusoids (Fig. 1e).


In the treated group. The cells of zona glomerulosa were increased significantly in their diameter (18.9±2.0 µm, Table 1) and possessed large nucleus with many nucleoli. The cytoplasm was full of small lipid droplets. These cells were surrounded by little connective tissues contained many TCs. TCs were small irregular cells stained deeply with toluidine blue and had many cell processes and oval to elongated nuclei (Fig. 1b). The cytoplasm of zona fasciculate was packed with small lipid droplets with lightly stained nuclei and the cells showed a significant increase in their diameter (19.1±0.89 µm, Table 1) and separated by


operators, in a double-blind operation, comparing the results obtained subsequently. The measurementswere carried out on 15 randomly selected sections of each gland per animal (five different areas were measured from each section) as follows:


∙ Diameter of adrenocortical cells (μm) using 100× objective.


∙ Number of chromaffin cells per 50 μm2 using 100× objective.


∙ Diameter of chromaffin cells (μm) using 100× objective. ∙ Diameter of ganglion cells (μm) using 100× objective. ∙ The diameter of secretory chromaffin granules (nm) in TEM images.


∙ Diameter of Schwann cells (μm) in TEM images. ∙ The number of TCs per 20 μm2 using 100× objective. ∙ The length of telopodes (Tps) (μm) of TCs inTEMimages. ∙ Diameter of secretory vesicles (nm) of TCs and their number per 20 μm2 in TEM images.


All data were expressed as means±SE, which were


statistically analyzed using “T-Test Graphpad Software” (Version 6.05, International Scientific Community) to com- pare different measurements of both control and treated animals. Differences were considered significant if p<0.05 (*) and highly significant if p<0.01 (**).


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