Histology and Ultrastructure of Transitional Changes in Skin Morphology in the Juvenile and Adult Four-Striped Mouse (Rhabdomys Pumilio)
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Hindawi Publishing Corporation The Scientific World Journal Volume 2013, Article ID 259680, 11 pages http://dx.doi.org/10.1155/2013/259680 Research Article Histology and Ultrastructure of Transitional Changes in Skin Morphology in the Juvenile and Adult Four-Striped Mouse (Rhabdomys pumilio) Eranée Stewart, Moyosore Salihu Ajao, and Amadi Ogonda Ihunwo School of Anatomical Sciences, Faculty of Health Sciences, University of the Witwatersrand, 7 York Road, Parktown, Johannesburg 2193, South Africa Correspondence should be addressed to Amadi Ogonda Ihunwo; [email protected] Received 7 August 2013; Accepted 19 September 2013 Academic Editors: C. Lucini and C. Tan Copyright © 2013 Eranee´ Stewart et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The four-striped mouse has a grey to brown coloured coat with four characteristic dark stripes interspersed with three lighter stripes running along its back. The histological differences in the skin of the juvenile and adult mouse were investigated by Haematoxylin and Eosin and Masson Trichrome staining, while melanocytes in the skin were studied through melanin-specific Ferro-ferricyanide staining. The ultrastructure of the juvenile skin, hair follicles, and melanocytes was also explored. In both the juvenile andadult four-striped mouse, pigment-containing cells were observed in the dermis and were homogeneously dispersed throughout this layer.Apartfromthesecells,thehistologyoftheskinoftheadult four-striped mouse was similar to normal mammalian skin. In the juvenile four-striped mouse, abundant hair follicles of varying sizes were observed in the dermis and hypodermis, while hair follicles of similar size were only present in the dermis of adult four-striped mouse. Ultrastructural analysis of juvenile hair follicles revealed that the arrangement and differentiation of cellular layers were typical of a mammal. This study therefore provides unique transition pattern in the four-striped mouse skin morphology different from the textbook description of the normal mammalian skin. 1. Introduction The four-striped mouse, Rhabdomys pumilio, is unique in that it is the only species of its genus [1]andparticularly The typical mammalian skin consists of an epidermis that is in interesting where pigmentation is concerned as it has mul- contact with the free surface and an underlying dermis. Deep tiple dark and light stripes on its dorsal surface [2] despite to the dermis is a layer of subcutaneous tissue known as the all other external parts of the skin being black. This common hypodermis. In mammals, skin or coat colour is essentially field mouse, widely distributed in Southern Africa, has a determined by melanocytes either situated in the epidermis grey to brown coloured coat with four characteristic dark or the hair bulb. These specialised cells synthesize melanin stripes running along its dorsal side. The dark stripes are in membrane-bound organelles termed melanosomes, which separated by three distinctive stripes of a lighter colour that are transferred through dendritic processes to their final des- may even be white [2]. We observed that the juvenile four- tination [8, 12, 13, 29]. Melanocytes are specialised cells found stripedmouseexhibitsitsstripesonboththeskinandcoat betweenthestratumbasalecellsandinhairfollicles[8–10, 12– hair, while the adult presents its stripes on the coat hair only. 14], although their precise distribution in mammalian skin The histology and ultrastructure of these stripes and their is different from one species to another [29]. These neural transition phase for this occurrence remain to be elucidated. crest-derived cells transfer melanosomes into surrounding Our investigations provide microscopic and ultrastructural cellsthroughcytoplasmicprocesses[8, 12, 13, 29]andare evidence of the transition in skin layers, unique pattern of predominantly responsible for fur, feather, and skin colour in the location of hair follicles, and melanocytes from juvenile mammals [29]. to adult skin. 2 The Scientific World Journal 2. Materials and Methods washed in 1% glacial acetic acid for 2 minutes. Sections were counterstained in picro-ponceau for 3 to 5 minutes 2.1. Experimental Animals. Twojuvenileandtwoadultfour- under microscopic control, differentiated in water, dehy- stripedmiceweretreatedandusedaccordingtotheguide- drated through a graded series of alcohols, cleared in xylene, lines of the University of the Witwatersrand Animal Ethics and mounted in Entellan. Screening Committee, which parallel those set down by the National Institute of Health (NIH) for use of animals in scientific experiments. The mice originated from the 2.6. Tissue Processing for Electron Microscopy. Small pieces ∘ ∘ Honeydew Grassland, Gauteng 27 55 S, 26 4 E, South offour-stripedmouseskintissuewerefixedin2.5%glu- Africa. A four-striped mouse with a body weight from 40 g taraldehyde in phosphate buffer, pH 7.4 followed by a wash [3] to 80 g with stripes present on both its skin and coat in phosphate buffer, pH 7.4 for 2 hours [4, 5]. Tissues were hair was defined as juvenile, whereas an adult was 80–95 g immersed and postfixed for 1 hour in 1% osmium tetroxide and then placed in 70% alcohol overnight in the refrigerator at and having the stripes on its coat hair only. Animals were ∘ euthanised intramuscularly with 20 mg/kg ketamine after 4 C. Tissues were dehydrated through 2 changes each of 95% which transcardial perfusion was carried out with 0.9% saline and absolute alcohol for 20 minutes, cleared, and infiltrated ∘ at 4 C, followed by 4% paraformaldehyde in 0.1 M phosphate with propylene oxide and Epon-Araldite resin solutions of buffer [4]. The shaved skin from the dorsal striped region varying ratios. First, in a solution of 3 parts propylene oxide to was harvested and processed either for light microscopy or 1 part resin, second, in equal parts propylene oxide and resin electron microscopy. solution, and third in a solution of 1 part propylene oxide to 3 parts resin, for a length of 30 minutes per solution. Lastly, the tissues were left overnight in resin, followed by embedding in ∘ 2.2. Tissue Processing for Light Microscopy. Tissues were fixed fresh Epon-Araldite resinat 60 Cfor48hours. in 10% buffered formalin5 [ ] and routinely processed for After polymerisation 1 msemithinsectionswascuton light microscopy in a Shandon Citadel 1000 automatic tissue a Reichert-Jung Ultracut ultramicrotome (Germany) and processor (UK). Paraffin wax tissue blocks were sectioned stained with Toluidine Blue-Pyronin Y for 30 seconds, dried, at 6 m using a Leica 1400 sledge microtome (Germany). ∘ and mounted in Entellan. Semithin sections with the area Prepared slides were placed in an oven at 60 Cfor30minutes of the dermis and its hair follicles, the hypodermis from the to ensure that the sections adhered firmly to the slides. All adult; the dermis and its hair follicles, the hypodermis and its sectionsweredewaxedin2changesofxylenefor5minutes hair follicles from the juvenile were selected. Ultrathin gold each, passed through 2 changes of absolute alcohol for 30 sections were cut and placed on copper grids and stained with seconds each, then transferred to 95% alcohol for 30 seconds, uranyl acetate for 3 minutes. Drops of lead citrate were placed and rinsed in gently running tap water. on strips of dental wax, and once stained, grids were rinsed first in dilute sodium hydroxide, followed by distilled water 2.3. Haematoxylin and Eosin Staining. Sections were stained andthendried. in a modified Mayer’s Haematoxylin5 [ ]for5minutes. Once removed from the Haematoxylin, they were left to 2.7. Evaluation of Slides. Light microscopy analysis of stained “blue” in running tap water and staining was controlled sections was done using a Zeiss Axioskop 2 plus Light microscopically. Sections were counterstained in Eosin for microscope (Germany), fitted with a Zeiss Axiocam HRc 30 seconds, followed by a brief wash in running tap water. camera (Germany). Ultrastructural examination and electron Sections were dehydrated through a graded series of alcohol: micrographs were taken, with a JEOL JEM-100S transmission 95% alcohol and then 2 changes of absolute alcohol. After electron microscope (Japan) at 80 kV, and negatives were dehydration, sections were cleared in 2 changes of xylene and scanned with an Epson Expression 1680 scanner (Japan). mounted in Entellan. 2.4. Masson Trichrome Staining for Connective Tissue. Sec- 3. Results tions were stained in Iron Haematoxylin [6]for10minutes, 3.1. Epidermis. In histological studies of the adult four- washed in running tap water, differentiated in 0.5% acid alco- striped mouse (Figures 1(a), 1(c),and1(e)), the dermal- hol, and washed thoroughly in running tap water. Sections epidermal junction was regular with an absence of dermal weretreatedwithsaturatedalcoholicpicricacidfor3minutes, papillae and epidermal ridges. Resting on a basement mem- dipped a few times in water, and stained with ponceau- brane is the stratum basale, consisting of a single layer of fuchsin solution for 10 minutes. The sections were treated cuboidal to columnar cells with large, round to oval nuclei with 2% phosphotungstic acid for 5 minutes, dehydrated and 1 or 2 prominent nucleoli. The stratum spinosum had a through a graded series of alcohols, cleared in xylene, and thickness of 1 to 2 polyhedral cells, each with a single centrally