Rat vibrissa became the follicle for investigation because red deer skin was unavailable due to the unusual winter weather condition. The major vibrissal follicles on the upper lip of rats (Fig.1) were seen. The size of the whisker hair shaft is much larger and thicker than the pelage hair shaft. The vibrissa follicle is also much deeper than the pelage hair follicles. The keratinized hair fibre tapers towards in the upper part of the hair shaft (Fig.1a).
Sacpic staining produced multicoloured sections allowing different parts of the hair follicle to be distinguished in the sections of rat vibrissal follicles. It showed remarkable diversity of staining colour and intensity (Fig.2). The keratinized part of the hair fibres were easily identified as they stained in bright yellow (Fig.2a).
The glassy membrane, the layer that surrounds the outer root sheath, and the capsule were stained in strong blue. The non-keratinising epithelial cells were grey-blue including the outer root sheath. Distinction could be made easily between yellow staining of fully keratinized hair fibres, the orange staining of the lower keratinising area and the blue grey non keratinised hair shaft (Fig. 2a, 2b). The Henle and Huxley's layers of the inner root sheath were easily recognized as they were stained in bright red.
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Although the epithelial structures of the pelage hair follicle and vibrissal follicle are essentially the same, the dermal tissues surrounding both types of follicles are very different. The vibrissal follicle is surrounded by a highly developed blood sinuses (Fig. 2a). These tissues are absent in the pelage hair follicle (Fig. 2c). Also the whisker follicle is at least 4 times the size of the pelage hair follicle vertically (Fig. 2a) and 20 times the size horizontally (fig. 2d). Sebaceous glands were difficult to see in the vibrissal follicle.
(a) The spatial arrangement photographed under the dissecting microscope of the rat whiskers on the rat's face is illustrated. The arrow pointed out two hair shaft is growing from the same follicle. Magnification Ã- 4
(b) In situ and micro-disectied rat vibrissae follicles. The arrow pointed out two hair shaft is growing from the same follicle. Magnification Ã- 8
(c) The side view of a rats facial skin tissue where the (BF) is showing the bottom of a whisker hair follicle. (D) is the dermal layer and (E) is the epidermis. Magnification Ã- 15
(d) Microdissected vibrissae attached vibrissal follicle. Photographed under the dissecting microscope. Magnification Ã- ????
Figure 2. Photomicrographs of rat facial skin stained with Sapic.
Figure 2a. Rat whisker skin, vertical section. Magnification Ã-40
The keratinized parts of the hair fibres which are yellow are easily distinguished. The epithelial cells including outer root sheath, are stained with a different blue from non-keratinizing epithelial cells.
Figure 2b. High power view of vertical section of rat vibrissal follicle. Magnification Ã-100. Showing the Henle and Huxley layers of the inner root sheath stained red. It also shows the changing of colour along the hair fibre. The lower part of the keratinising hair fibre is reddish orange and it changed to yellow when it is fully keratinised at the upper part.
Figure 2c. High power view of a pelage hair follicle with Saptic stain, vertical section. Magnification Ã-100
Figure 2d. Rat whisker skin sample stained with Sapic stain, transverse section. Magnification Ã-40
Sections shows 1 vibrissal follicle, 4 pelage hair follicles and sebaceous gland of pelage hair follicles stained grey. The green part surrounded the hair follicles are the dermis.
Legend: C, capsule; CS, cavernous sinus; CT, cuticle; DP, dermal papilla; GM, glassy membrane; HB, hair bulb; HEN, Henle's layer; HS, hair shaft; HUX, Huxley's layer; IRS, inner root sheath; KG, keratinising hair shaft; KT, keratinised hair shaft; M, medulla; NK, Non-keratinised hair shaft; ORS, outer root sheath; PF, pelage hair follicle; RS, ring sinus; RW, ringwulst ;SG, sebaceous gland ; VF, vibrissal follicles.
Localisation of cytokeratin-6 protein using Immunohistochemistry
Immunohistochemistry experiments to investigate the localization of the highly expressed intermediate filament cytokeratin-6 in the rat whisker was carried out .
Results of immunostaining showed that antibody to cytokeratin-6 protein immunoreacted specifically to the outer root sheath. Cytokeratin-6 protein expression was only seen in the outer root sheath hair cone. Immunostaining was not observed in the other cells of the hair follicle and the epidermis (Fig. 3).
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Initial attempts ??? large amount ?? non specific red staining and a series of experiment were carried out to clarify why this was occuing
Figure 3. Immunohistochemical localisation of cytokeratin-6 in rat whisker cryosections. Photomicrographs of rat facial skin photographed at Ã- 40 magnification.
(a) A control sample processed without the primary antibody. In place of the antibody, the sample was treated with 5% bovine serum albumin
(b) A control sample processed without the biotinalyted secondary antibody. In place of the secondary antibody, the sample was treated with 5% bovine serum albumin
(c) A control sample processed with 5% bovine serum albumin instead of avidin, a high affinity biotin-binding protein.
(d), (e) cytokeratin-6 expression was only present in the outer root sheath.
red: positive staining ;blue: Mayer's haematoxylin counterstained.
localisation of KATP channel component using Immunohistochemistry
Immunohistochemical investigations of KATP channel component of hair bulb region is positively expressed with KATP channel subunit SUR 1 protein in the hair matrix. Results of immunostaining showed that SUR 1 protein is specifically located in the hair matrix. SUR 1 protein expression was only seen in the epithelial cells of the hair matrix but not in the dermal papilla and dermal sheath. (Fig. 4)
Figure 4 . Immunolocalization of KATP channel subunit SUR 1 in the rat whisker hair follicle bulb. Original magnification Ã-100
(a), (c) No staining occurred in the hair bulb with the negative control that omitted primary antibody.
(b), (d) SUR1 expression was only presented in the hair matrix, with no staining evident in the dermal sheath or papilla
red: positive staining ; blue: haematoxylin counterstain. Mayer's haematoxylin
The original aim of this project was to localise KATP channel subunit SUR1 in red deer follicles. This is because it is a large follicle and rat whiskers are good for learning the structure of hair follicle.
Sapic staining successfully stained the sections of both rat vibrissal follicles and pelage hair follicles. It provided a multicoloured stain on the sections and allowed the identification of the different structures and layers of the hair follicles. This staining pattern has previously be used to examine sheep (Nixon, 1993) and human (Nutbrown & Randall 1996) follicles.
Talk abu vibrassa V. human follicles + pelage follicles
From the experiment carried out, the size of whisker hair shaft is much larger and thicker than the pelage hair shaft (Fig. 1) as well as the follicle it is relatively much deeper than pelage follicles (Fig. 2). May be this is because it is a sensory organ for rats where they have got sensory neuron attached. (Richard Robinson, Motion, Contact, or Both: Three Paths Convey Whisker Sensation in the Rat)
(Yu C, Derdikman D, Haidarliu S, Ahissar E (2006) Parallel thalamic pathways for whisking and touch signals in the rat)
From the results, it shows that cytokeratin-6 was positively expressed on the outer root sheath of the hair follicle (fig. 3c, 3f). It is due to learning the techniques of immunohistochemistry. Interestingly, transgenic mice expressing a mutant cytokeratin-6 have been shown to develop progressive cicatricial alopecia. It was suggested that hair loss is due to the destruction of the outer root sheath (Rothnagel et al., 1994). It could be because from anagen V to the mid-catagen phase, the inner root sheath moves up the follicle ahead of the cortical layer, the cortex forming the brush end of the club hair by interdigitating with the transformed outer root sheath. (Aoki et al., 2001)
The results confirm that SUR1 is localized in the hair matrix (fig. 4b, 4d). From other published papers concluded that novel drug NNC 55-0118, a selective opener of kir6.2/ SUR1 potassium channels (Dabrowski et al., 2003; Hansen, 2006). When kir6.2 and SUR1 were detected both together in the matrix. This shows that these are physiologically relevant. Kir6.1/SUR1 channels haven't been detected together in the other tissues (Ashcroft and Gribble, 2000; Jahangir and Terzic, 2005; Nichols, 2006).Therefore SUR 1 is always coupled with kir6.2 only (Koster et al., 1999). In rat vibrissa follicles, whisker growth permanently ceases when more than the lower third of the follicle base is removed (Oliver, 1966). So the dermal papilla is very important in the formation of hair follicles.
The length and size of hair depends on the anagen phase in its cycle. The growth factors are induced the proliferation of cells in the matrix, dermal papilla and dermal papillary vascular system. The mode of action of hair growth effect of minoxidil is not completely elucidated, but the most plausible explanation proposed here is that minoxidil works as a sulfonylurea receptor (SUR) activator and prolongs the anagen phase of hair follicles. Since it has been proved that SUR1 is expressed in the hair matrix, this means that the KATP channel is present on the hair matrix.
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It is now clear that the Kir6.2 subunits can generate ATP-sensitive K channels in the complete absence of expressed SUR subunits(Mikhailov et al., 1998).But the mechanism is still unknown. Previously, some drugs that stimulate SUR1 receptors for pancreatic use would also produce hair growth as side effect therefore the potassium channel regulators affect the KATP channels in the hair follicle by vascular effects (Davies et al., 2005). However, some of potassium channel openers including monixidil oppose the entry of calcium into the cells and induce hair growth (Buhl, 1991). It is suggested the minoxidil sulfate induced low concentration of cytosolic calcium might play an important role in the regulation of growing hair (Ohtsuyama et al., 1994).