Expression Pattern Of Draxin In Follicle Cycle Of Synchronized Mouse Vibrissa And The Function Of Draxin During Vibrissa Growth

Objective: Vibrissa is a kind of hair, the hair follicle cyclictransformations from phases of anagen, via catagen to telogen. The hairfollicle is a complex mini-organ, including mainly connective tissuesheath (CTS), dermal papilla (DP), inner root sheath (IRS), outer rootsheath (IRS), hair bulb(HB), hair shaft (HS) and hair root (HR). Theformation of the embryonic period of hair follicles, and after the birth ofcyclical changes of shape and structure of hair follicles, are the result ofleather-skin cell interactions. Their DP cells secrete a variety of activeprotein and cytokines, which induce differentiation of epidermal stem cellproliferation and cause hair follicle cycle stages morphology change.Draxin is newly discovered axon guidance factor, in the process of thedevelopment of the central nervous system, repulsiving commissuralneuronal axons from dorsal to ventral projection. DCC is a receptor ofdraxin and expressed in hair follicle epidemal stem cell height, so thisstudy aims at investigating whether draxin, in DP parts, plays a role andparticipates in the structural changing of the hair follicle forms at allstages of hair follicle cycle. Using HE staining, immunofluorescence, anddetecting of vibrissa growth length after depilation, the authors observedthe hair follicle cycle structural changes of normal mice vibrissa hairfollicle at all stages, the draxin distribution patterns within the hairfollicle, and the relationship between draxin and vibrissa growth.Through this study we can analysize the expression pattern and the effectof draxin in synchronized mouse vibrissa follicle cycle.Methods:1Experimental animals and grouping:42healthy,7-8weeks female and clean level wild type mice (draxin+/+) and draxin knockout(draxin-/-) mice (Hideaki Tanaka by Japanese kumamoto universityprofessor friendship gift) for this study. The36wild type mice weredivided into6groups at random, each group contains6. Five were usedfor5,10,16,20and25days group after depilation; One group as thecontrol group and together with6draxin-/-mice were used for measuringthe vibrissa length of1-25days after depilation. All the mice wereregularly feed in accord with the standard of clean laboratory (T22-25,50-70%humidity).2Hair follicle cycle synchronization building: according to the Pausand other methods,7-8weeks female, draxin+/+and draxin-/-mice wereused. A bee wax/rosin mixture was employed to remove the vibrissa ofdraxin+/+and draxin-/-mice. Repeat this process until vibrissa wascompletely pulled out. The day was counted as0days of the hair cycle.3Experimental tissue obtaining: wild type mice at the time points of5,10,16,20and25days after depilation were selected and anesthesiawas performed by intraperitoneal injection of1%sodium pentobarbitalanesthesia(50mg/kg). The mice was executed by breaking neck. Cut theskin by scissors and take about1cm x1cm for fixation. After cleaning inthe0.1MPBS buffer rapidly, flat out the samples on the filter and put itinto4%paraformaldehyde, on ice shake table fixed for3h. After fixationthe samples were put into30%sucrose, on ice shake shaking bedovernight. The second day the skin was cut into small pieces using asurgical blade along the vibrissa. Put the sample in the four orifice platecontaining O.C.T, with the repaired side down and froze rapidly into-80℃refrigerator.4The preparation of frozen sections, HE and immunofluorescencestaining:10um slice along the direction of the hair follicles was cut andstick the slices to prevent adhesion on the slide and bake the slides on37℃for30minutes. HE staining was used for observing the changes of thehair follicle morphology at the5time points of5,10,16,20and25days after depilation of wild type mice; Immunofluorescence technique wasused to detect the draxin expression pattern in the hair follicles.5The growth length after depilation: The vibrissa growth length invibrissa parts of wild type and draxin knockout mice was observed byOlympus universal microscope.6mice were used for each type of miceand measurement of vibrissa growth length was performed at differenttime points. When vibrissa was relatively short, measure was done usingthe scale under the microscope. When the vibrissa grew up to a certainlength, measure was done using a ruler. The vibrissa length between thewild type and draxin knockout mice is compared at the time points of5,10,16,20and25days after depilation and the results was statisticalanalyzed.Results:1HE staining:5,10and16days after depilation were at thecharacteristics stages of anagen,20d at catagen and25d at telogen. Onthe5th day after depilation, HB surrounded the DP and the DP made upmore than one-third of the bulb volume. The melanin granules appearedwithin the IRS and the HS was entirely surrounded by the IRS. The tipof the IRS and the HS resided just below the level of the SG. Comparedwith5d, the DP was still round, and, the diameter of the DP was alsostill more than one-third of the bulb diameter on the10th day. The distalend of the IRS and the tip of the HS had already entered the hair canal.On the16th day, the tip of the HS emerged through the epidermis. TheDP and the bulb became narrow. On the20th day, the shape of the DPbecame oval and was not completely enclosed by the HB. The folliclewas shorter, the bulb and the DP located close to the panniculuscarnosus. On the25th day, the DP was upward to the epidermis and leftbehind a long tail of CTS.2Immunofluorescence testing: The immunofluorescence resultsdemonstrated that draxin expression was localized in the DP through thefollicle cycle. The draxin expression was intense in anagen (5,10and 16d), moderate in catagen (20d) and weak in telogen (25d). On the5thday, the draxin expression was also in CTS, but rarely expressed on the10th,16th,20th and25th day after depilation.3Length of vibrissa measurment: No growth of the vibrissa of twotypes of mice was oberserved on the5th day after depilation. The lengthof vibrissa of draxin-/-mice was longer than draxin+/+mice on the10th,16th,20th and25th day after depilation. The result has significantdifference.Conclusion:1Time points5,10,16,20and25d are the characteristic stages ofanagen, catagen and telogen.2The draxin expression exists dynamic change in follicle cycle.3The vibissa length in driaxin knockout mice was longer than wildtype, which suggested that draxin might have inhibition in follicle cycle.