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Exogen, Shedding Phase of the Hair Growth Cycle: Characterization of a Mouse Model

2002; Elsevier BV; Volume: 119; Issue: 3 Linguagem: Inglês

10.1046/j.1523-1747.2002.01842.x

1523-1747

Yoram Milner, Michael Kashgarian, James Sudnik, Mario Filippi, Menas Kizoulis, Kurt S. Stenn,

Dermatologic Treatments and Research

The hair growth cycle is generally recognized to comprise phases of growth (anagen), regression (catagen), and rest (telogen). Whereas, heretofore, the hair shedding function has been assumed to be part of the telogen phase, using a laboratory mouse model and newly developed techniques for quantitative collection and spectroscopic determination of shed hair, we found that shedding actually occurs as a distinct phase. Although some shedding occurs throughout the growth cycle, the largest peak is coupled to anagen. Using hair dye and rhodamine labeling we established that the shafts that shed arise during the previous hair cycle. We found that over the cycle the ratio of shed overfur to shed underfur hair shafts varies with the cycle phase and that the shed shaft base is unique morphologically, having a cylindrical shape with scalloped or "nibbled" edges. By electron microscopy the mooring cells of the exogen root show intercellular separation suggesting a proteolytic process in the final shedding step. This is the first report describing a distinct shedding, or exogen, phase of the hair cycle. This study supports the notion that this phase is uniquely controlled and that the final step in the shedding process involves a specific proteolytic step. The hair growth cycle is generally recognized to comprise phases of growth (anagen), regression (catagen), and rest (telogen). Whereas, heretofore, the hair shedding function has been assumed to be part of the telogen phase, using a laboratory mouse model and newly developed techniques for quantitative collection and spectroscopic determination of shed hair, we found that shedding actually occurs as a distinct phase. Although some shedding occurs throughout the growth cycle, the largest peak is coupled to anagen. Using hair dye and rhodamine labeling we established that the shafts that shed arise during the previous hair cycle. We found that over the cycle the ratio of shed overfur to shed underfur hair shafts varies with the cycle phase and that the shed shaft base is unique morphologically, having a cylindrical shape with scalloped or "nibbled" edges. By electron microscopy the mooring cells of the exogen root show intercellular separation suggesting a proteolytic process in the final shedding step. This is the first report describing a distinct shedding, or exogen, phase of the hair cycle. This study supports the notion that this phase is uniquely controlled and that the final step in the shedding process involves a specific proteolytic step. Hair growth occurs over a cycle consisting of growth (anagen), regression (catagen), and resting (telogen) phases (Stenn and Paus, 2001Stenn K.S. Paus R. Hair follicle growth controls.Physiol Rev. 2001; 81: 449-494Crossref PubMed Scopus (1033) Google Scholar). At the end of hair growth the inferior portion of the follicle regresses. During this catagen phase the lower follicle moves up to the level of the arrector pili muscle and the base of the fully formed shaft differentiates, assuming the morphology of a brush, formed by cornified cells apparently arising within the region of the sheath (Ryder, 1965Ryder M.L. Wool fibre shedding and seasonal variation in wool growth.Proc R Soc Med. 1965; 58: 806-808PubMed Google Scholar). Some time after the follicle enters the resting phase, the hair shaft sheds. Because this shedding process was conceived to have its own definition and controls, it was recently referred to as the "exogen" phase (Stenn et al., 1998Stenn K.S. Parimoo S. Prouty S. Growth of the hair follicle: a cycling and regenerating biological system.in: Chuong C.-M. Molecular Basis of Epithelial Appendage Morphogenesis. RG Landes Company, Austin, TX1998: 111-130Google Scholar). The mechanism and control of hair shedding is unknown; moreover, in the absence of a good laboratory model and rigorous techniques the process remains ill defined. Nevertheless, some relevant work has been done. The timing of hair shedding appears to vary between sites and follicle types. In animals, molting appears to be influenced by genetics, photoperiod (Bissonnette, 1935Bissonnette T.H. Relations of hair cycles in ferrets to changes in the anterior hypophysis and to light cycles.Anat Rec. 1935; 63: 159-168Crossref Scopus (18) Google Scholar), and hormones (pituitary, thyroid, and adrenal) (Ryder, 1965Ryder M.L. Wool fibre shedding and seasonal variation in wool growth.Proc R Soc Med. 1965; 58: 806-808PubMed Google Scholar). Of the hormones, estrogens allegedly inhibit the loss of club hairs, high levels of ACTH and adrenal hormones stimulate shedding of club hair, and low levels of adrenocortical hormones retard shedding (Johnson, 1958Johnson E. Quantitative studies of hair growth in the albino rat. 2. The effect of sex hormones.J Endocr. 1958; 16: 351-359Crossref PubMed Scopus (50) Google Scholar). Mechanistically, the notion that the new shaft (arising from the underlying anagen follicle) somehow pushes the resting shaft out of the pilary canal would appear to be too simplistic (Dry, 1926Dry F.W. The coat of the mouse (Mus Musculus).J Genet. 1926; 16: 281-340Crossref Scopus (230) Google Scholar) as it cannot explain the many examples found in normal and abnormal states where hair shafts are retained in the pilary canal adjacent to growing anagenic follicles and shafts. Therefore, as shedding appears to occur independently of the follicle growth phase (Johnson, 1972Johnson E. Moulting cycles.Mammalian Rev. 1972; 1: 198-208Crossref Scopus (12) Google Scholar), the shedding phase appears to have its own unique set of controls. We have initiated a long-term study to discover and define the controls of this very important phase of the cycle. In this paper we describe studies on the occurrence and mechanistic details of hair shedding using the laboratory mouse. We report a method for collecting and quantifying shed hair, define the morphology of the exogen (shed) shaft versus the resting (telogen) shaft, and describe the pattern of shedding in the first spontaneous and induced cycles. Mice (Balb/c, C3H, C57Bl/6, female, 6–8 wk old, or pregnant dams) were purchased from local vendors (Charles River, Kingston, NY, or Taconic Laboratories, Germantown, NY) and housed as described previously (Stenn et al., 1994Stenn K.S. Lawrence L. Veis D. Korsmeyer S. Seiberg M. Expression of the bcl-2 protooncogene in the cycling adult mouse hair follicle.J Invest Dermatol. 1994; 103: 107-111Abstract Full Text PDF PubMed Google Scholar). Hair growth was induced in telogen skin mice by depilatory cream stimulus (Nair Lotion Hair Remover, Carter Wallace; 10 min exposures), by daily topical cyclosporinee applications (Paus et al., 1989Paus R. Stenn K.S. Link R.E. The induction of anagen hair growth in telogen mouse skin by cyclosporine A administration.Lab Invest. 1989; 60: 365-369PubMed Google Scholar) (CsA, Novartis Pharm, Basel, Switzerland; 2.5 mg per ml suspension in ethanol:propylene glycol, 7:3 vol/vol, respectively, applying approximately 300 µl to the back of unshorn mice, for 10–12 d) or by four daily intraperitoneal injections of 3 mg CsA in corn oil (adapted fromPaus et al., 1996Paus R. Bottge J.-A. Henz B.M. Maurer M. Hair growth control by immunosuppression.Arch Dermatol Res. 1996; 288: 408-410Crossref PubMed Scopus (33) Google Scholar). Animals were judged to be in telogen or anagen by their pink or gray skin color, respectively. A telogen hair shaft is defined as one that arises from a telogen follicle. An exogen shaft is defined as one easily collected by combing with gentle vacuum. For telogen hair collection, a tuft of hair in telogen skin (central mid back) was plucked using blunt forceps. For exogen hair collection, a modified pet comb (the "vacomb", a device specifically built for this work; see Figure 1) collects loose hairs on a fiberglass filter (Gelman Sciences Extra Thick Glass Fiber Filters, 25 mm, Gelman Sciences, Michigan) supported by a plastic grid holder (Corning Swin-Lok filter holder, 25 mm) by means of vacuum suction created by a laboratory pump (290 mmHg Savant VP 100) attached to the vacomb via a siliconized rubber tube. At each collection time combing was repeated 12–24 times (depending on the experiment) in a cephalad direction, over the vertebral line starting from the iliac crest and progressing to the scapular region. The shed hairs collected daily were quantified by either manual counting on adhesive glass slides (dissecting microscope, Leica MZ12), by weighing (Microbalance, Mettler, AE240) or by spectrophotometry. The spectrophotometric method for quantifying hair was by alkaline solubilization (2 ml of 1 N NaOH, overnight at 54°C) read by spectrophotometry (350 nm). Hair shafts were collected as described above and immediately fixed (10% phosphate buffered formalin) (VWR, West Chester, PA). For light microscopy the fibers were stained by the SACPIC method (Nixon, 1993Nixon A.J. A method for determining the activity state of hair follicles.Biotech Histochem. 1993; 68: 316-325Crossref PubMed Scopus (82) Google Scholar;Nutbrown and Randall, 1996Nutbrown M. Randall V.A. Recognition of cellular differentiation in the human hair follicle at the light microscope level using SACPIC staining.in: Van Nest D. Randall V.A. Hair Research for the Next Millenium. Elsevier, Amsterdam1996: 161-166Google Scholar) and for electron microscopy they were treated as previously described (Kveton and Kashgarian, 1994Kveton J.F. Kashgarian M. Particulate matter within the membranous labyrinth: pathologic or normal?.Am J Otol. 1994; 15: 173-176PubMed Google Scholar). Hair shafts were metabolically labeled by injecting 1 ml portions of rhodamine isothiocyanate (Sigma Chemical, St. Louis, MO) (mixed isomers, 4 mg per ml in phosphate-buffered saline) intraperitoneally, thrice over 15 h intervals, into 8-wk-old, anagen-induced C57Bl/6 mice. Hair fluorescence was observed microscopically from plucked and shed hair samples. For hair dying, Balb/c white mouse fur was exposed to commercial hair dye ("Just for Men", COMBE) using supplier's instructions (two applications over a 24 h interval). The apparatus constructed to collect hair adapts and connects a comb to a suction device via tubing and a filter trap. The loose hairs are then collected on a fiberglass filter. The collection device ("vacomb") is shown in Figure 1. We have empirically defined the loose hairs collected in this way as shed, exogen phase, shafts, which are shown in the figure on the filter. Hair was collected from each experimental mouse no more frequently than once per day. Initially, to quantify the sample we weighed or counted the number of hairs. Later, we found that spectrophotometric measurements of alkaline-dissolved hair preparations facilitated the determination and correlated very well with weight and number (correlation coefficient for weight, R = 0.96, and for hair counts, R = 0.99) (see Materials and Methods). We observed that the alkaline solubilization method is sensitive enough to detect three zigzag (fine, underfur) hairs and one nonzigzag (coarse, overfur) hair. Telogen and exogen hair shaft bases are distinguishable by their unique morphology, which is revealed best by light microscopy using the SACPIC stain (see Figure 2). The plucked telogen shaft base is somewhat rotund and club-shaped (Peereboom-Wynia, 1997Peereboom-Wynia J.D.R. Trichogram.in: Camacho F. Montagna W. Trichology, Diseases of the Pilosebaceous Follicle. Aula Medica Group, Madrid1997: 113-117Google Scholar). Its outline is smooth and its base consists of a centrally lying brush of keratinized cells surrounded by apparent mooring cells (which sometimes obscure the central lying brown-colored brush) containing easily found, discrete nuclei and abundant cytoplasm. The shed exogen shaft base, as shown in Figure 2(c), on the other hand, is shrunken, and has a more elongated shape and a scalloped and pitted margin ("nibbled"). At the final stage of exogen (not shown in the figure), it consists predominately of the keratinized brush devoid of cytologic detail. Within this shaft base there is little associated cytoplasm and very few shrunken and fragmented nuclei. The latter changes suggest an apoptotic process (Vaux and Strasser, 1996Vaux D.L. Strasser A. The molecular biology of apoptosis.Proc Natl Acad Sci USA. 1996; 93: 2239-2244Crossref PubMed Scopus (894) Google Scholar). In any shed hair specimen intermediate structures are found, between the telogen and the fully shrunken exogen club, suggesting a progressive and dynamic process of exogen shaft formation (Figure 2b). Electron microscope studies of the telogen shaft base show considerable cytologic details including desmosomes and distinct nuclei (Figure 3b); in contrast this detail is lost in the exogen shaft base (Figure 3a). It is notable that the telogen shaft appears to separate from its mooring cells by a cleft within the cytoplasm of the cells making up its club base (see open arrows in Figure 3b). On the other hand, the exogen shaft appears to separate from its cellular mooring between the cells that make up the club base (see arrow of Figure 3a), which might indicate a proteolytic process. To test the validity of the morphologic differences between the exogen and telogen shaft bases, a differential count was made of plucked and shed shafts taken from telogen and late anagen (stage VI) skin. As shown in Table I hair shafts shed from anagen skin have roots with exogen morphology ("nibbled" edge, elongated contour) (66%). Hairs plucked from anagen skin show predominately smooth contours and relatively intact mooring cell envelope (64%), but even at this time of the cycle about 19% of the shafts show early and late exogen morphology. Clearly, in this model telogen and exogen shafts/follicles are present in anagen skin. We attribute the rare anagen shaft forms found in this specimen to the plucking conditions, which lead to broken shafts. Shed shafts from telogen skin consist predominately of exogen forms (81%) with some residual telogen forms (8%). Shafts plucked from telogen skin consist of telogen (64%) and exogen (37%) forms. It is very likely that some of the shafts classified as telogen in the shed samples are, in fact, early exogen where the cell-cell adhesion is loosened before the diagnostic morphologic changes can be observed.Table IDistribution of various hair base forms in shed and plucked hair taken from skin with predominately telogen or anagen follicles. The data represent hair shafts collected from seven to eight mice.Total # hairsHairs with Telogen baseHairs with Exogen baseHairs with Anagen baseBroken hairs#%#%#%#%Telogen follicle skin Hairs shed7568618100811Hairs plucked31019864822720.6289Anagen follicle skin Hairs shed40457142646530.78020Hairs plucked319204646019247.53110 Open table in a new tab In order to establish when in the spontaneous cycle hair shafts are shed we collected by vacomb hair from day 10 through day 60 after birth of the mice. We correlated the number of hairs collected (shed) with the hair cycle phase using skin color (gray-black skin indicating anagen and white skin indicating telogen hair). From Figure 4(a) it can be seen that, whereas there is some background shedding (within the telogen phase) and variation between animals, the greatest amount of hair shedding occurs in anagen. It is important to appreciate that when we say "shedding occurs in anagen" we are describing a follicle with at least two shafts in two separate phases (though in one follicle): one shaft is growing (and thus is part of an anagen follicle) and one shaft is not growing (and thus is part of a telogen follicle) (see Figure 6). So, apparently, the shedding shaft leaves the pilary canal (undergoes exogen) as the new shaft enters and egresses the outer pilary canal (anagen VI). It is notable that the shedding peak is relatively broad in the newborn mouse. We interpret its breadth as due to the movement of the synchronized hair cycle wave along the back skin and thus the movement of the shedding peak. Because the hair growth wave moves over the mouse's back (Chase, 1954Chase H.B. Growth of hair.Physiol Rev. 1954; 34: 113-126Crossref PubMed Scopus (335) Google Scholar), what we assume is that we are measuring the average of the migrating exogen peak. Although by collecting hair in a smaller area we should have been able to sharpen the peak, in fact, for reasons that are not immediately obvious, we did not achieve this result.Figure 6Histology of late telogen/early exogen follicles. Histology of mouse skin (C57Bl/6) at the end of a growth cycle. (A) Late anagen/catagen form with telogen follicle from previous cycle. Note that the telogen shaft is separated from the out-moving anagen shaft by its own epithelial wall. Arrows indicate possible cleft formation of telogen/early exogen hair shaft base. (Magnification 200x.) (B) Horizontal section of follicle with three silos one of which contains a full anagen/catagen shaft, a second containing a possible telogen shaft and an empty silo (arrow). (Magnification 400x.) (C) Follicle with two silos, one representing a late catagen follicle and the other telogen (or early exogen showing an epithelial cleft at the base, arrow) (SACPIC stained, magnification 200x).View Large Image Figure ViewerDownload (PPT) Figure 4(b) shows the kinetics of hair shedding when anagen is induced (by cyclosporinee treatment). In contrast to the spontaneous cycle, as this technique induces essentially synchronous growth of all the hair follicles the shedding phase is somewhat sharper. These experiments corroborate the notion that the greatest extent of shedding is, in some way, coupled to anagen. We found that the proportion of overfur (guard, awl, auchene) to underfur (zigzag) varied slightly, but significantly, over the whole cycle. As shown in Figure 5, during the telogen stage of the cycle (during which all follicles of the skin are in morphologic telogen) a greater number of shed hairs are underfur (a ratio of 4:6 overfur:underfur) whereas during the anagen-coupled peak a greater number of hairs are overfur shafts (5.5:4.5 overfur:underfur). This finding supports the notion that smaller follicles tend to retain their shaft longer than larger follicles (Dry, 1926Dry F.W. The coat of the mouse (Mus Musculus).J Genet. 1926; 16: 281-340Crossref Scopus (230) Google Scholar) and that follicles differ not only in their size but also in their periodicity of shedding. The fact that the large-based anagen shaft is housed in the same follicle as the thin-based telogen shaft might suggest that the outwardly moving anagen shaft might drag the telogen shaft out with it, thus playing a role in the shedding mechanism. It is notable to recall that this phenomenon is unlikely as (i) the cuticle structure of the shafts would allow them to slide freely by one another, and (ii) the base of each shaft sits in its own epithelial-lined silo. Histologic examination documents the latter relationship. In Figure 6(a) the base of the telogen follicle shaft shows an epithelial wall separating the two shafts. Early cleft formation (arrows) between the proximal shaft and the mooring epithelial sheath cells is interpreted to be the first step of exogen. This is further shown in a transverse section of the skin in the anagen stage at a level close to the sebaceous gland in Figure 6(b). Here one can see a follicle with a cluster of three hair canals: one with hair in anagen (shaft with defined medulla), one in catagen/telogen (round and colorless shaft), and one enlarged, vacated canal without a shaft, the putative site of a recently shed hair (arrow). Figure 6(c) shows a vertical section of a late catagen and early exogen shaft base. The follicle on the right shows separation and some scalloping of the shaft base from the sheath (arrow) in what seems to be an early preparatory step for shedding. That there is a major shedding peak in anagen raises the question which shaft sheds in the coupled exogen phase: the newly formed shaft or the shaft formed in the previous cycle(s). In order to ascertain which hair shaft is shed in association with anagen, we labeled hair shafts with a fluorescent tag (rhodamine isothiocyanate) during induced anagen and then examined plucked and shed hairs for fluorescence with time (Figure 7). By this procedure a fluorescent tag is incorporated into the newly forming hair shafts. Fluorescent-labeled shafts can be seen in whole skin 7 d after the label injection (Figure 7b, white arrows). Two weeks later labeled shafts with a heavily labeled red fluorescence band, distal to the shaft base, were found in the plucked hair sample (not shown here; at this time the skin follicles were in the telogen phase). Concomitantly, very few labeled hairs were found in the shed hair sample (although 1%-2% labeled fragmented hair shafts were found). When anagen was again induced in this rhodamine-labeled skin, the shed hairs collected this time were heavily labeled (over 60% of the hair, Figure 7a). In a parallel experiment Balb/c white mouse fur was dyed black. The plucked and shed sample after dying showed only pigmented shafts; however, when anagen was induced (CsA treatment), a band of newly formed, white hair appeared over the mouse back after 12 d (Figure 7c, arrow). Although the plucked sample contained both white and black shafts, the shed sample consisted entirely of black hairs (Figure 7d). Both of these experiments show that the hair shaft that sheds arises in a previous anagen phase and that the newest formed shaft is retained. In this paper we have presented studies on the shedding, or exogen, phase of the hair cycle in the laboratory mouse. Using a novel system for collecting and quantifying hair, we found that exogen is coupled to anagen, that a shed hair base differs morphologically from a telogen hair base, and that the hair fiber shed is the one that has been in telogen phase for the longest time. The challenge of collecting and quantifying shed hair has not been an easy one. In previous studies hair shafts were collected from a brush, a comb, a sink, or a bath/shower drain; the hair was quantified by weighing or counting (e.g.,Randall and Ebling, 1991Randall V.A. Ebling F.J. Seasonal changes in human hair growth.Br J Dermatol. 1991; 124: 146-151Crossref PubMed Scopus (169) Google Scholar;Guerrara et al., 1997Guerrara M. Semino M.T. Rebora A. Quantitating hair loss in women: a critical approach.Dermatology. 1997; 194: 12-16Crossref PubMed Scopus (36) Google Scholar). Moreover, no one animal model has been put forth for analyzing this phase. The mouse model and methods presented here promise to fulfill these needs. One of the most surprising observations in this study is that shed and plucked telogen hairs differ at their base: the former has sculpted edges and is bordered by cell membranes with deteriorating nuclei whereas the latter has a smooth-edged border rich in cells with intact nuclei and with a separation fracture within basal cell cytoplasm. Although this distinction is not usually noted (Peereboom-Wynia, 1997Peereboom-Wynia J.D.R. Trichogram.in: Camacho F. Montagna W. Trichology, Diseases of the Pilosebaceous Follicle. Aula Medica Group, Madrid1997: 113-117Google Scholar), in fact Kligman, 1961Kligman A.M. Pathologic dynamics of human hair loss.Arch Dermatol. 1961; 83: 175-198Crossref PubMed Scopus (211) Google Scholar recognized this unique shed shaft before but ascribed it to a telogen follicle club hair. In clinical conditions where there was extensive hair loss he referred to the conditions as telogen hair loss or telogen effluvium, terms that we believe are misnomers as our studies suggest that shedding is post-telogenic. What is the mechanism of hair loss? Kligman, 1961Kligman A.M. Pathologic dynamics of human hair loss.Arch Dermatol. 1961; 83: 175-198Crossref PubMed Scopus (211) Google Scholar states: "the club hair is shed because it is forced out by the new hair". At the same time Kligman notes that there are several states in man and animals where multiple shafts may be present in one outer pilary canal and that growing shafts may grow past an adherent (resting) telogen follicle/shaft. The hypothesis that the growing shaft either pushes or pulls the resting hair shaft out appears to be unlikely for several reasons. First, the growing shaft is actually in its own sheath separate from the resting hair shaft. Although this morphology is well illustrated in the literature (Johnson, 1972Johnson E. Moulting cycles.Mammalian Rev. 1972; 1: 198-208Crossref Scopus (12) Google Scholar), it is important to reemphasize that each telogen shaft sits in its own epithelial silo quite separate from the anagen portion, or other telogen forms, of the same follicle (Figure 5). Histologically, then, exogen develops in a portion of the follicle that is quite separate from the anagen machinery and most of the outwardly migrating and newly forming shaft. Second, as mentioned above, the cuticle patterning should allow easy slippage. Third, animal follicles (e.g., dog, sheep) often normally house several shafts in the "same" follicle (Johnson, 1972Johnson E. Moulting cycles.Mammalian Rev. 1972; 1: 198-208Crossref Scopus (12) Google Scholar;Spearman, 1977Spearman R.I.C. Hair follicle development, cyclical changes and hair form.in: The Physiology and Pathophysiology of the Skin. 4. Academic Press, London1977: 1255-1292Google Scholar). Fourth, the trichostatic conditions (e.g., trichostasis spinulosus) illustrate that multiple shafts can rest pathologically in the "same" follicle (Dawber, 1997Dawber R. Diseases of the Hair and Scalp. Blackwell Science, Oxford1997: 258-259Google Scholar). So, for these reasons an outward moving anagen shaft probably does not "drag" or "push" a resting shaft out. Furthermore, our findings suggest an active intercellular separation process in the shedding shaft base, which would lead one to believe that physical contact or a push-pull force on the shedding shaft is not necessary. In this study we found that, though exogen also occurs in telogen follicles, the largest peak of exogen is coupled to anagen, though we do not yet know how. We have no data to suggest that exogen occurs with every anagen; in fact, there must be anagens in which exogen is suppressed, such as the trichostatic conditions and in the cycling of some animals' follicles, as mentioned above. We believe that the process of exogen has at least two steps (Figure 8). After a variable time in telogen the follicle base cells receive a signal, or series of signals, that initiate exogen. The latter turns on the effectors of shedding. Our preliminary work suggests that the effector step involves a proteolytic separation of the mooring cells. This conclusion is based on the following observations. (i) Shed hairs show a sharp intercellular separation of their basal cells (Figure 2, Figure 3). To achieve such a clean cell-cell separation we suspect a proteolytic mechanism is at play and that the responsible enzymes must be capable of lysing intercellular adhesions and/or desmosomes. Relevant to this finding is that desmoglein 3, a component of desmosomes, has been demonstrated to be important to telogen hair shaft mooring (Koch et al., 1998Koch P.J. Mahoney M.G. Cotsarelis G. Rothenberger K. Lavker R.M. Stanley J.R. Desmoglein 3 anchors telogen hair in the follicle.J Cell Sci. 1998; 111: 2529-2537PubMed Google Scholar). (ii) Preliminary studies show that the exogen shaft base expresses significant chymotryptic- and tryptic-like proteolytic activities (Milner and Stenn, unpublished). It is notable that other groups have identified proteolytic activities in this region of the follicle as well (Ekholm and Egelrud, 1998Ekholm E. Egelrud T. The expression of stratum corneum chymotryptic enzyme in human anagen hair follicles: further evidence for its involvement in desquamation-like processes.Br J Dermatol. 1998; 139: 585-590Crossref PubMed Scopus (38) Google Scholar;Meier et al., 1999Meier N. Dar T.N. Boehm T. A novel serine protease overexpressed in the hair follicles of nude mice.Biochem Biophy Res Commun. 1999; 258: 374-378Crossref PubMed Scopus (30) Google Scholar;Jensen et al., 2000Jensen P.J. Yang T. Yu D.-W. Baker M.S. Risse B. Sun T.-T. Lavker R.M. Serpins in the human hair follicle.J Invest Dermatol. 2000; 114: 917-922Crossref PubMed Scopus (26) Google Scholar). Despite the presence of proteolytic activity and enzymes within this locus, the proteolytic control may instead be in the form of a released proteolytic inhibitor. To this point the PAI-2 inhibitor has been found in the postmitotic cells of the outer root sheath directly abutting the club (Lavker et al., 1998Lavker R.M. Risse B. Brown H. Ginsburg D. Pearson J. Baker M.S. Jensen P.J. Localization of plasminogen activator inhibitor type 2 (PAI-2) in hair and nail: implications for terminal differentiation.J Invest Dermatol. 1998; 110: 917-922Crossref PubMed Scopus (39) Google Scholar). Although the final step in shedding may be proteolytic, we do not yet know what initiates this process. We observed here that the greatest amount of shedding in the highly synchronized mouse cycle is associated with late anagen and that, as long as the follicle is kept in telogen, significant shedding does not occur (Table I, Figure 4). Because hair is a protective covering in most animals, it is critical that hair loss does not occur before a new replacement hair fiber has formed, so it is logical that the animal would not shed its old hairs before new hairs are in place. By such an argument it would be optimal for exogen to occur not before the new growth is in mid to late anagen. In summary, recognizing that the telogen compartment is separate from the anagen compartment we found that the largest exogen component is coupled to anagen, that the exogen shaft base differs from the telogen shaft base, and that the shedding shaft is the earliest one formed. Above all, these studies indicate that there is a distinct shedding phase, exogen, and it merits critical scrutiny. With the methods to collect and quantify shedding and the defined animal model presented here we would expect greater insights into exogen in the near future. The authors would like to thank Alain Khaiat, Johnson & Johnson, SE Asia, for material support, Rick Ouellette, Don Wells, and Walter Markulec for help in constructing the vacomb, and Mary Deubler for help with the manuscript. Corrections to "Exogen, Shedding Phase of the Hair Growth Cycle: Characterization of a Mouse Model"Journal of Investigative DermatologyVol. 120Issue 6PreviewIn the September issue of JID the following figures, accompanying the article "Exogen, Shedding Phase of the Hair Growth Cycle: Characterization of a Mouse Model" by Milner et al should have appeared in color. Full-Text PDF Open Archive