/ . Embryol. exp. Morph. Vol. 36, 3, pp. 597-607, 1976
Printed in Great Britain
597
Morphological changes associated with the growth
cycle of vibrissal follicles in the rat
By R. D. YOUNG 1 AND R. F. OLIVER 1
From the Department of Biological Sciences, University of Dundee,
Scotland
SUMMARY
Morphological changes which occur in the growth cycle of the rat vibrissal follicle during
the transitional period between consecutive anagen phases are described. In contrast with
pelage hair follicles, there is no shortening of the follicle, no formation of a papilla 'rest' and
no close synchrony between club differentiation and follicle regression. Telogen is therefore
considered to occur after loss of the matrix of the hair bulb and maximal diminution of the
dermal papilla to a small aggregation of cells. These differences are discussed in relation to
current nomenclature of the hair cycle and the function of the vibrissal follicle.
INTRODUCTION
The characteristic periodicity of hair growth is an intriguing and still little
understood feature of the proliferation of mammalian skin. Many investigations have been made of the cyclic activity of the pelage hair follicles of
rodents, and the morphological changes which occur in the progression from
anagen (growing phase) through catagen to telogen (resting phase) are now
well-established (Chase, Rauch & Smith, 1951; Straile, Chase & Arsenault,
1961; Ebling & Johnson, 1964). A vast temporal diversification has long been
recognized in these hair cycle schedules, not only between species, but also
between follicle types and even anatomical locations within any one animal. In
addition, sex, age and diet form intraspecific variables on which cycle and
individual phase durations may depend.
Our present understanding of follicle activity has arisen almost exclusively
from observations of the pelage. There are, however, several comprehensive
accounts of the gross morphology of anagen vibrissal follicles in rats (Vincent,
1913) and mice (Davidson & Hardy, 1952; Melaragno & Montagna, 1953).
Vibrissal follicles, which constitute the largest fibre-producing structures of
adult rats, are characterized by an outer collagen capsule which contains the
ring and cavernous sinuses surrounding the central follicular core. These
features, probably associated with the sensory function of the follicle, contribute
1
Authors' Address: Department of Biological Sciences, Dundee University, Dundee,
DD1 4HN, Scotland, U.K.
598
R. D. YOUNG AND R. F. OLIVER
to its large circumference and are absent from the follicles of the pelage.
While there has been an increasing use of vibrissal follicles in experimental
studies since the pioneering work of Cohen (1961), comparatively little is known
of the hair cycles of these large tactile follicles. There is evidence (Ling, 1965)
that in certain species of marine mammal they undergo extensive resting phases.
More recently, the work of Lyne, Downes & Chase (1974) on the marsupial
Trichosurus vulpecula has shown that vibrissal follicles may retain whiskers for
more than one year after the completion of the growth phase. This contrasts
strongly with the situation in rodents where vibrissae appear to be in a state of
constant proliferation. However, these follicles do cease fibre production, but
for only a short time, and Ibrahim & Wright (1975), on the basis of measurements of whiskers, have suggested that the resting phase lasts less than 3 days
in the rat.
Fundamental problems of the control of the follicle cycle centre around the
initiation and termination of the growth phase. Vibrissal follicles, by virtue of
their superior size, prominent disposition and remarkably constant distribution (Danforth, 1925; Oliver, 1966), would seem to be ideally suited to investigations of these critical events, but for the lack of evidence that they undergo a
telogen phase at all (Garrett & Hashimoto, 1974).
In view of the increasing use of the vibrissal follicle as an experimental model,
a fuller understanding of morphological events throughout the growth cycle is
essential. The present study describes the sequence of events which occur in
mystacial vibrissal follicles of the rat during the period between the production
of successive generations of whiskers.
MATERIALS AND METHODS
Vibrissal follicles were obtained from the mystacial region of animals from an
inbred strain of hooded rat (Colony, Dundee University). They are embedded
in the musculature of the upper lip in a regular array of horizontal and vertical
rows; the largest, most posterior follicles produce the longest vibrissae which
commonly measure 45-60 mm in males and 40-50 mm in females.
The routine location of follicles in the intergrowth period proved to be difficult. Direct measurement of whisker growth from the skin surface was not
sufficiently accurate for the detection of declining bulbar proliferation. Moreover, follicles at this stage, exposed by reflexion of the whisker pad, could not
be easily recognized macroscopically as there was no dramatic shortening of the
follicle as occurs in the pelage.
The following observations derive therefore from the study of 27 follicles
in the intergrowth period. These were obtained by histological examination of
more than 150 follicles, dissected individually from the mystacial pads of
freshly killed rats. Excised follicles were fixed in formol-saline, embedded in
paraffin wax and sectioned serially at 7-8 /.im. These sections were then stained
Growth cycle of vibrissal follicles in the rat
599
with haematoxylin and eosin, Cason's one-step Mallory method or a combination of alcian blue, Weigert's haematoxylin and Curtis's Ponceau S. Alcian
blue stains specifically for acid mucopolysaccharides. The cyclic fluctuations in
metachromasia of pelage follicle dermal papillae, revealed by staining with
toluidine blue, were recognized as a characteristic feature of the hair growth
cycle as early as 1950 (Sylven), but the significance of the underlying polysaccharide material to the production of hair remains uncertain.
OBSERVATIONS
In the anagen vibrissal follicle, the bulb-like appearance of its proximal
region (Fig. 1) results primarily from a distension of the collagen capsule by
vigorously dividing epidermal cells and the enclosed dermal papilla. The
epidermal component, often termed the matrix, exhibits a pronounced basophilia throughout the germinal region and especially along the basement membrane where it is associated with a population of dendritic melanocytes. Maturing
epidermal cells move distally to keratinize and form the hair and hair
sheaths. The papilla, invested by matrix, appears as a richly vascularized plug
of dermal cells, with a high affinity for alcian blue, which extends distally into the
medulla of the hair and terminates proximally in a well-defined stalk, contiguous with the follicle mesenchyme layer and collagen sheath below.
The transition from this anagen condition to catagen and subsequent events
are shown in Figs. 2-8 and diagrammatically in Fig. 9. As the hair approached
its terminal length, the first detectable morphological change in the bulb,
associated with the onset of catagen, appeared as a decrease in the volume of the
matrix (Fig. 2), accompanied by a decline in the proliferation of matrix cells
and by continued upward movement of the hair. The disappearance of basophilia occurred first in epidermal cells around the papillary stalk and progressed
distally. These cells subsequently appeared identical to those of the lower outer
root sheath. A progressive reduction in the extent to which the papilla was
enclosed proximally by epidermal cells and an apparent increase in the size of
its stalk were also seen. In addition, the stalk became surrounded by a translucent collar with a high affinity for alcian blue, clearly shown in Figs. 2-6.
The well-ordered, radial arrangement of maturing epidermal cells became
obscure early in the transitional sequence, but at this stage, the germinal region
still appeared very active. Epidermal cells continued to move upward and the
formation of inner root sheath, cortex and medulla persisted via a normal
keratogenous zone. During the initial decline in epidermal mitosis, there was
a gradual increase in the density of nuclei in the apical region of the dermal
papilla (Figs. 3 and 4), a process which represented a reduction in cytoplasmic
volume of the papillary cells.
The decline in proliferation of epidermal cells was followed by the commence-
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R. D. YOUNG AND R. F. OLIVER
Growth cycle of vibrissal follicles in the rat
601
ment of club root formation. Less frequent mitosis and declining basophilia in
the matrix preceded the cessation of medulla and inner root sheath cell production and the disappearance of the keratogenous zone. Subsequently,
cortical cell lines ceased division as the whisker continued to emerge distally,
removing at its origin the majority of the upper epidermal cell population from
the bulb. These cells underwent keratinization and contributed to the manypronged club root, which ultimately held the dead fibre in the follicle at the
level of the sebaceous glands.
As the processes causing the separation of the dermal papilla and the maturing
whisker neared completion (Fig. 5), the papilla appeared stretched between its
tenuous attachments with the disengaging club and its connexion with mesenchymal cells and collagen capsule below. At this stage, papillary cytoplasm no
longer stained well with alcian blue although the pedicular collar of acid
mucopolysaccharides persisted. The glassy membrane became more prominent,
a feature which could be recognized throughout the subsequent changes, and
remained as a supporting tube to contain the regressing tissues which collapsed
in the wake of the distally moving club root. Basophilia of epidermal cells
finally became restricted to a cell monolayer apposed to the basement membrane around the papilla while remaining cells became indistinguishable from
those of the lower outer root sheath (Fig. 6). The continued diminution of the
dermal papilla eventually resulted in a small, compact ball of cells with much
reduced cytoplasmic volume and less abundant capillary supply, enclosed
laterodistally in a shallow inverted cup of epidermal cells (Fig. 7). Substantial
thickening of the basement membrane accompanied this reduction in papillary
size while epidermal mitotic figures were not seen. However, a crescent-shaped
group of basophilic epidermal cells was retained above the papilla. No residual
affinity for alcian blue was detectable within the dermal papilla or its stalk by
the time this total cessation of whisker production was attained.
FIGURES
1-4
Fig. 1. Bulb region of anagen vibrissal follicle surrounded by cavernous sinus (CS)
showing active epidermal matrix (M) investing dermal papilla (DP) and giving rise to
keratinizing hair cortex (Co), cuticle (Cu) and inner root sheath (IRS). Outer root
sheath (ORS). The papilla is confluent with the follicle mesenchyme layer {ML) via
its stalk (PS). (See Fig. 9 A.) Haematoxylin and eosin. x 160.
Fig. 2. Early catagen showing decline in matrix volume and loss of epidermal cell
basophilia proximally (arrows). Note vacuole adjacent to papillary stalk. (See
Fig. 9B.) Haematoxylin and eosin. x 160.
Figs. 3 and 4. Subsequent stages in mid-catagen: further decline in the matrix is evident and reduction in papillary size is emphasized by increased cellular density,
especially in the apical region. Melanocytes (Me) along the basement membrane
and capillaries (Ca), within the papilla, are clearly visible. Haematoxylin and eosin.
xl60.
602
R. D. YOUNG AND R. F. OLIVER
I
Growth cycle of vibrissal follicles in the rat
A
603
I)
Fig. 9. Diagram of vibrissal follicle growth cycle. (A) Metanagen. (B, C) Stages in
catagen: removal of matrix to form the club and regression of the papilla. (D) Telogen: the bulb is maximally regressed and the club has disengaged from the papilla.
(E) Proanagen: regeneration of the bulb occurs while the club continues to differentiate. (F) Metanagen: mature club and rapidly growing whisker present together.
At that time, and during the ensuing emergence of the developing club,
the basic structure of the follicle remained unchanged. There was no discernible
modification of the blood supply and the collagen capsule, as would be expected,
remained in toto as a permanent feature. Moreover, the central epidermal core
FIGURES 5-8
Fig. 5. Advanced stage of catagen showing proximal region of the developing club
hair (CH), prominent glassy membrane (GM) and 'stretched' dermal papilla. The
mucopolysaccharide collar is still conspicuous. (See Fig. 9C.) Alcian blue, Weigert's
haematoxylin and Curtis's Ponceau S. x 240.
Fig. 6. Late catagen: the club root is disengaging from the papilla which is much
reduced in size. Note the prominent papillary stalk. Alcian blue, Weigert's haematoxylin and Curtis's Ponceau S. x 320.
Fig. 7. Telogen: papillary diminution is maximal. The proximal aspect of the completely disengaged club is just above the field of the photograph. The epidermal cells
are identical to outer root sheath (O7?5),although residual basophilia is present along
the basement membrane. (See Fig. 9D.) Alcian blue, Weigert's haematoxylin and
Curtis's Ponceau S. x 320.
Fig. 8. Lower region of follicle in proanagen showing regenerating matrix, keratinizing inner root sheath and incompletely invested papilla. The club hair is immature
and below its definitive anchorage. (See Fig. 9E.) Cason's trichrome. x 140.
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R. D. YOUNG AND R. F. OLIVER
A
B
Fig. 10. Diagram of pelage follicle growth cycle.
(A) Metanagen.
(B-D) Stages in catagen showing follicle regression and club formation.
(E) Telogen: a mature club hair and papilla 'rest' are present. The inner root
sheath is absent and follicle shortening is at a maximum.
(F) Proanagen: follicular downgrowth and regeneration of the bulb occur.
underwent none of the dramatic shortening which occurs in pelage follicles.
The dermal papilla was therefore retained at its anagen locus in the proximal
extremity of the follicle.
Subsequently, upon the resumption of anagen, increased basophilia and
mitosis of epidermal cells resulted in the re-investment of the papilla which
simultaneously expanded. Keratinization of epidermal cells was resumed before
the full matrix complement was restored and therefore preceded the complete
proximal inclusion of the dermal component. The latter, too, attained its full
anagen configuration only after keratinization had recommenced. The earliest
stage in anagen yet observed was present in three follicles examined, and revealed a cone of inner root sheath cells capping the regenerating matrix, while
the dermal papilla already exhibited a high affinity for alcian blue. At this time,
the proximal aspect of the club root was not far removed from the base of the
follicle (Fig. 8). Keratinizing inner root sheath and cortical cell lines very
rapidly gave rise to a slender whisker, the un-medullated tip of which was
observed at the level of the skin surface while the developing club was still below
its definitive anchorage in the follicle.
Growth cycle of vibrissal follicles in the rat
605
DISCUSSION
A major problem associated with our understanding of the production of
vibrissae has arisen through a failure to detect and describe the intergrowth
period. However, the general supposition has been that catagen and telogen do
occur, but that they occupy only a very limited period of the growth cycle
(Chase, 1954; Oliver, 1966; Ibrahim & Wright, 1975).
The intergrowth period of vibrissae essentially contains changes within the
matrix and papilla (Fig. 9). Observations made in the present study support the
suggestion made by Straile et ah (1961), from a study of pelage follicles, that
catagen should be regarded as a phase of terminal differentiation rather than one
of degeneration, as the reduction in matrix results not only from declining
mitosis but also from the removal of these cells to form the hair club. In vibrissal
follicles, while remaining epidermal cells reverted in appearance to outer
root sheath cells, basophilia persisted in those adjacent to the basement
membrane which suggests that there is never a complete cessation of RNA
synthesis. Accompanying changes in the dermal papilla culminated in its
reduction to a small ball of cells (Fig. 9D). The appearance of the papillary apex
seemed to follow closely the synthesis of the hair medulla although it is unlikely that the former is directly responsible for medullary cell production
as this feature of the papilla is absent in many follicle types which produce
medullated fibres.
An extensive reduction in the number and size of papillary blood vessels was
observed during the period of maximal regression, but functional capillaries
could still be recognized. Vascular changes are almost certainly secondary events
in the precipitation of a follicle into catagen, as indicated by Ellis & Moretti
(1959). A distinctive fluctuation in the presence of acid mucopolysaccharides in
the papilla was also recognized throughout the growth cycle although the
present work sheds no light on the possible significance of these macromolecules
to the growth of hair. Due to the difficulty in locating follicles during the
earliest stages of anagen, it could not be ascertained whether mucopolysaccharide synthesis preceded the regeneration of the matrix or vice versa.
These processes seemed to follow an even shorter time-course than those
involved in club disengagement.
Comparison of changes undergone by vibrissal and pelage follicles
during the intergrowth period
As a result of physiological considerations, nomenclature describing the
pelage follicle cycle has been suggested (Chase, 1965) which emphasizes the
continuous nature of the regeneration process. Five distinctive phases, designated proanagen (anagen I-1V), mesanagen (anagen V), metanagen (anagen
VI), catagen and telogen are recognized. The critical stages in terms of the
initiation and termination of the intergrowth period are illustrated in Fig. 10.
606
R. D. YOUNG AND R. F. OLIVER
It is especially significant that in pelage follicles of the rat and mouse, regression
of the dermal papilla, and the formation of a hair club, accompanied by follicle
shortening, proceed through catagen to a simultaneous climax. At this point
a morphological situation defined precisely by the term telogen is established
(Fig. 10E). The follicle remains in this state for up to several weeks.
In marked contrast, this phase is extremely brief in rat vibrissal follicles and
terminal differentiation of the club hair is not associated with either extensive
shortening of the follicle or with the formation of a papilla 'rest'. Moreover,
this variation is not exclusive to the vibrissal follicle since a similar situation has
been described by Ling (1965) for the pelage follicles of the southern elephant
seal. In spite of these morphological differences, the definitive phases of the
hair cycle, with the possible exception of telogen, can still be recognized. However, vibrissal follicles exhibit another temporal variation from the pelage in
that the proanagen and mesanagen phases both occur before club differentiation
is completed (Fig. 9). So far as we are aware, this occurrence of anagen before
the completion of club formation is unique in hair follicle cycles and these
differences cannot be accurately accommodated by existing nomenclature for
the growth cycle. The problem arises as to whether vibrissal follicles undergo a
telogen phase. This term, by definition, refers to the termination of the formative phase which can be considered in general terms as a cessation of the
production of cells destined to become keratinized and incorporated into hair.
In vibrissal follicles, while a brief period exists during which no new cells are
formed for this purpose, there is simultaneously a continuation of controlled
synthetic processes involved in the maturation of cells already produced. Thus,
keratinization persists in cells of the proximal regions of the club although
proliferation has ceased at their site of origin. This period of club maturation
could be regarded as a sub-stage of catagen (compare Figs. 9D and 10D), in
which case, a telogen phase would be absent from the growth cycle of vibrissal
follicles. Pre-determined events concerned with the final development of a hair
would then receive more emphasis than mechanisms involved in the control of
follicle periodicity. We feel that the designation of the phase of maximal
papillary regression as telogen is reasonable, not only on morphological and
histological grounds, but more significantly on physiological considerations in
terms of the interactions between epidermis and dermis, which are now believed
to be of major importance in the growth and maintenance of skin and its appendages (Cohen, 1969; Oliver, 1971).
The rat vibrissal follicle undergoes an unusual overlapping of phases in the
growth cycle during the terminal differentiation of a hair. As a result, catagen
and, in particular, telogen phases make only a brief contribution to the cycle, in
comparison with the pelage follicle. One is tempted to regard them both as
transitional phases in a process wholly directed towards the rapid production
and replacement of a specialized mechanotransducer.
Growth cycle of vibrissal follicles in the rat
607
The authors gratefully acknowledge the support by a grant from the Science Research
Council.
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{Received 21 May 1976, revised 27 July 1976)