J. Embryol. exp. Morph., Vol. 17, 1, pp. 27-34, February 1967
With 1 plate
Printed in Great Britain
27
Ectopic regeneration
of whiskers in the hooded rat from implanted
lengths of vibrissa follicle wall
By R. F. OLIVER 1
From the Department of Zoology and Comparative Physiology and
The Medical Research Council, Unit for Research on the
Experimental Pathology of the Skin, The University of Birmingham
INTRODUCTION
Previous studies relating to the effect on whisker growth of removal of
various components of the whisker follicle have shown that both after removal
of the dermal papilla alone and after removal of as much as the lower third of
the follicle, regeneration of new dermal papillae and then the generation of
whiskers may occur (Oliver, 1966 a, b).
Histological studies of this regenerative phenomenon revealed that the outer
root sheath became a solid cord or rod of cells and that dermal cells, derived at
least in part from the mesenchymal layer, aggregated over the proximal surface
of the rod and eventually became the new dermal papilla.
To determine whether the regenerative process was dependent on factors
present in the local follicle environment on the upper lip, lengths of isolated
follicle wall ('follicle tubes') composed of the outer root sheath and the adherent
mesenchymal layer were transplanted as autografts to ascertain whether they
could regenerate dermal papillae and fibres in another site.
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MATERIALS AND METHODS
Operations
All operations were performed on animals 2-3 months old from an inbred
strain of Hooded rat. Anaesthesia was induced by the intraperitoneal injection
of Nembutal (Abbott), 0-065 cc/100 g body weight. The whisker follicles on the
upper lip were exposed by making an incision below the most ventral row of
whiskers and reflecting the whisker pad. The proximal (i.e. bulbar) ends of the
follicles were dissected free of connective tissue and lengths of the lower end of
the follicle, 'root ends', were then removed. The level at which the root ends
were removed determined the most proximal level of the follicle tubes since
1
Author's address: Medical Research Council, Unit for Research on the Experimental
Pathology of the Skin, The Medical School, The University, Birmingham 15, U.K.
28
R. F. OLIVER
these were obtained from the lengths of follicle remaining. Growing whiskers in
the follicles were plucked either before or, more usually, after the removal of the
root ends. The follicle tubes were isolated by making two opposing incisions in
the capsule, along the long axis of the follicle, with a pair of iridectomy scissors.
The incisions were taken up to about the level of the ring sinus, and the dermal
trabeculae in the cavernous sinus severed. The two capsule flaps were then cut
off and discarded.
The implantation site was then prepared and each tube removed by a transverse cut and implanted. All but one of the follicle tubes were implanted into
ear skin in pockets made in the dorsum of the ear with a fine scalpel. One
follicle tube was implanted into an ovarian capsule from which the ovary had
been removed after ligaturing the ovarian blood supply.
After operation the whisker pad was stitched back in position. The wound
healed within a week, and at no time did the animals have difficulty in drinking
or feeding.
Design
Transplantation operations were performed on eleven rats. Sixteen follicle
tubes were implanted into ear skin, seven singly and nine in groups of three. A
further tube was implanted into an ovarian capsule.
The growing whiskers were plucked either before or after the removal of the
root ends in the preparation of sixteen of the implants; one of these was divided
into a proximal and a distal length before implantation. The single unplucked
tube was divided into two longitudinally. The remaining fifteen were implanted
intact. Of these, four tubes were dissected out so that the most proximal aspect
of the implants had been derived from within the inferior enlargement; a further
nine were prepared so that the most proximal level of each tube was obtained
from just above the level of the follicle bulb. The other two, including the one
implanted into ovarian capsule, were obtained from between these two regions
as were the two divided implants.
The implantation sites were examined at intervals and then biopsies were
performed at various times after operation.
Six additional tubes, from two other rats, werefixedimmediately after removal
and prepared for histological examination. Three of these were obtained after
plucking before the removal of root ends, and three after removing the root ends
before plucking.
Histology
All material was fixed in formol saline, serially sectioned at 8 /i and stained
with Weigert's haematoxylin and alcian blue and counterstained with Curtis's
Ponceau S.
Transplantation of vibrissa follicle wall
29
RESULTS
Five of the six follicle tubes fixed immediately after removal and sectioned
contained the keratinized inner root sheath as well as the outer root sheath and
mesenchymal layer (Plate 1, fig. A). Matrix tissue from the bulbar region had
lodged in one of the three tubes obtained after first plucking the growing shaft
before removing the root end.
Nine tubes obtained by first removing just the end bulb and then plucking the
growing shaft had been implanted, in groups of three, into single pockets in the
left ears of three rats. A single whisker was observed growing from each of
the three implantation sites, after 43 days, at which time their lengths were 3,
3 and 4 mm.
Sections of one of the ears, biopsied 43 days after operation, revealed the
presence of dermal papillae in association with all three of the implants including, of course, the one which had made contact with the ear epidermis and
which had produced a whisker above this level (Plate 1, fig. G). One of the
papillae, flattened distally, was present in a very short 'follicle', which was
confluent with the ear epidermis, and producing keratin not as a fibre but in
layers parallel to the skin surface (Plate 1, fig. G). A few melanocytes were
present in the 'matrix', in the proximal region of which were several mitoses,
and a small ductless sebaceous gland was attached to the neck of the follicle.
This follicle was considered to have been derived from the second of the implants
because immediately below and to one side of the follicle was an isolated glassy
membrane. No ectodermal cells could be seen within the membrane, which was
surrounded by dermal cells which had also apparently passed through the
membrane into its lumen. A developing dermal papilla was present at the
proximal end of the third implant. Cells from the mesenchymal layer appeared
to be passing through the glassy membrane into the position occupied by the
developing papilla (Plate 1, fig. H).
The two other ears were biopsied 119 days after operation. A white whisker
of 6-5 mm length was present above skin level in one of these ears (Plate 1,
fig. J) and a pigmented whisker coil was discernible under the skin surface of the
same ear. Histological examination revealed that a fine non-pigmented whisker
had also been produced, from the third implant. The pigmented whisker was
probably produced from the only tube, very short in length, removed from a
follicle producing pigmented whiskers.
Sections of the third ear, from which a whisker of 5 mm length had been
produced, revealed the presence of the two other implants which had persisted
but showed no evidence of papilla formation or whisker growth.
The tube which had been plucked before root end removal and implanted into
an ovarian capsule was biopsied after 71 days. A coiled whisker, at least 5 mm
in length, had been produced. The follicle was in a resting condition and contained a small dermal papilla which had not been stained with alcian blue; there
30
R. F. OLIVER
were no mitoses at the proximal end of the outer root sheath rod. The whisker
shaft was medullated and had grown out of one side of the implant (Plate 1,
fig. F).
One tube, which had been plucked before root end removal, had been divided
into distal and proximal lengths which were implanted separately under ear
skin. External examination at 61 days revealed a whisker 2 mm in length, doubled
at its base, growing above skin level from the proximal segment. Sections of this
material demonstrated that the follicle was continuous with the ear epidermis
distally and that in fact three separate fibres, enclosed in a common outer root
sheath, were being produced associated with three distinct dermal papillae
(Plate 1, figs. C, D). No fibre growth was detected from the distal segment,
which was unfortunately lost during histology.
PLATE 1
All sections stained with Weigert's haematoxylin, alcian blue and
Curtis's Ponceau S.
Fig. A. Longitudinal section of a long, plucked follicle tube removed from a follicle in early
anagen. The inner root sheath, which is becoming keratinized, is sectioned obliquely to the
left; peripheral to this is the outer root sheath, glassy membrane and mesenchymal layer.
x75.
Fig. B. Longitudinal section of a tube implant 33 days after implantation into ear skin. The
outer root sheath has become solid; the basophilic proximal region contains cells in division.
An aggregation of dermal cells is present at the proximal surface of the rod. x 140.
Fig. C. Oblique, nearly transverse, section through the bulbar region which has developed
in an implant 61 days after implantation into ear. Three dermal papillae, two of which are
very small, have regenerated (arrows), x 185.
Fig. D. Transverse section through the distal region of the implant shown in fig. C. Three
whisker fibres, enclosed in a common outer root sheath, can be seen (arrows) corresponding
to the three dermal papillae present in the bulb. The largest whisker had grown 2 mm above
the skin, x 185.
Fig. E. Longitudinal section of a tube implant, originally removed from above the lower
third of a follicle, 74 days after implantation into ear. The proximal end of the implant is to
the right; cells are keratinizing into a lumen, probably the persisting follicle cavity, x 60.
Fig. F. Longitudinal section of an implant 71 days after implantation into an ovarian capsule,
after which time a whisker (right arrow) of over 5 mm had been produced. The follicle is in
a resting condition; the dermal papilla is present as a small indentation (left arrow), x 45.
Fig. G. Section through two implants 43 days after implantation into ear. The very short
'follicle' at the top, considered to have been derived from the outer root sheath alone, is
producing keratin not as a fibre but in layers parallel to the skin surface. The dermal papilla
has a flattened distal aspect. The lower implant, sectioned obliquely through the bulbar
region, is confluent with the epidermis in other sections. A fibre, 3 mm in length, was being
produced from this implant, x 75.
Fig. H. Longitudinal section through the proximal end of the third implant implanted into
the ear shown infig.G. Cells from the mesenchymal layer, apparently in process of passing
through the glassy membrane, are indicated, x 45.
Fig. J. Left ear of a rat 119 days after implantation of three follicle tubes. A white whisker
(arrow) 6-5 mm long has been produced from one of the implants and is probably second
generation.
/. Embryo]'. exp. Morph., Vol. 17, Part 1
R. F. OLIVER
PLATE 1
facing p. 30
Transplantation of vibrissa follicle wall
31
The four tubes plucked after removing the root ends so that their most
proximal level was within the inferior enlargement were implanted separately
into four ears. No whiskers were produced from these implants, which were
biopsied 54, 72, 74 and 74 days after operation. Sections of these implants
showed that the outer root sheath cells had coalesced proximally although a
lumen into which cells were keratinizing was present in the more distal region.
There were no indications of dermal papilla formation or fibre production
(Plate 1, fig. E).
Of the two other tubes implanted into ear skin, one was unplucked and had
been divided into two longitudinally. It was biopsied 21 days after operation.
Mitoses were present in the proximal region of the outer root sheath but no
dermal papilla formation had occurred in either piece of material. The last tube
had been plucked before root end removal and was biopsied after 33 days. The
proximal outer root sheath cells had coalesced to form a rod and there was a
slight aggregation of dermal cells, confluent with the cells of the mesenchymal
layer, over the proximal surface of the rod which may represent an early stage
in dermal papilla regeneration (Plate 1, fig. B). The outer root sheath just distal
to the dermal aggregation was basophilic and contained many cells in division.
DISCUSSION
The results clearly demonstrate that proximal lengths of follicle tube, completely devoid of dermal papilla cells, can regenerate dermal papillae and short
whiskers when transplanted under ear skin or into ovarian capsule. However,
none of the implants developed those dermal features typical of vibrissa follicles:
attenuated apex to the dermal papilla, ringwulst and a sinus system enclosed
within a capsule. It is worth noting here that transplanted 'end bulbs' and
'whole papillae' (dermal papillae with their ectodermal matrices) which can
produce short whiskers also do not develop the typical vibrissa dermal features
mentioned above (Cohen, 1961).
In the tube implants the regenerating dermal papillae appear to derive mainly
from the mesenchymal layer, with which they are always confluent; they develop
as after the removal of dermal papillae and root ends (Oliver, 19666), at the most
proximal level of the outer root sheath after this has become a rod of cells.
None of the lengths of tube obtained so that their most proximal aspect was
from within the inferior enlargement produced whiskers, nor were there any
discernible signs of papilla formation. These implants did, however, maintain
their typical histological character. They behaved, therefore, in a very similar
fashion to lengths of follicle left in situ after removal of more than the lower third
of the follicle; these also do not regenerate dermal papillae or whiskers (Oliver,
19666).
The ectopic regeneration of papillae and fibres by the tube implants logically
isolates the outer root sheath and/or mesenchymal layer as minimal require-
32
R. F. OLIVER
ments for the regeneration process. The development of 'derived' follicles from
'original' follicles in the bandicoot (Lyne, 1957) from the upper region of the
outer root sheath and adherent dermal layer, which is equivalent to the mesenchymal layer, presents a naturally occurring example of dermal papilla and hair
formation from these two tissues.
A number of questions are raised by the above results. It has been shown that
only lengths of follicle wall of which the most proximal aspect was derived from
the lower third of the follicle can regenerate whiskers, but the minimum length
of wall required for regeneration is not known; nor is it known whether the
cylindrical arrangement of the outer root sheath and mesenchymal layer is an
essential feature for the regeneration process to occur.
It is also not yet clear whether only cells from the mesenchymal layer are
capable of forming dermal papillae or whether local dermal tissue can form
dermal papillae in association with transplanted lengths of outer root sheath
ectoderm isolated from the mesenchymal layer.
The transplant work of Van Scott & Reinertson (1961), Billingham & Silvers
(1965) and Cohen (1965) suggests that the organization and maintenance of
adult mammalian epithelia, including the skin appendages, is dependent on the
nature of the local dermis. If this is the case one can only assume that the
presence of the ensheathing mesenchymal layer and perhaps the relatively thick
glassy membrane in the tubes implanted into ear skin overrides the influence of
the local dermis, since where fibres were regenerated they were whiskers and not
ear-type hairs. Similarly, in those implants which did not produce whiskers the
outer root sheath maintained its typical histological character for at least
119 days.
There is, indeed, some evidence that the mesenchymal layer may have a
maintaining influence on the outer root sheath. In one tube implant the outer
root sheath had become separated from the glassy membrane and mesenchymal
layer. The outer root sheath had apparently made contact with the ear epidermis
to form a short 'follicle', which did not produce whisker, and whose ectoderm
was indistinguishable in histological appearance from ear epidermis. Van
Scott & Reinertson (1961) found that segments of outer root sheath alone from
human scalp hair follicles, unlike segments of vibrissa outer root sheath and
mesenchymal layer, usually showed degenerative changes when examined 7 or
9 days after implantation as autografts under back skin. However, it is not clear
whether the glassy membrane or even the basal cell layer was present in their
segments of outer root sheath, which were obtained from plucked hairs. They also
found that the ectodermal bulbar region of the hair follicles usually degenerates
after transplantation under back skin, while Cohen (1964) has reported that
the ectodermal matrix only of the vibrissa bulb becomes fully keratinized, but
without forming whisker, when transplanted under ear skin in the rat.
Further information about the regeneration process and a possible influence
of the local dermal environment on implants may be obtained in the future
Transplantation
of vibrissa follicle wall
33
from current experiments involving the implantation of outer root sheath and
mesenchymal layer separately under ear skin.
SUMMARY
1. A method is described for isolating and removing lengths of follicle wall,
composed of the inner root sheath, outer root sheath and mesenchymal layer,
from vibrissa follicles. These segments of follicle wall were transplanted into ear
skin and, in one case, into an ovarian capsule.
2. Seven of the thirteen implants originally dissected from the lower third
of the follicle produced short whiskers up to 6 mm in length. Two further
implants had also regenerated dermal papillae but not whiskers.
3. The dermal papillae regenerated at the proximal end of the implants and
appeared to derive from cells from the mesenchymal layer.
4. None of the four implants obtained from above the lower third of the
follicle regenerated dermal papillae or produced whiskers.
5. The outer root sheaths of all of the implants which did not regenerate
papillae or whiskers did, however, maintain their typical histological character.
6. The possibility that maintenance of the implants was dependent on the
presence of the mesenchymal layer is discussed.
7. Whisker growth from transplanted lengths of follicle wall logically isolates
the outer root sheath and/or the mesenchymal layer as the essential prerequisites
for papilla regeneration.
RESUME
Regeneration ectopique depoils de moustache apartir d'implants defragments de la
paroi dufollicule de vibrisse chez le rat a capuchon
1. On decrit une methode de prelevement de segments longitudinaux de
paroi folliculaire, comprenant la gaine interne, la gaine externe et la couche
mesenchymateuse du follicule de vibrisse. Ces segments de paroi folliculaire
sont implantes dans la peau de l'oreille et, dans un cas, dans la capsule de
l'ovaire.
2. Sept des treize implants preleves dans le tiers inferieur du follicule ont
forme un poil de moustache d'une longueur allant jusqu'a 6 mm. Deux autres
implants ont aussi regenere des papilles dermiques, mais non des poils.
3. La papille dermique se reconstitue a l'extremite proximale de l'implant et
provient apparemment des cellules de la couche mesenchymateuse.
4. Aucun des quatre implants preleves au-dessus du tiers inferieur du follicule n'a regenere de papille dermique ni forme de poil.
5. Neanmoins la structure histologique typique de la gaine externe est
conservee dans tous les implants n'ayant regenere ni papille ni poil.
6. II est possible que le maintien des implants dependent de la presence de la
couche mesenchymateuse.
3
JEEM 17
34
R. F. OLIVER
7. La croissance des segments longitudinaux de paroi folliculaire transplantes indique que la gaine externe ou la couche mesenchymateuse ou l'un et
l'autre de ces constituants sont des elements essentiels de la regeneration de la
papille.
I wish to express my gratitude to Dr C. N. D. Cruickshank and Dr J. Cohen for their
encouragement and advice.
REFERENCES
R. E. & SILVERS, W. K. (1965). Some unsolved problems in the biology of skin.
In Biology of the Skin and Hair Growth (ed. A. G. Lyne and B. F. Short), pp. 1-24. Sydney:
Angus and Robertson.
COHEN, J. (1961). The transplantation of individual rat and guinea-pig whisker papillae.
/. Embryol. exp. Morph. 9, 117-27.
COHEN, J. (1964). Transplantation of hair papillae. Symp. zool. Soc. Lond. 12, 83-96.
COHEN, J. (1965). The dermal papilla. In Biology of the Skin and Hair Growth, ed. A. G. Lyne
and B. F. Short, pp. 183-99. Sydney: Angus and Robertson.
LYNE, A. G. (1957). The development and replacement of pelage hairs in the bandicoot
Perameles nasuta Geoffroy (Marsupialia: Peramelidae). Aust. J. Biol. Sci. 10,197-216.
OLIVER, R. F. (1966a). Whisker growth after removal of the dermal papilla and lengths of
follicle in the hooded rat. / . Embryol. exp. Morph. 15, 331-47.
OLIVER, R. F. (19666). Histological studies of whisker regeneration in the hooded rat.
/ . Embryol. exp. Morph. 16, 231-44.
VAN SCOTT, E. J. & REINERTSON, R. P. (1961). The modulating influence of stromal environment on epithelial cells studied in human autotransplants. /. invest. Derm. 36, 109-31.
BILLINGHAM,
{Manuscript received 23 May 1966)