/. Embryol. exp. Morph. Vol. 72, pp. 209-224, 1982
Printed in Great Britain © Company of Biologists Limited 1982
A quantitative study of hair growth using
mouse and rat vibrissal follicles
I. Dermal papilla volume determines hair volume
ByLAMEES IBRAHIM1 AND E. A. WRIGHT1
From the Department of Morbid Anatomy King's College Hospital
Medical School, London
SUMMARY
The volumes of the dermal papilla, matrix and fully grown hair of vibrissal follicles of rats
and mice were measured.
Dermal papillae were surgically removed from rats' vibrissal follicles and a small new
dermal papilla was always reformed. The lower third of rats and mice follicles, containing
the whole of the dermal papilla was amputated and very small dermal papillae were reformed.
Follicles containing small dermal papillae produced small hairs and a constant positive
linear relationship of 7-0 was found between the volume of the dermal papilla and the volume
of the full-grown hair for all cases studied.
INTRODUCTION
The hair produced by any follicle is an orderly mass of complexly layered
keratinized epithelial cells forming a distinct geometrical shape. Normal mouse
and rat vibrissae and some other hairs approximate to the shape of an elongated
cone. In normal circumstances the rate of increase in length of the vibrissa is
constant for nearly the whole of the growing period (e.g. mouse F whisker
1-0 mm/day and rat F whisker 1-5 mm/day). However, the diameter of the tip
of the whisker is very small (2-5 fim for the mouse F and 3-5 /an for the rat F)
and the diameter of the last part of whisker to be produced is large (mouse
80-90/tm and rat 160-180 /tm). Thus the amount of hair being produced in
unit time varies by a large factor (of several hundreds or even thousands) from
the beginning to the end of the growing period. It is therefore clear that 'growth'
of a hair cannot be characterized solely by rate of increase in length but must
include width at all times so as to compute rate of increase in volume and total
volume to be calculated. Changes in thickness of the inner root sheath also
must be taken into account for some hairs (Priestley & Rudall, 1965). We have
attempted to produce measurements of these volume changes in connexion with
1
Authors'1 address: Department of Morbid Anatomy, King's College Hospital Medical
School, Denmark Hill, London, S.E. 5, U.K.
209
210
L IBRAHIM AND E. A. WRIGHT
hairs produced by the reformed dermal papillae as well as the normal. Van
Scott & Ekel (1958) measured the volume of the dermal papilla and its surrounding matrix in normal human scalps and those with alopecia areata using serial
cross-sections of the follicle and found a relationship between the size of the
dermal papilla and the size of the matrix.
Dermal papillae have been shown to be the inductive stimulus for hair growth
(Ibrahim & Wright, 1977) but when dermal papillae were excised from rats'
vibrissal follicles new dermal papillae were reformed and hairs reaching the
normal final length were produced (Oliver, 1966). Oliver (1966) also amputated
the bottom third of rats' vibrissal follicles; regeneration took place and vibrissae
nearly half as long as the normal controls were produced.
The aim of this paper is to confirm Oliver's findings and examine the relationship between the volume of the dermal papilla in normal follicles and those
reformed after amputating the bottom third (containing the whole of the
original dermal papilla) and that of the hair produced.
MATERIALS AND METHODS
Animals
Young adult C3H mice inbred at King's College Hospital and randomly mated
closed-colony Wistar-derived rats were used. All experiments were carried out
on the vibrissal follicles of the mystacial group.
Whisker measurements
(a) Length measurements
The direct method using a graduated capillary glass tube has been used
throughout this investigation. A detailed description is given in Ibrahim &
Wright (1975).
(b) Width measurements
Normal E, F, G, H whiskers were found to increase in diameter almost
linearly starting from the tip (2-5 fim in mice and 3-5 /tm in rats) to the widest
part (excluding the club) which is 80-90/*m in young adult mice and 160—
180 fim in young adult rats (fuller details are given in Ibrahim & Wright, 1975).
(c) Volume measurements
(i) Volume of the hair. As width was found to increase steadily from the tip
to the widest part at the end, all vibrissae were in the shape of an elongated cone,
their volumes were therefore measured applying the formula:
volume =
where L is the length and r the radius.
Growth of mouse and rat vibrissal follicles
211
(ii) Dermal papilla and matrix volume measurements. Van Scott & Ekel (1958)
measured the volume of human hair dermal papilla and the surrounding matrix.
They cut 8 ju,m transverse sections. Every section was in the shape of a frustum.
The volume was measured applying the following formula:
volume = $nA(R2 + Rr + r2),
where A is altitude, R is larger radius and r small radius. The volume
of the dermal papilla was the sum of the volumes of these frusta. Because a
longitudinal section was required in this work (and most other studies of hair
growth) the method was applied to longitudinal sections, using one section
passing through the middle. To test the accuracy of this method it was tested
against Van Scott's (transverse sectioning) method. A 1-month-old C3H mouse
was killed and its eight follicles E, F, G and H of both sides were fixed immediately in Bouin's fixative. The four follicles of the right side were sectioned
longitudinally and the four on the left side were sectioned transversely. To
assure comparable volumes the timing of every step of the histological process
from the fixative to staining was standardised. All the dermal papillae volumes
were measured applying the formula above. A longitudinal section passing
through the middle of the dermal papilla including the stalk and the tip was
used. The section was projected, enlarged 600 times and drawn on paper.
A line was drawn representing the longitudinal axis that passes through the
centre of the dermal papilla. Parallel lines were drawn perpendicular to this
axis and parallel to each other from the bottom end of the stalk to the tip of the
dermal papilla. These lines represent the diameters of the succesive frusta which
compose the dermal papilla.
Operations on follicles
The most posterior column of vibrissal follicles on both sides of the face was
used. The operations were carried out on the right side using the left side of the
face as a control. An incision was made posterior and parallel to the E, F, G
and H follicles. The skin was closed by interrupted sutures of black silk (4/0
sterile suture). All operations were carried out under a dissecting microscope
x 160 magnification. The animals never had any difficulty in eating or drinking
after these operations.
(a) Removal of the dermal papilla
Rats were anaesthetized and E, F, G, H follicles were exposed by making an
incision in the skin posterior and parallel to their whiskers, and the follicles were
cleared of the surrounding tissue. Using a pointed scalpel a longitudinal incision was made along the bottom third of the follicle. Then using two watchmaker's forceps the dermal papilla was everted from the follicle and plucked
from the exposed stalk; slight damage to the bottom of the follicle always took
0-667
0-689
0-553
0-710
Cross
Longitudinal
Cross
Longitudinal
Cross
Longitudinal
Cross
Longitudinal
E
F
G
H
0-831
0-703
0-456
0-464
Sectioning direction
Follicle
Dermal papilla
volume
(mm3 x 103)
6-13
800
5-20
5-56
4-73
4-94
3-98
4-30
Matrix
volume
(mm3 x 103)
6-96
8-70
5-76
6-79
5-39
5-58
4-44
4-77
0-135
0-088
0106
0-128
0141
0139
0114
0107
0119
0081
0096
0104
0-124
0-123
0-102
0-109
0-880
0-918
0-903
0-818
0-876
0-887
0-896
0-901
Matrix and
Matrix
Dermal papilla
dermal papilla
Dermal papilla
Matrix and
Matrix and
volume
dermal papilla dermal papilla
Matrix
(mm3 x 103)
(Cross sectioning was applied to the left side and longitudinal sectioning to the right. The ratios between the volumes calculated
using the two methods of sectioning are presented to show accuracy and substitutional possibility.)
5d
o
en
Z
1—1
X
5
>d
Table 1. Volumes of dermal papillae and matrices of eight vibrissal follicles taken from a C3H mouse at the beginning of the 2nd
cycle of their cyclic growth when vibrissae were 2-3 mm long
P
to
to
H
HOI
Growth of mouse and rat vibrissal follicles
213
Table 2. Representing the final length, mean value and standard error of the
vibrissae produced by rats'1 (E, F, G, H) follicles after surgical removal of the
dermal papilla (final lengths of their control vibrissae on the left side of the rats
are also presented)
No. of
samples
Treatment
Range of vibrissae
final length (mm)
20
Control vibrissae
22
Reformed vibrissae
Mean
(mm)
S.E.
43-8-58-3
50-68
1-23
6-2-57-7
28-45
2-455
place. The two sides of the incised capsule were brought together and the skin
was closed.
(b) Amputation of the follicles
The posterior column of mystacial vibrissal follicles, namely E, F, G and H of
Wistar derived albino rats and C3H mice, were amputated. The animal was
anaesthetized and E, F, G, H follicles were exposed. Using fine iridectomy
scissors the bottom third of each follicle was amputated just below the nerve
entrance to the follicle, which is at the junction between the bottom third and
the proximal two-thirds of the follicle. The amputated lower portion was
immediately placed in Bouin's fixative, processed and sectioned for histological
studies.
OBSERVATIONS AND RESULTS
(1) Volume measurements
Volumes of the bulb components of eight (E, F, G, H) vibrissal follicles of a
C3H mouse were measured and two different methods of sectioning (cross and
longitudinal) were used.
Each of the four follicles on one side was compared with its analogue on the
other side of the face (Table 1). All the follicles were at the beginning of the 2nd
growth cycle, therefore, volumes of the matrices measured were slightly smaller
than if they were measured at the middle or towards the end of the growing phase
(anagen). By calculating the ratios between the components measured using the
two methods, the volumes were found to be very close (Table 1).
(2) Removal of the dermal papilla
Twenty-six (E, F, G, H) follicles in nine 5- to 6-week-old albino Wistarderived rats were operated on and the dermal papilla removed. The first observation was made about 4 weeks afterwards. Twenty-two follicles were found to
have a new vibrissa, but the other four failed to produce a hair.
Histological examination at the end of the observation period showed a
214
L. IBRAHIM AND E. A. W R I G H T
Fig. 1. The amputated part of one of the rat's follicles. H & E, x 125.
normal-looking dermal papilla present at the bottom of the follicle. Those
which failed to produce a whisker were found to have an epithelial or keratogenous cyst and there was no dermal papilla.
The 22 new whiskers were observed and measured once a week for at least
10 weeks. Their final lengths are listed in Table 2. They all had a new dermal
Growth of mouse and rat vibrissal follicles
215
Table 3. Representing the final length, the mean values and s.E. of the vibrissae
produced by {E, F, G, H) vibrissal follicles after amputating their bottom third
compared with the normal vibrissae on the left side of each animal
Animal/no, of
vibrissae
Treatment
Range of vibrissae
final
length (mm)
Mouse/14
Mouse/14
Rat/13
Rat/13
Control
Amputated follicles
Control
Amputated follicles
22-9-30-0
6-5-19-6
42-9-52-5
13-5-26-1
Mean
s.E.
26-86
12-81
47-77
20-66
0-52
1-02
0-87
1-19
Because of technical reasons, the final length of some of the vibrissae measured were not
included in the table.
papilla at the bottom of the follicle and except for the variations in their sizes
they all looked histologically alike.
The new vibrissae varied in their final length which is probably an indication
of the extent of damage inflicted during the operation, but a few were nearly as
long as the controls. Their rate of growth varied between 0-9 and 1-2 mm/day,
while the controls were 1-5 mm/day. Thus, when volumes of the new dermal
papillae and the vibrissae produced were measured the largest were found to be
only one quarter to one third that of the normal control (Fig. 8).
(3) Amputation of the lower third of the vibrissal follicle
Five 6-week-old Wistar-derived rats and four 5- to 6-week-old C3H mice
were operated on. Thirty-six follicles were amputated. The amputated pieces
were fixed and histologically examined and in all the cut was made above the
tip of the dermal papilla (Fig. 1). This proves conclusively that the dermal
papillae were completely removed. Thirty-two amputated follicles (88 %) grew
new vibrissae which erupted above the skin 3-4 weeks later in mice and 5-7
weeks in rats. They were measured twice weekly during the first few weeks, then
once a week until the end of the experiment. Mice vibrissae were measured
weekly for 5 months while rats were killed after 2-5 months. Their final lengths
are tabulated in Table 3. The final length and rate of growth were measured
for all the new vibrissae and each compared with its control on the left side
of the face. (Graphs illustrating the growth of three vibrissae are shown in
Fig. 2.) The bottom part of two follicles (a, a rat and, b, a mouse) are shown
in Fig. 3. The four follicles which failed to regenerate were found to have
developed an epithelial or keratogenous cyst extending down to the bottom
of the follicle. In Table 4 are shown measurements of the components of some
of these follicles and the hair produced compared with those of the normal
controls.
Although the whiskers produced by these follicles were approaching half the
full length of a normal whisker, they were only 1/6-1/12 the volume of the
216
L. IBRAHIM AND E. A. WRIGHT
30
20
10
30
20
...-©••• 0--V—0— O
10
0
•
30
20
10
20
40
60
80
100
120
140
Days after amputation
Fig. 2. The growth curves of E (top), F (middle), G (bottom graph) vibrissae produced by mouse follicles after amputation of their lower third (O). Normal control
vibrissa on the left side of the mouse (#).
normal, and to illustrate that the diameter of a normal vibrissa and that produced after amputation were measured every mm starting from the tip to the
bottom. The radius representing the linear increase in width and the squared
radius reflecting the increase in volume were calculated and both presented
alongside in Fig. 4.
No blood capillaries were seen in the newly formed dermal papillae. They
therefore probably have a poor blood supply. It was also noted that none of the
0078
4-20
64-59
743-89
Vibrissa max. diameter (mm)
Fibre volume
mm 3 x 103
Papilla volume
mm 3 x 105
Matrix volume
mm 3 x 105
Eleft
normal
26-3
...
Length of vibrissa
(mm)
Vibrissa
E right
reform.
11-3
0-042
0-52
13-98
107-38
Gleft
normal
29-5
0090
6-25
87-47
275-33
18-27
3-31
0-60
0-042
13-5
G right
reform.
A.
Mouse
4-10
1-86
0-55
0-042
12-0
H right
reform.
152-9
12-40
0-93
26-93
5-62
0-88
0064
8-2
12-2
0-054
G right
reform.
H right
reform.
—
430-50
31-00
0-162
45-5
Fleft
normal
69-22
5-51
•1-51
0066
12-8
F right
reform.
A
Rat
(Components of the normal follicles on the left side of the animal were measured and presented for comparison.)
62-69
22-06
5-15
0-087
260
F right
reform.
Table 4. The vibrissal length, its maximum diameter, volume, dermal papilla volume and the matrix volume for some of
the follicles after surgical amputation of their lower third
I
§
^»
a
a
oj mouse
ucies
Fig. 3. Two F-follicles; (a) from a rat, (6) from a mouse 5 months after amputating their lower third. They produced three
generations of hair. The newly formed dermal papillae lacked a stalk and a pointed tip. H & E, x 125.
3(b)
J %
H
X
w
o
>
z
o
•
w
t-H
r
00
219
Growth of mouse and rat vibrissat follicles
29-5
13-5
2025
Fig. 4. A sketch representing linear and geometrical dimensions of a 13-5 mm long
vibrissa produced by a mouse follicle after amputation, and those of the control
vibrissa (29-5 mm) on the other side of the face. A linear relationship is represented
on the left half of the diagram and a geometrical on the right, (r = radius.)
40
r
30
20
10
20
40
60
80
100
120
140
155
Days after amputation
Fig. 5. The growth curves of the giant vibrissa produced by an amputated H-follicle
on the right side of a mouse and its small second cycle. Growth curves of the normal
H on the left side of the face is also presented. • , Normal control H-vibrissa; CD,
vibrissa produced after amputation.
220
L. IBRAHIM AND E. A. W R I G H T
Fig. 6. A histological microphotograph of G follicle on the right side of a C3H mouse
containing two small dermal papillae reformed after amputating the lower third of
the follicle H & E, x 125.
Growth of mouse and rat vibrissal follicles
221
30-
20
10-
10
40
70
100
Days after amputation
130
Fig. 7. The growth curves of the two small vibrissae produced by the follicle shown
in Fig. 6 and the growth of the normal G vibrissa on the left side of the face. # ,
Normal control. O, Two vibrissae produced after amputation.
reformed dermal papillae had a stalk (Fig. 3) and in most cases the pointed tip
was missing.
Two exceptionally interesting cases occurred after amputating the bottom
third of the vibrissal follicles. They are:
(a) A giant hair was produced by an amputated H follicle of a C3H mouse.
Its growth curve is shown in Fig. 5. The vibrissa grew for 82 days reaching a
final length of 38 mm. The second cycle was only 16 mm long and histology at
the end of this cycle showed a small dermal papilla resembling the others.
(b) In a follicle of another C3H mouse, two small dermal papillae were reformed and two whiskers were produced. The histological appearances are
shown (Fig. 6). It seems possible that mesenchymal cells migrating centrally
failed to merge. The two whiskers were 11-8 and 12-3 mm long. Their total
volume was about one-fifth that of the normal single whisker while total volume
of both dermal papillae was about one quarter that of the normal. The growth
was followed for two successive cycles (Fig. 7).
(4) The relationship between the volume of the dermal papilla
and the hair produced
The volume of a fully grown hair produced by any mouse or rat follicle was
found to be related to the volume of its dermal papilla.
Smaller hairs produced after excision of the dermal papilla and after amputa8
EMB J2
222
L. IBRAHIM AND E. A. WRIGHT
50
10
•a l
01
10
100
500
D.P. vol. mm3 X 10s
Fig. 8. Final hair volume plotted against dermal papilla volume of some normal
mice and rats vibrissal follicles and those produced after excision of dermal papilla
and after amputating the lower third of the follicle. A, Normal rat follicles; A,
rat follicles after excision of their dermal papillae; A, amputated rat follicles;
# , normal mouse follicles; O, amputated mouse follicles.
tion of the lower third of the follicle were also related to the volume of their
dermal papillae. (Table 4). Hairs and dermal papillae of eight normal follicles
and twelve experimental follicles were measured and all volumes drawn on a
scatter diagram (Fig. 8). A logarithmic scale was used to allow accommodation
of all points illustrated. A positive relationship was clearly displayed. Regression
analysis of the data showed the slope of the line to be 0-78 and the coefficient
of correlation as high as 0-936. The ratio of the variability of X values (dermal papilla volumes) to the variability of rvalues (hair volumes) was a highly
significant value of 133-3. The probability of the data deviating from the mean
was 0-00000065.
DISCUSSION
Volume measurements of the dermal papilla and its surrounding matrix in
relationship to that of the hair produced using a convenient longitudinal section
of the hair follicle is a step towards establishing the quantitative relationship
between these important components of the hair follicle. Although there was a
constant equal increase in length per unit time during the growth period, there
was also a constant increase in width making the increase in the amount of hair
produced per unit time increase by a factor of hundreds or even thousands
between the tip and the bottom of the fully grown hair. Therefore, the mechanism controlling the growth of hair must contain at least two different factors
acting simultaneously.
Growth of mouse and rat vibrissal follicles
223
When the volume of hair was measured, it was assumed that it is in the shape
of an elongated cone which does not account for variations in width that might
occur in some types of hair. On the other hand it is very difficult to obtain
absolute volumes of the dermal papillae and the histological preparations being
the most convenient method available were used. Technical errors involved in
the processing of tissue were reduced to the minimum by standardization, and
a strong positive linear relationship between the volume of dermal papilla and
the volume of hair produced was displayed, and as the aim of this work is a
quantitative relationship rather than absolute figures then this method (as has
been shown) is applicable. No positive relationship between the volume of hair
produced and the length of the growth cycle can be drawn because many factors
may affect the length of cycle.
Van Scott and Ekel (1958) concluded from observing human scalp hair
follicles in normal and those with alopecia that the dermal papillae lacked a
stalk which they believed to be related to the loss of hair in alopecia. In the
present study all reformed dermal papillae lacked a stalk and were still capable
of inducing generations of hair.
Oliver (1966) removed dermal papillae from rat vibrissal follicles and reported
observing a new hair growing to a normal. Although the same results were
achieved here, the volume of the new dermal papilla and the hair produced were
less than half of normal controls. He also amputated the bottom third of some
other rat vibrissal follicles and vibrissae nearly half as long as the normal were
produced. The experiments presented here confirm Oliver's findings. However,
these vibrissae were found to be up to 12 times smaller in volume. But because
these operations had little effect on the rate of growth of vibrissae produced then
the result was a long fine hair. Their dermal papillae were often minute bodies
of mesenchymal cells only 1/10 or even 1/25 of the normal volume. Follicles
which failed to reform a new dermal papilla did not produce a hair.
Wessells & Roessner (1965) found that in embryonic mice dermal papilla
cells of the dermal condensation fail to incorporate [3H]thymidine and do not
contain significant numbers of mitotically active cells. Pierard & Brassine
(1975) confirmed this in the adult follicles of the rat. The dermal papilla cells
have not been seen to divide and under normal circumstances there is a constant
number of cells (Ibrahim, 1976; Young, 1980). It also is not affected histologically or physiologically by high doses of X-irradiation (Ibrahim, 1976; Ibrahim
& Wright, 1977). The process of reformation may best be explained as due to
migration of cells from the adjacent mesenchymal sheath to form a small body
of dermal cells. The mesenchymal sheath had been shown to be capable of
forming a follicle when transplanted into other parts of the body (Oliver, 1967);
this ability is restricted to the bottom third of the follicle. The higher the cut the
smaller the new dermal papilla. By amputating more than a third no dermal
papilla was reformed.
8-2
224
L. IBRAHIM AND E. A. W R I G H T
We would like to thank Dr R. Littleton for his help with the statistical analysis and the
Iraqi Government for their financial support.
REFERENCES
(1976). A study of hair growth with particular reference to epithelial-mesenchymal
interactions. Ph.D. thesis, London University.
IBRAHIM, L. & WRIGHT, E. A. (1975). The growth of rats and mice vibrissae under normal
and some abnormal conditions. /. Embryol. exp. Morph. 33 (4), 831-844.
IBRAHIM, L. & WRIGHT, E. A. (1977). Inductive capacity of irradiated dermal papillae.
Nature, Lond. 265, 5596, 733-734.
OLIVER, R. F. (1966). Whisker growth after removal of the dermal papilla and lengths of
follicle in the hooded rat. /. Embryol. exp. Morph. 15, 331-347.
OLIVER, R. F. (1967). Ectopic regeneration of whiskers in the hooded rat from implanted
lengths of vibrissa follicle wall. /. Embryol. exp. Morph. 17, 27-34.
PIERARD, G. E. & De la BRASSINE, M. (1975). Modulation of dermal cell activity during hair
growth in the rat. /. Cutan. Path. 2, 35-41.
PRIESTLEY, G. C. & RUDALL, K. M. (1965). Modifications in the Huxley layer associated with
changes in fibre diameter and output. The Skin and Hair Growth 165-170.
VAN SCOTT, E. J. & EKEL, T. M. (1958). Geometrical relations between the matrix of the hair
bulb and its dermal papilla in normal and alopecic scalp. /. Invest. Dermat. 31, 281-289.
WESSELLS, N. K. & ROESSNER, K. D. (1965). Non-proliferation in the dermal condensations
of mouse vibrissae and pelage hairs. Devi Biol. 12, 419-433.
YOUNG, R. D. (1980). Morphological and ultrastructural aspects of the dermal papilla
during the growth cycle of the vibrissal follicle in the rat. /. Anat. 13 (2), 355-365.
IBRAHIM, L.
(Received 4 May 1982, revised 14 June 1982)