A quantitative study of hair growth using mouse and rat vibrissal follicles

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1 /. Embryol. exp. Morph. Vol. 72, pp , 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 IBRAHIM 1 AND E. A. WRIGHT 1 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 /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.

2 210 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 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.

3 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(r 2 + Rr + r 2 ), 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

4 to to 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 (Cross sectioning was applied to the left side and longitudinal sectioning to the right. The ratios between the volumes calculated 5 using the two methods of sectioning are presented to show accuracy and substitutional possibility.) >d Follicle E F G H Sectioning direction Cross Longitudinal Cross Longitudinal Cross Longitudinal Cross Longitudinal Dermal papilla volume (mm 3 x 10 3 ) Matrix volume (mm 3 x 10 3 ) Matrix and dermal papilla volume (mm 3 x 10 3 ) Dermal papilla Matrix Dermal papilla Matrix and dermal papilla Matrix Matrix and dermal papilla X 1 1 Z o en 5d HOI H

5 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 Range of vibrissae Mean samples Treatment final length (mm) (mm) S.E. 20 Control vibrissae Reformed vibrissae 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

6 214 L. IBRAHIM AND E. A. WRIGHT 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

7 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 Range of vibrissae vibrissae Treatment final length (mm) Mean s.e. Mouse/14 Control Mouse/14 Amputated follicles Rat/13 Control Rat/13 Amputated follicles 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

8 216 L. IBRAHIM AND E. A. WRIGHT V 0 O 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

9 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 (Components of the normal follicles on the left side of the animal were measured and presented for comparison.) I oj mouse Rat Mouse A A. F right reform. F right reform. Fleft normal G right reform. H right reform. H right reform. G right reform. E right reform. Gleft normal Eleft normal Vibrissa... a a Length of vibrissa (mm) Vibrissa max. diameter (mm) Fibre volume mm 3 x 10 3 ^» Papilla volume mm 3 x 10 5 ucies Matrix volume mm 3 x 10 5

10 J % 3(b) 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 r t-h w z ow > o X H

11 Growth of mouse and rat vibrissat follicles 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.) r 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.

12 220 L. IBRAHIM AND E. A. WRIGHT 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.

13 Growth of mouse and rat vibrissal follicles Days after amputation 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 amputa- 8 EMB J2

14 222 L. IBRAHIM AND E. A. WRIGHT a l D.P. vol. mm 3 X 10 s 500 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 The ratio of the variability of X values (dermal papilla volumes) to the variability of rvalues (hair volumes) was a highly significant value of The probability of the data deviating from the mean was 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.

15 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 [ 3 H]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

16 224 L. IBRAHIM AND E. A. WRIGHT We would like to thank Dr R. Littleton for his help with the statistical analysis and the Iraqi Government for their financial support. REFERENCES IBRAHIM, L. (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), IBRAHIM, L. & WRIGHT, E. A. (1977). Inductive capacity of irradiated dermal papillae. Nature, Lond. 265, 5596, OLIVER, R. F. (1966). Whisker growth after removal of the dermal papilla and lengths of follicle in the hooded rat. /. Embryol. exp. Morph. 15, OLIVER, R. F. (1967). Ectopic regeneration of whiskers in the hooded rat from implanted lengths of vibrissa follicle wall. /. Embryol. exp. Morph. 17, PIERARD, G. E. & De la BRASSINE, M. (1975). Modulation of dermal cell activity during hair growth in the rat. /. Cutan. Path. 2, 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 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, WESSELLS, N. K. & ROESSNER, K. D. (1965). Non-proliferation in the dermal condensations of mouse vibrissae and pelage hairs. Devi Biol. 12, 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), (Received 4 May 1982, revised 14 June 1982)