A quantitative study of hair growth using mouse and rat vibrissal follicles: I. Dermal papilla volume determines hair volume

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 μm for the mouse F and 3–5 μm for the rat F) and the diameter of the last part of whisker to be produced is large (mouse 80–90 μm and rat 160–180μm). 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 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.

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.

The direct method using a graduated capillary glass tube has been used throughout this investigation. A detailed description is given in Ibrahim & Wright (1975).

Normal E, F, G, H whiskers were found to increase in diameter almost linearly starting from the tip (2–5 μm in mice and 3–5 μm in rats) to the widest part (excluding the club) which is 80–90 μm in young adult mice and 160— 180 μm in young adult rats (fuller details are given in Ibrahim & Wright, 1975).

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.

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 × 160magnification. The animals never had any difficulty in eating or drinking after these operations.

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 place. The two sides of the incised capsule were brought together and the skin was closed.

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.

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).

Twenty-six (E, F, G, H) follicles in nine 5- to 6-week-old albino Wistar- derived 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 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 kerato-genous 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 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).

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 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 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).

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 amputation 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 Y values (hair volumes) was a highly significant value of 133 · 3. The probability of the data deviating from the mean was 0 · 00000065.

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.

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.

We would like to thank Dr R. Littleton for his help with the statistical analysis and the Iraqi Government for their financial support.