Hair growth in the rat occurs in a series of waves, which start ventrally and pass over the flanks to the back (Dry, 1926; Butcher, 1934; Johnson, 1958a). The activity of the hair follicle is cyclic; when the hair has been fully formed there is a period of quiescence during which the dead hair is retained as a ‘club’. The duration of the complete cycle varies with site and age, ranging from 24 to about 100 days (Ebling & Johnson, 1964).
When hair follicles are translocated, they continue to maintain the periodicity characteristic of their sites of origin (Ebling & Johnson, 1959). On the other hand, when skin is exchanged between rats of different ages and thus with their hair growth waves out of phase, follicular activity in the graft skin in some circumstances comes into line with the activity of the host (Ebling & Johnson, 1961). The idea of systemic control of follicular activity is further supported by the fact that although it is possible by various treatments to alter the speed of passage of the waves of hair growth, the pattern remains intact (Johnson, 1958a,b; Ebling & Johnson, 1964). If such a controlling systemic influence exists, whether it be an inhibitor or an accelerator, the apparent selfwise behaviour of autografts could be explained on the basis of different reaction times for dorsal and ventral skin.
We here describe experiments to find out whether a cross action due to transmissible systemic factors can be demonstrated in parabiotic rats having their hair growth cycles out of phase.
Animals and operative technique
The experimental animals were female rats from a randomly mated colony which had been used for many previous studies (Ebling & Johnson, 1964). In homograft experiments over 60 per cent of grafts survived for at least 20 days; most of these were still healthy at 40 days, and a number at 80-100 days, after grafting (Ebling & Johnson, 1961).
Parabiosis was performed by the method of Bunster & Meyer (1933) with these modifications: the scapulae were scraped to expose raw bone before suturing; the abdominal muscles were sutured without opening the peritoneal cavities; the femora were exposed by separating the lateral thigh muscles, gently scraped and sutured together at two points, with care not to injure the sciatic nerves. This method gives a mechanically sound union anteriorly and posteriorly, and avoids the risk of cross-herniation of abdominal viscera; it has the drawback of causing some disability of the inner hind legs. Pentobarbitone sodium, approx. 6 mg./ 100 g. body weight given intraperitoneally, was used as anaesthetic.
Parabiosis was performed between (a) pairs of rats both 28 days old; (b) pairs of rats, one of which was 28 days and the other 49 days old; (c) pairs both 49 days old. About half the parabiotic pairs of equal age were litter-mates. No difficulty was encountered in making the unequal pairs. Litter-mates of the animals used for parabiosis were kept as single control animals.
All the rats were periodically clipped and dyed, and, as far as possible, observations were made at regular intervals of 2 days throughout the whole period of the experiment. For each wave of growth the times of eruption of hair in the ventral, mid-side and anterior mid-dorsal regions were recorded.
After the completion of the observations of hair growth, the rate of exchange of plasma was measured in fourteen pairs, by the method used by Hervey (1959), except that the animals were maintained lightly anaesthetized throughout with pentobarbitone.
The effects of parabiosis
Seventy-nine parabiotic pairs were made for studies of hair growth. Of these, six died during the operation, or later from intercurrent causes; forty-three were lost as a result of parabiotic disharmony; thirty pairs were available for study. Three of the last group showed signs of parabiotic disharmony at some time. The successful pairs gave an impression of being less healthy than pairs which were made concurrently from another stock, in which the incidence of disharmony was zero. The high incidence of disharmony is surprising in view of the successful use of the same colony for homograft studies. There was no difference in its incidence between 28–28-, 28–49- and 49-49-day pairs, and approximately equal numbers in these three groups survived. There was also no difference in the incidence between sib and non-sib pairs. The same incidence of disharmony and impression of ill-health were seen in an additional series of twenty female 28-28-day pairs, made from the strain used for the hair growth studies but not clipped or dyed; these effects were thus not caused by the procedures used to study hair growth. Of the forty-three pairs which died from disharmony or were killed when obviously suffering from it, thirty-seven showed marked inequality of body size, (the proportion was the same whether or not the initially unequal 28–49-day pairs were taken into account); fourteen showed a marked difference in colour of eyes, ears, paws and tail, indicating unequal distribution of blood; only six showed poor union or breakdown of the anastomosis, as far as could be judged by naked-eye examination. Thirty-one of the pairs which died from disharmony did so within 14 to 23 days (mean 18 days) after parabiosis.
In the animals which survived in the 28–49-day group, difference in body size could no longer be discerned 1 to 2 months after parabiosis. The individuals in successful 28–28-day pairs attained body weights as young adults 71 to 89 per cent (mean 80 per cent) of the weights of the single controls, agreeing with Hervey’s (1959) finding for a hooded strain. The 49-49-day pairs attained slightly higher weights relative to their controls (mean 88 per cent); the 28–49-day pairs were intermediate.
The fourteen determinations of rates of plasma exchange gave a mean result of 1·5 per cent of one animal’s plasma volume exchanged per minute (s.e. ±0·2 per cent). A concurrent series of determinations made on thirteen pairs from the disharmony-free strain also gave a mean result of 1·5 per cent (s.e. ±0-2 per cent). In both series the mean result was made up from a majority of values centred around 1·1 per cent, and two or three higher values, and so the difference from Hervey’s (1959) result of 1·1 per cent mean (s.e. ± 0·2 per cent) is probably not significant. The extent of cross-circulation obtained in parabiotic pairs which survive does not appear to be affected by the incidence of disharmony in the strain.
In each wave of growth, eruption always occurred symmetrically on the individual rats in the parabiotic pairs, new hair appearing dorsal to the parabiotic suture at the same time as on the outer flanks (Text-fig. 1). The skin of each partner remains intact in front of and behind the parabiotic suture. Unless we are to believe that effects can spread forwards and backwards for about 5 cm. without any time lag, this observation would appear to be evidence, additional to that of Ebling & Johnson (1959), that the wave of hair-growth is not propagated.
The mean age at eruption of hairs in each wave is shown for each exper imental group and for each respective control group in Table 1; in Text-figs. 2, 3 and 4, and in Table 2, which shows the duration of the follicular cycles, all the control results have been grouped together.
Parabionts of equal age
In rats joined in parabiotic pairs at 28 days of age the time of eruption of hairs of the first wave of growth, due about 12 days later, was unaffected in the ventral and mid-side regions, though there was a significant delay in the mid-dorsal region (Tables 1 and 2; Text-figs. 2,3 and 4). Subsequent eruptions in all regions were significantly delayed in comparison with normal fitter mate controls. In rats joined in parabiotic pairs at 49 days of age, there was a slight delay in the ensuing (second) wave of growth due to start ventrally about 14 to 16 days later, and a significant delay in the mid-side and dorsal regions. Subsequent eruptions were significantly delayed in all regions.
If there are transmissible systemic factors influencing activity of hair follicles, a tendency to synchrony of the waves should be demonstrable even between parabiotic rats of equal age. To test whether each rat resembled its partner more than it would another chosen at random, the variance ratio within and between twelve pairs of non-sib parabiotic rats was examined (Table 3). It can be seen that the rats become, more or less progressively, synchronized with their parabiotic partners.
Parabionts of unequal age
It is possible to test whether any cross-action has occurred between members of a pair by analysing the differences between their ages at eruption of the hairs in the different regions. If there is no cross-action there should be no significant difference. If, on the other hand, the follicular cycles become synchronized due to cross-action, a mean difference of 21 days would be expected. It can be seen from Table 4 that there is a progressive and significant cross-action as the waves proceed, and that the most clear-cut results were seen in the ventral region. By the fourth wave the mean difference, for all regions, was not significantly different from 21 days.
The pattern of this cross-action may be seen by referring to Tables 1 and 2 and Text-figs. 2, 3 and 4. First, one can compare rats having parabiotic partners 21 days older with those having parabiotic partners of the same age. The following points may be noted :
The first wave of growth, due to start ventrally at about 12 days after parabiosis, was completely unaffected.
In the second wave, the ventral eruption was significantly earlier than in parabionts of equal age; thus the follicular cycle 1–2 was shorter (Table 2). The midside eruption was slightly later and the dorsal eruption significantly later.
In the third wave, the ventral and mid-side eruptions occurred at about the same age as in parabionts of equal age. The dorsal eruption was significantly later, but this did not mean that the duration of the cycle was greater (Table 2); the delay was merely a consequence of that already established in the second wave.
In the fourth wave, the ventral eruption was significantly later than in parabionts of equal age with a probable lengthening of the cycle (Table 2). The midside eruption was unaffected. Although the dorsal eruption occurred at about the same age as it did in parabionts of equal age, the follicular cycle 3–4 was, in fact, significantly shorter.
We may now consider the pattern of hair growth in rats having parabiotic partners 21 days younger and compare them with rats having parabiotic partners of equal age. The main points can be summarized thus :
The ventral eruption of the second wave of growth, due about 19 days after parabiosis was carried out, was unaffected. In the mid-side and dorsal regions, however, eruption of hair was significantly retarded.
The third wave of growth was significantly retarded in the ventral region, involving a longer follicular cycle 2–3. There was also delay in the mid-side and in the dorsum, but this was a consequence of delay already established in the previous wave; the durations of the follicular cycle did not differ.
The fourth wave of growth was retarded in all regions, but this was in consequence of previously established delays; there were no significant differences in the duration of cycle 3–4.
In summary, cross action seems to occur to accelerate or retard; there were two examples of significant shortening of the follicular cycle and four of lengthening.
These experiments show that in parabiotic pairs having their hair cycles out of phase cross action occurs which tends gradually to synchronize them; this is achieved by acceleration or retardation of the waves in the individual members of the pair. The effect is not rapid enough to suggest that follicular activity is intimately controlled by systemic factors, but rather that inherent rhythms are slowly brought into phase.
This result appears to contradict those of Zeckwer (1953) and of Jensen (1958). Zeckwer, however, did not design her experiments having regard to the cycles of hair growth, but merely observed the regrowth of clipped hair in a series of parabiotic pairs of rats of widely varying ages. She adrenalectomized one rat of each pair and noted that the resulting acceleration of the onset of hair follicle activity was not transmitted across the parabiotic union. She observed, further, that hair regrowth in the adrenalectomized partner was delayed in comparison with single adrenalectomized rats, but she does not say whether any such delay occurred in intact parabiotic pairs. The effect could be explained solely by a passage of adrenocortical hormones, which are known to delay initiation of activity, from the intact rat to its adrenalectomized partner.
Jensen (1958) used five pairs of mice, each with a disparity in age of 11 to 19 days, for parabiotic unions. He stated that the cyclic nature of hair growth was maintained in both partners, that the times of onset and completion of hair growth were within normal range and that there were no alterations in pattern. There was, however, a wide scatter in his few results. The differences in age at onset of the wave ranged from —23 to +22 days in third wave (based on four available records) and from —63 to + 32 in the fourth wave (based on only three records).
In all our parabiotically joined rats, even those of equal age, we found that the waves of hair growth moved more slowly than in single controls. This fact is not accounted for by any immediate surgical shock due to parabiosis, from which the rats might recover, since the waves are unaffected at first and the delay is progressive. It could be that stress arising from antigenic or other disharmony causes increased adrenocortical secretion which in turn delays the onset of follicular activity. Johnson (1958c), for example, has shown such an effect after administration of ACTH. Retardation of bodily growth as compared with single controls is also consistently seen in parabiotic rats. This is not due to surgical stress, for a sham operation of comparable severity does not produce it. It may be nutritional in origin, due perhaps to interaction of the animals’ food intake regulations (Hervey, 1959). This might affect hair growth.
Other explanations are, however, possible. Even in rats of equal age the hair waves are not exactly in phase, and we have already seen that synchrony tends to occur between such parabionts. If there are periodic changes in the concentrations of accelerators or inhibitors, such changes will be shared with the parabiotic partner to an extent depending on the cross-circulation developed, and on the physiological life of the substances concerned, as compared with the mixing time of the two circulations (Hill, 1932; Finerty, 1952). In the pairs used for the present investigation the exchange of plasma was at least as great as is usually found in parabiotic rats. In the normal animal, we might postulate that the concentration of any hormone will not fluctuate much beyond the threshold level for producing its effect since, whether the evolution of a hormonal mechanism involves an increase in concentration or potency of the hormone, or the development of sensitivity in the reacting tissue, selection pressure is removed once the result is achieved. The sharing with a parabiotic partner may thus reduce concentration so that the threshold is not reached in either animal until that partner also contributes; this must result in a delay of the hair wave. The model is illustrated diagrammatically in Fig. 5, and will be referred to as the hypothesis of threshold delay.
If the results are to be fully explained on the hypothesis of threshold delay, the average amount of delay in parabiotic rats ought to be predictable from their normal variability. In an attempt to test this the figures for control rats were paired at random. Then, using figures for either rat, the maximum possible interval between each successive eruption was calculated. Means and standard errors of these were computed and compared with means and their standard errors of the intervals in parabiotic rats. The results are given in Table 5. The usual /-test is not applicable because the within group variances are different : in the control rats they increase as, in succeeding intervals, the means increase; in the parabiotic rats they diminish because, as already shown, the partners tend to become synchronized with each other. Nevertheless, the means for the parabiotic rats are consistently larger. Bearing in mind that the mean for the control rats is based on the greatest delay which could occur in one member of each randomly selected pair and is thus a maximum rather than an average estimate, it can be seen that the interval between eruptions in parabiotic rats is greater than could be accounted for on the postulated model. Some other mechanism, possibly an adrenal stress reaction, must be involved. The explanation of immunological stress appears likely from the extent of parabiotic disharmony and would be supported if the delay in non-sib pairs were greater than in sib pairs. It is clear from the comparison made in Table 6 that there is, in fact, no such significant difference. But equally there was no difference in the incidence of parabiotic disharmony between siblings and non-siblings. Irrespective of the lack of positive evidence it would thus be unwise to rule out immunological incompatibility as a contributory factor.
Parabiosis was performed between pairs of rats both 28 days old, pairs of rats one 28 days old and the other 49 days old, and pairs both 49 days old. In parabiotic pairs of unequal age it was demonstrated that the waves of hair growth tend to become synchronized and that both shortening and lengthening of the follicular cycles in different regions contribute to this process. A significant tendency to synchronization was shown even in parabiotic pairs of equal age. The hair waves were substantially retarded in comparison with groups of single control rats.
L’activité des follicules pileux chez les rats en parabiose
On a réalisé des parabioses entre rats ayant tous deux 28 jours, entre rat de 28 jours et rat de 49 jours, et entre rats ayant tous deux 49 jours. Chez les couples de parabiontes d’âge différent, on a démontré que les ondes de croissance pileuse tendent à devenir synchrones et que le raccourcissement et l’allongement des cycles folliculaires dans les différentes régions contribuent à ce processus. Une tendance significative à la synchronisation a été mise en évidence même chez des couples de parabiontes de même âge. Les ondes pileuses étaient retardées considérablement par comparaison avec les groupes de rats témoins isolés.
It is a pleasure to acknowledge the help given by Dr Elizabeth Hervey, Miss K. White and Mr John Skinner. We are very grateful to Miss Hilda E. Davies, of the Department of Statistics, University of Sheffield, for discussing statistical procedures with us, and for the analyses shown in Tables 3 and 5. Financial assistance was received from the Sheffield University Research Fund and from Beecham Toiletry Division.