Relationship of the Spread of India Ink to the Hair Cycle in Rabbit Skin1

The known factors which influence the spread ol insoluble dyes injected into the skin of experimental animals are: sex (1), heredity (1), age (2), body site (2), temperature (3), and hormonal status (4, 5, 6). This report considers an additional factor, the influence of the hair cycle. This applies specifically to animals such as the rat, mouse and rabbit in which the hair coat exhibits a regional pattern of growth. The hairs in a given region of these animals, unlike humans and guinea pigs, are all in the same phase of growth. Periodic and regu lar waves of hair growth (anagen period) are succeeded by periods of quiescence during which the hairs remain inactive as “club” hairs (telogen period). Plucking telogen hairs initiates a new growth cycle in the plucked area (7, 8, 9). It is thus possible in the same animal to prepare areas in which the hairs will be at a definite desired stage in the cycle. During the anagen period in animals with a regional pattern of hair growth, the cutis and subcutaneous tissues are markedly thickened, the follicles are considerably elongated and the vascular supply greatly increased in contrast to the much smaller volume and extensiveness of these tissues during telogen (10, 11); thus, there are prominent changes in the skin during the various phases of the hair cycle. Indeed, the hair cycle is simply the externally observable reflection of a more fundamental cyclic phenomenon in the skin. One could thus speak quite appropriately of “skin cycles". METHOD White male rabbits weighing 2.0 to 2.5 kilograms were used. The hair was shaved from a small area on the side. The area was judged to be in telogen if no hair growth occurred during the next five days. Then, immediately adjacent to this, the hair was plucked in an area about 2.5” square to start a new hair cycle. The prepared sites were considered satisfactory for study, if 10-12 days after plucking, a new hair coat appeared on the plucked area and the original shaved area still remained hairless. On the 15th day, an intracutaneous injection of 0.5 cc of Higgins India ink diluted 5 times with physiological saline was made into the adjacent anagen and telogen areas. The two greatest diameters of spread at each site were recorded ninety minutes later and the approximate area of spread calculated from these. (The results were approximately the same 24 hours later but the borders were less distinct). The injected areas were totally excised after 90 minutes in several animals. The tissue blocks were bisected and subjected to histologic study. 425 426 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY TABLE I Comparison of Areas of Spread in India Ink in Anagen and Telogen Areas of Rabbit Skin ANIMAL TELOGEN, (MM*) ANAGEN, (MM2) 1 2 3 4 5 6 7 8 9 10 11 616 596 682 707 707 531 732 488 381 133 239 416 434 113 381 314 188 364 330 330 202 133 Mean 528 291* * These data are statistically significant (P < 0.01). Fig. 1. Surface appearance of spread of India ink in an anagen (left) and telogen (right) area. RESULTS AND DISCUSSION Table I shows that the spread of India ink as measured from the surface is considerably less in anagen areas (Fig. 1). This is, however, entirely misleading. It is difficult to assess the meaning of the reduced spreading observed in anagen areas when the observations are made from the surface. It is necessary to point HAIR CYCLE IN RABBIT SKIN 427 out that the corium is at least twice as thick in anagen as in telogen. Thus, the ink is being injected into a larger volume of tissue. If the volume of tissue were equal, it is even possible that the spread as measured from the surface would be greater in anagen than in telogen. Another consideration, in addition to the above, is that there is a marked difference in the pattern of spread in anagen and telogen areas. This is readily seen on gross observation of the cross sections of the tissues. In the former areas the ink is distributed throughout the corium in a more or less sharply bordered narrow column with convex sides. At the base of the column, however, the ink T ..41.....i ~5T......j ~W~T~ir “i~5T ; HT \ 110 j~~-j[f fliiTlTTfT ri 111111 m 111 Itti 11111 ti ii Fig. 2. Cross sections demonstrating the difference of pattern of spread of India ink in anagen (above) and telogen (below) areas. has spread out in a distinct thin plane parallel to the skin surface (Fig. 2). The diameter of spread of this narrow band is almost twice that of the column of ink above it. The surface measurements evidently record only the diameter of the column and not the deeper lying band which is too deep to be clearly visualized from the surface. In the telogen areas, the cross sectional view shows the ink in a dome shaped pattern which flattens out laterally and below but does not form a distinct band. Histologic study shows that the band in the anagen areas corresponds to the upper surface of a fascial sheet lying just above the muscle and below the level of the bulbs of the hair follicles. In anagen areas the hair bulbs are extremely deep and are almost at the level of the fascial sheet. This fascial layer is grossly prominent in the rabbit and lies between the corium and the muscle. In anagen areas it is easy to see that ink descends in a column through the corium until it meets the fascial sheet which it cannot penetrate. It then spreads out along the surface of this sheet in the lowermost region of 428 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY the corium. Perhaps, then, it is a collecting lymphatic plexus in this area which furthers the movement of ink in a band-like pattern. In telogen areas, there is some evidence that some of the ink has spread out along this same plane, but this is not conspicuous. Thus, the greatest diameter of spread obviously depends on whether observations are made from the surface or from cross sectional visualizations. The diameter of greatest spread in an anagen area is at least as large as that in telogen areas if it is the limits of the lower band which are being recorded from a cross section. Therefore, the surface measurements give results which are more apparent than real. SUMMARY The spread of intra-cutaneously injected India ink as measured at the surface in the skin of rabbits is less in anagen than in telogen. Since the corium in anagen is much thicker than in telogen, this difference in spread at the surface must simply represent a difference consequent upon the injection of similar quantities of ink into different volumes of tissue. Also, the patterns of spread in anagen and telogen are found to be distinctively different. It cannot be concluded that the spread of India ink is less in anagen than in telogen. However, for comparable results in experiments dealing with the spreading phenomenon, the stage of the life cycle must be definitely considered.