Observation of a System of Linear Loops Formed by Re-Growing Hairs on Rat Skin.код для вставкиСкачать
THE ANATOMICAL RECORD 291:858–868 (2008) Observation of a System of Linear Loops Formed by Re-Growing Hairs on Rat Skin LI-YUAN LIU,* DONG-SHENG GUO, XIU-YU XIN, AND JIN FANG Beijing Normal University, College of Life Science, Beijing, People’s Republic of China ABSTRACT This paper details linear hair re-growth patterns observed in rats. Adult rats were shaved and observed. The first wave of hair re-growth did not distribute everywhere, but along specific craniocaudally-oriented lines. The hair-lines were 2–15 mm wide and ran from the head, through the torso to the limbs, and were symmetrical along the left and right sides of the body. The symmetric hair-lines from both sides of the body converged around the mouth, nose, and at the pubic region or ventral midline to form a system of hair-loop-lines (HLLs). The loops can be differentiated into four main patterns. The Dorsal Loop and the Lateral Dorsal Loop run along the dorsum and hindlimb. The Ventral Loop and Lateral Ventral Loop travel along the thorax, abdomen, and forelimb. These hair-lines coincide with our previously observed sympathetic-substance lines (SSLs) in the rat’s skin. Histological observation indicates that rat hair follicles along the hair-lines were at anagen phase. The catecholamine histofluorescent check showed abundant sympathetic nerve fibers beneath the hair-lines. After the rats’ hairs were dyed, and selected portions shaved, re-growth was only observed on the shaved portions, indicating that the linear hair growth closely correlated with the shaving. Lastly we examine the cause of the preferential re-growth and briefly discuss the purpose and physiological role of the HLL. Anat Rec, 291:858– 868, 2008. Ó 2008 Wiley-Liss, Inc. Key words: hair-loop-line; sympathetic-substance line; environmental stimulation The presence of hair is a common characteristic of all mammals. Primary data on hair growth come from the fundamental studies by Dry (1926) on hairy mice. Since then, hair growth in skin regions has been characterized by growth phases succeeded by a resting phase. Hair change occurs in three to four growth cycles (G1–G4) up to the 126th day of the life in a normal hairy mouse. A normal G1 cyclic is completed between the 8th and 10th day in hairy mice (Dry, 1926; Andreasen, 1953; Borum, 1954). The G2 cycle ends on approximately the 45th day of a normal hairy mouse (Andreasen, 1953; Borum, 1954), in contrast to the rat, whose G2 cycle ends between the 44th and 50th day of its life (Butcher, 1934). The G3 cycle ends on approximately the 100th day. No other distinct regular hair pattern can be noted in normal hairy-pigmented mice after the G3 cycle (Dry, 1926). Hair cycles in rodents may be different, dependÓ 2008 WILEY-LISS, INC. ing on the species. It is especially true in mice (Johnson, 1958; Eaton, 1976; Militzer, 2001). In one of our previous studies, using whole body macro-autoradiography, with an iodine 125-labeled tyrosine, we localized the distribution of iodine-125-catecholamine in the rat skin. The images on the film showed Grant sponsor: NSFC. *Correspondence to: Li-yuan Liu, College of Life Science, Beijing Normal University, Beijing, 100875, China. Fax: +86-1013693121193. E-mail: firstname.lastname@example.org Received 16 August 2007; Accepted 27 February 2008 DOI 10.1002/ar.20702 Published online in Wiley InterScience (www.interscience.wiley. com). SYSTEM OF LOOPS FORMED BY RE-GROWING HAIRS ON RAT SKIN various pairs of symmetrical lines running from the head, through the back of the animal, and to the hind limbs, that we termed sympathetic substance-lines (SSLs). Furthermore, we proved that the substance-lines came from dense nets of noradrenergic nerve fibers innervating the arrector pili muscles beneath the line (Liu et al., 2005). In an accidental case involving observations of skin hyperaemia to NA released from the SSL, an adult rat’s hairs were shaved in a large region (this was researched and reported in a separate study). After 2 days, we noticed that the first wave of hair regrowth was located along a line from the head through the back to the hind limbs. Thus, we researched such hair-lines in detail, and the cause of growth. MATERIALS AND METHODS Wistar white rats, 100–120 days old, weighing 200– 280g were selected. There were 20 males and 10 females. The animal experiments described in this study were performed in accordance with the ‘‘Principles of Laboratory Animal Care’’ (NIH publication No. 86–23, revised 1985). Hair-Line Observation The rats were anesthetized with pentobarbital sodium (40 mg/kg, intraperitoneal injection). All of the rats’ hairs were carefully shaven by razors. The rats were fed in an air conditioned room (approximately 22–248C) in summer. Hair growth rates were observed daily. When adequate hair-lines appeared, the rats were anesthetized again and photographed or scanned by a scanner. Some rats were recorded every 3–5 days to observe the various phases of hair re-growth. After all shaved hairs grew to normal length (approximately 30 days), the rats were killed with an overdose of pentobarbital sodium, and the skins were made into specimens. Histological Check In the four rats anesthetized with pentobarbital sodium, skin tissue samples were cut from the hair-lines and shaved bare areas of the rat’s back 3–5 days after the hairs were shaved. Frozen sections, 15 mm thick, were sliced vertically along the direction of the hair root. The sections were stained by the general hematoxylin–eosin method (H&E). The sampled rats were killed with an overdose of pentobarbital sodium. Catecholamine Histofluorescent Check When the rats’ re-growth hair-lines appeared significantly after shaving, skin tissue samples were obtained from more than 10 rats anesthetized with sodium pentobarbital. Samples were cut from the hair-lines and the shaved bare region of the rat’s back. Frozen sections, 25 mm thick, were sliced obliquely, in which the section was parallel with the transverse plane and along the direction of the hair shafts, at approximately 45 degrees to the skin surface, so as to obtain the entire section of the hair follicle’s AP muscle group. Sections were then processed with our modified sucrose-phosphate-glyoxilic acid (SPG) method (De La Torre, 1980; Liu et al., 2005). After covering the specimens with paraffin oil, catecholamine histofluorescence was observed and photographed 859 with an Opton fluorescence microscope (excitation range: 355–425, emission suppression filter: 460 nm, 50 W mercury vapor lamp). Shave and Growth To study the cause of growth, several methods have been performed. Dyeing hairs. The hairs of the five rats were dyed with 1% methyl violet liquid. Methyl violet is a nontoxic cudbear, or dye. After 10 days’ observation, the dyed hairs were shaved in alternating rows of skin to form both shaved and unshaved semicircuitous zones. The shaved rats were photographed. After 10 days’ additional growth, photographs were taken again. Chemical sympathectomy. Six shaved rats (three males and three females) were be intracutaneously injected immediately with the neurotoxin 6-hydroxydopamine (6-OHDA, 1 mg in 0.2 ml) along the dorsal-lateral line (a hair-line always passed) at the upper right back when the hairs were shaved. Control injection is equal volume saline at the left upper back. The regrowing hairs were observed as above. Temperature. Ten rats (five males and five females) were shaved and observed as above, but the room temperature was keep above 328C in summer. Otherwise, six rats (three males and three females) were shaved and observed as above, but the thorax were wrapped with a circle of thick cotton to preserve heat. RESULTS Hair-Line Observations One to 2 days after the hairs were shaved, new hairs erupted in 25 of 30 rats. In some rats, hairs began to erupt 3–6 days after shaving. The first wave of hair regrowth did not distribute evenly, but grew along craniocaudally oriented lines or zones, which we have termed hair-lines (Fig. 1, 2). The hair-lines were approximately 2- to 15-mm wide and symmetrical along both left and right sides of the body. The hair-lines usually began to appear at the lateral back, extending toward the head and limbs after 3–10 days. During this time, no hair regrew on other areas of the skin. The boundary between the hair-line and shaved areas were clear and welldelineated. Hairs began to grow in those shaved regions after 10–20 days. After all hairs arrived at normal length, the boundaries disappeared. The wider hair-lines were mainly distributed along the lateral dorsal skin, decreasing in size along the limbs and head. The hair-lines always formed several close loops, which we have termed hair-loop-lines (HLLs). HLL are distinguished into four patterns, according to courses and numbers of the loop. Finally, all patterns converge with each other around the mouth and nose. Due to the presence of these loops, we have to suggest a starting point for sake of description. Dorsal lateral loop. The first pattern of the HLL starts along the posterior axillary line of the rat (Fig. 2 2). In the cranial direction, the hair-line runs up 860 LIU ET AL. Fig. 1. Hair-loop-lines appeared 4 days (left, right) and 9 days (middle) after shaving a rat’s hair (No. 20). A small circle appeared on the top of the head. (1) dorsal loop; (3) ventral lateral loop. Scale bar 5 20 mm. to the face, terminating at the upper lip just above the mouth, and joining the hairs around the nose where the hair-lines from both side connect together. In the caudal direction, the hair-line travels along the anterior lateral portion of the hindlimb. In the middle of the crus, the hair-line coils around the front to the medial portion, then courses upward along the posterior medial portion of the hindlimb toward the groin. Thus a U-shape hairline forms along the front surface of the crus. Finally, the hair-line transverses across the pubic region and merges with the opposing symmetric hair-line. Thus, the hair-line consists of a loop around the lateral surface of the back, hindlimb, and pubic region. We refer to this loop as the dorsal-lateral loop (DLL), because it is more proximal to the back (Fig. 3 DLL). This was the largest loop, observed among almost all rats. Dorsal loop. The second pattern of the HLL starts along the scapular median line on the rat’s back (Fig. 1, 2 1). Near the cranial direction, the hair-line runs upward toward the underside of the ear, coiling toward the cheek, just below the eye. The hair-line then terminates above upper lip, where the hair-lines from both sides connect together. In the caudal direction, the hair- line runs downward around the root of the tail and anus, merging along both sides at the posterior spermary (male) or perineum (female). This hair-line also gives a fine branch at the hip. This branch runs along the lateral posterior aspect of the hindlimb toward the calcaneal tendon, where the hair-line surrounds the ankle, forming a small loop. Thus one large loop and two small loops are formed around the head, back, tail, and hindlimbs. We refer to these loops as the dorsal loop (DL; Fig. 3 DL). The small loop around the ankle we termed the ankle loop. Sometimes, the two hair-lines along both sides of the back converge in the mid-dorsal line over the anterior dorsal region forming an H- or Xshaped pattern in the latter growth (not first appeared). In the case of the DL, a small circular hair-line always exists at the top of the head between the ears, which we have termed the top loop (TL; Fig. 1). Another hair loop pattern, a wide hair-band distributed along the dorsal midline of the rat’s back, can be regarded as a variety of the DL. At the neck, the hair-band divides into left and right branches. The two branches coil around the root of the ears and unite to form one hair-line between the eyes. A local loop is then formed around the head and neck. The united hair-line runs along the midline of the nose, terminating above the nose. At the sacral region, SYSTEM OF LOOPS FORMED BY RE-GROWING HAIRS ON RAT SKIN Fig. 2. New growth hairs distributed along hair-lines and hair-looplines after shaving rat’s hairs. c: No. 24 rat after shaved 6 days showing three hair-lines in the lateral view. d: No. 16 rat 11 days after shaving showing the part of the ventral loop in thorax. a: No. 20 rat show- 861 ing the wrist loop. b: No. 14 rats 9 days after shaving showing the part of the dorsal lateral loop in the pubic region. (1) Dorsal loop; (2) dorsal-lateral loop; (3) ventral-lateral loop; (4) ventral loop. Scale bar 5 20 mm. 862 LIU ET AL. Fig. 3. The schematic presentation of hair-loop-lines along a rat’s skin. DLL, dorsal-lateral loop; DL, dorsal loop; DL1, DL2, the two varieties of DL; VLL, ventral-lateral loop; VL, ventral loop; VL1, VL2, the varieties of VL. the hair-band branches into two ramifications, which coil around both sides of the tail and anus, and merge to form a small narrow loop. Similar to the previous DL, two hair-lines branch from the hip and run toward the ankle, forming the Ankle Loop. These may be regarded as the two separate hair-lines along both sides of the back in the above DL combine together. Therefore, the hair-line along the dorsal midline is always wider (approximately 20 mm). The H- and X-shaped hair-line in the above DL may be regarded as a transitional stage from the two lines to the single band line (Fig. 3 DL1, DL2). In a specific case, only one hair circle was observed on the top of the head with a short hair-line on the sacral region. Ventral lateral loop. The third HLL pattern starts from the rat’s underlip below the mouth and runs down through the lateral neck to the shoulder (Fig. 1, 23). The hair-line coils around the posterior shoulder and travels along the anterior axillary line to the lateral groin. The hair-line then obliquely crossed the hypogastric region to the front of the urethra, where the hair-lines from both sides merge with each other. A hair-line or hair-band always branches from the shoulder and runs obliquely from the anterior lateral portion of the forelimb to the dor- sal lateral wrist, where the hair-line circles the wrist, forming a small loop. Under the lip, the hair-lines of both sides connect to each other, thus forming loops around the thorax, abdomen, and forelimb. We refer to these as the ventral-lateral loop (VLL; Fig. 3 VLL). The small loop around the wrist we call the Wrist Loop, a phenomenon observed in the every rat. In many cases, the inner boundary of the VLL at the abdomen was unclear, because shorter hairs always grew at the abdomen. Otherwise, the various parts of this loop usually united with part of the previous DLL. The united hair-lines, therefore, become wider. In a few cases, the hair-line at the hypogastric region protrudes downward toward the front thigh, a Ushape-like protrusion of hairs which appears to be formed by the merging of the DLL with the VLL (Fig 3). Ventral loop. The fourth HLL pattern remains unclear in many rats, usually growing later than others. This hair-line begins at the small protrusion below the chin and runs obliquely through the upper thorax to the anterior medial portion of the forelimb (Fig. 2 4). At the distal forelimb near the wrist, the hair-line turns toward the posterior medial portion of the forelimb and back toward the lateral axilla, forming a U-shape. The hair-line then coils upward slightly, before curving SYSTEM OF LOOPS FORMED BY RE-GROWING HAIRS ON RAT SKIN 863 TABLE 1. Appearance rate of the type of the hair-lines in all 30 rats Type DL21(DLL1VLL)1VL DL11(DLL1VLL)1VL DL11DLL1VLL1VL DL1(DLL1VLL)1VL1TP DLL1VLL1VL1TP Fig. 4. Number Characteristic 14 3 2 7 4 A long dorsal zone 1 A pair of wide lateral lines A short dorsal zone 1 A pair of wide lateral lines A short dorsal zone 1 Two pairs of lateral lines Two pairs of parallel lines on the back Most of the back is bare The histological photo of hair follicles along the hair-line (a) and in the bare area (b). Scale bar 5 40 mm. downward toward the thorax, descending along the clavicular midline toward the umbilical region of the abdomen, where the hair-lines from both sides merge with each other. Below the chin, the hair-lines of both sides begin at the same point. Thus forming a loop around the thorax, the medial portion of the forelimb, and the upper abdomen (Fig. 3 VL). We refer to it as the ventral loop (VL). This loop is described representationally, because the outer hair-line boundaries more or less follow the VL routes, while the inner boundaries remain unclear due to the short hairs spread the abdomen and thorax. In some rats, the hair-line is distributed as a rhombic or cruciate shape on the thorax (Fig. 3 VL1, VL2). The hair-lines on both sides are closed and combine together at the midline of the abdomen and thorax, similar to the hair-lines at the midline of the rat’s back. We drew the schematic figures of all HLL patterns in Figure 3. All four HLL patterns (DL, DLL, VLL, and VL) typically appear in all rats. However, in many rats, some parts of the DLL and VLL unite together. Both DL1 and DL2 patterns are found in many rats in place of DL patterns, and can be thought of as variants of DL. The same is true for the VL1 and VL2 varieties of VL. The DL and VLL are analogous, because the DL runs toward the hindlimb, forming a circle around the ankle, while the VLL runs toward the forelimb, forming a circle around the wrist. Also the DLL and VL are similar, as the DLL runs toward the hindlimb, turning back toward the pubic region forming a U-shape, while the VL runs toward the forelimb, turning back to the thorax, forming another U-shape. The DL and DLL are mainly distributed around the dorsal skin and hindlimb. The VLL and VL are mainly distributed around the ventral skin and forelimb. The dorsal hair-lines are always clearly delineated, whereas the ventral hair-line are often hard to recognize. The emergence of hair-line types of all 30 rats are listed in Table 1. The use of parentheses (DLL1VLL) indicates that parts of the DLL and VLL united together in some rats. The VL1 and VL2 are not listed as the hair-line types are difficult to judge. Regarding the difference between the sexes, only the hair re-growth in female rats somewhat were latterly than the males. No different pattern of HLL has been observed. Common Histological Check When new hair-lines appear in shaved rats, the hair follicles in the new hair-lines are always in anagen phase. The bulb of the hair follicles is extremely enlarged and deeply inserted into the hypodermis (Fig. 4a), but the hair follicles in the shaved bare areas are short, small, and in the phases of telogen, proanagen, or catagen (Fig. 4b). Catecholamine Histofluorescent Check In the sections from the re-growth hair-lines, numerous radiant clusters or parallel fascicles of catechol- 864 LIU ET AL. Fig. 5. Photomicrographs of catecholamine-containing fluorescent sympathetic fibers beneath the regrowing hair-line of the rat skin. The fibers form clusters and fascicles in the arrector pili muscles. Scale bar 5 40 mm. amine-positive nerve fibers could always be seen (Fig. 5). But in the sections from the shaved bare region, the catecholamine positive nerve fibers are seldom and usually appear in simple small bundles. Shave and Growth Dyeing hairs. When rats were shaved in semicircuitous patterns, the purple hairs are easily photographed and distinguished. Among the alternating hair and skin patterns, no white hairs could be found anywhere in any length (Fig. 6a). At 3 to 5 days after shaving, longitudinal hair growth was observed along the hairline patterns as described above, but only in the shaved regions. Ten days after shaving, the new hair growth nearly reached the same length as the purple, unshaved hairs (Fig. 6b), yet the purple, unshaved hairs along this same hair-line did not grow at all, nor did the shaved areas outside of the hair-line. Chemical sympathectomy. In five of six rats, the hair-line appeared through the injected point. The new hairs grew around the injected point. There did not show any visible differentiation between the druginjected point and the saline-injected point, and between the injected area and noninjected area. Temperature. The appearance of hair-line of rats fed in the 328C room were approximately 5 days (5.1 6 2.5). When compared with previous rats fed in lower temperature (2.5 6 1.9) by t-test, the results is significant (P < 0.05). The results from the cotton-wrapped rats were difficult to judge, because it is quite difficulty in keeping the cotton attached steadily to the bodies of active rats. DISCUSSION HLL and SSL We have observed a series of new hair re-growth phenomena in rats, with postshaving hair re-growth rates differing significantly from one body region to another. The first obvious feature of the hair-line is that it passes throughout the entire surface of the body. The second significant feature is that these patterns occur in loops. We may conclude that a system of linear loops occurs inherently in the rat’s skin. The HLLs encompass all body surfaces and occur in regular and geometrical forms. Given these results, the question remains: Why does hair re-growth emerge in lines, and why do these lines form loops? Although line-like hair growth has been illustrated by Dry in hairy mice and by Militzer’s photos in nude mice, the linear hairs are short, local, irregular, and in unstimulated states (Dry, 1926; Militzer, 2001). In Johnson’s observation, the hair growth of the waves in albino rats first erupted at the ventral (whereas in this study, the hair-lines usually began to appear at the lateral back), and spread laterally to the neck, mid-side and flank regions during its first and second week. The growth waves from each side converge in the mid-dorsal line over the anterior dorsal region 3–4 weeks after the first growth wave (Johnson, 1958). From the dorsal view, it seemed like a pair of hair zones running along the dorsal-lateral line of the rat and a H-shape formed (similar to the dorsal DLL in this study). Because the DLL appeared in all rats, Johnson’s dorsal inner border should be the homology with the DLL. But the H-shape only appeared in some rats in this study. Also in Johnson’s observation, the top of the head was finally covered by the hair growth of the waves (the uncovered region is similar to the inner area of the Top Loop in this study). The hair-lines described in this study occur in regular loops and throughout the entire body surface. Growth SYSTEM OF LOOPS FORMED BY RE-GROWING HAIRS ON RAT SKIN 865 Fig. 6. The influence of shaving on hair growth in a rat. The head, leg, and the left body were not dyed. a: At 10 days after dyeing, the hairs were semi-circuitously shaved. No new hairs grew. b: At 10 days after shaving, white hairs grew longitudinally along only the hair-line, and only in the shaved regions. Scale bar 5 20 mm. from several loops and bare areas (as opposed to waves from ventral to whole body) is a new phenomenon. They do not follow any known nervous, vascular, or lymphatic structure in the skin. Although the procedure of shaving have accomplished carefully, partially skin injury also occurred as visible scratches in rats where partial regrowth had occurred. Thus those scattered small injury did not shown any influence on the integral hair re-growth, especially the linear pattern of hair re-growth. According to our preliminary experiments, postshaving linear hair growth could be observed in mice and rabbits. The linear courses of hair-lines coincide with the arrays of previously mentioned SSLs (Fig. 7; Liu et al., 2005). In Figure 7, A1 and A2 are a pair of labeled linear arrays that run from the head, through the posterior axillary lines down to the medial portion of the feet, that correspond with the DLL. B1 and B2 are another pair of labeled linear arrays that run from the head through the scapular median line to the lateral portion of the hind limbs, that correspond with the DL. C1 and C2 are a third pair of labeled arrays that begin at the head, pass around the shoulder, and then run along the anterior axillary lines, corresponding with the VLL. The C1 and C2 expanse at the shoulders occur as wide hairlines in all rats. The expanse at the lateral groin coincides with the protrusion of the VLL hair-line in only a 866 LIU ET AL. Fig. 7. Autoradiograms of the whole rat skin show labeled arrays of sympathetic substance-lines (SSL) after intraperitoneal injection of 125 I-tyrosine. The dashed line is the dorsal midline. a: A male rat with two pairs of SSL (A1-A2 and B1-B2). b: A female rat with three pairs of SSL (B1-B2, C1-C2, and D1-D2). Note the hindlimb portion of C1C2 is not continuous to the line in the torso with a small break. c: The schematic representation of all SSL in the rat skin. few rats (Fig. 1). The short line at the hindlimb labeled C1 and C2 had a small break with a line from the back (Fig. 7b), which we erroneously regarded as part of C1 and C2. Now we know that C1 and C2 are really the VLL and do not belong to the hindlimb. Therefore, the label of C1 and C2 at the hindlimb in Figure 7 is mistaken. The D1 and D2 are labeled linear arrays in the forelimbs, which correspond with VLL in the forelimb. M1 corresponds with DL2, in which the two hair-lines of both sides combine together along the dorsal midline. M2 corresponds with part of the VL along the ventral midline. The SSL samples come from the incised skin decorticated from the rat, and are spread out into a plane. Some lines appear at the edge of the incision. Some lines might be disturbed. The skin in the distal part of the limb and the spermary are always imperfect. Here, the hair-lines displayed many details in the live rats, which were unseen in the autoradiography films. For example, the U-shape on the hind limb is difficult to confirm in the film. The lines in the pubic area are always damaged in the decorticated skin samples. The ankle loop and the wrist loop are almost unrecognizable, due to dense, irregular images at the distal limb in the film. We can confirm that the dense areas at the distal limbs are the wrist and ankle loops; and that the expanded region at the perineum is where both sides of the hair-line meet (Fig. 7a,b). We did not try to correct the details of the SSL according to the HLL, because the SSL lines represent our previous judgment, in which we have done our best. Thus the HLL form is very clear and more intact than the SSL. In comparison with complex, expensive, and dangerous autoradiographic treatment, hair-line observations are quite easy, allowing us to gather much data from many rats. The HLL helps us understand the SSL perfectly. But regretfully, the hair-line is obviously wider than the SSL. The catecholamine histofluorescent check showed abundant sympathetic nerve fibers beneath the re-growth hair-lines, identical with our previous observation in the SSL, indicating that the hairline grows along the SSL. Because the SSL arrays coincide with hair-lines, the hair follicles (or the arrector pili muscles along the hairlines) are innervated by a great number of sympathetic endings. We are observing the phenomenon that when the sympathetic innervating a local hairs along the hairline is stimulated and the local hairs stand up, these pilomotor will spread along the hair-line and finally form a pilomotor-line, which include some unknown physiological function (for example, pain). These will be reported latter in details. Here, we just want to say that the HLL not only a superficial phenomenon of hair regrowth, but also involve the internal function of the body. Cause of Linear Hair Growth Age and sex may be an important variable affecting hair growth. It was well established by Dry in 1926 that the synchronous hair growth patterns in rodents change dramatically from hair morphogenesis. In this extensive study, hair pattern development in the first three generations was provided in great detail for the mice. Because the HLL patterns were already very complex, all rats used in our experiment had already past their first 100 days of hair growth. We do not account for differences involving age in this study. However, primary data suggest the numbers of HLL patterns in younger rats were less than the older rats. In the old rats (>6 month), the re-grown HLLs were delayed and discontinuous. SYSTEM OF LOOPS FORMED BY RE-GROWING HAIRS ON RAT SKIN One may ask why we never noticed that the hairs along the hair-line were longer than those along the side. The reason for this is because we believed that cutting, shaving, or plucking hairs would not alter growth rate or hair character. Roersma and Veldhuis (2001) have observed that plucking of an anagen hair is not limited to reset of the follicle to telogen or continued hair growth, but that a temporary reduction of the mitotic activity is very likely to occur. Here, when the rats’ hairs were shaved, linear hair patterns noticeably appeared. Furthermore, the histological observation proved that the hair follicles along the hair-lines remained in active anagen growth states. Thus, the hair grew more quickly after being shaved than we expected. Why then, did hair growth along the line appear after being shaved? We have explored these questions in many ways. Hair growth and density are influenced by many factors, but most importantly by nutrition and hormones. But in our unpublished experiments, the injection of testosterone to the shaved female rats seemed to have no influence. The clinical and experimental observations have long suggested that the skin sympathetic nerves have ‘‘trophic’’ functions in the hair follicle development, growth, and/or cycling (Paus et al., 1997). Schotzinger and Landis (1990) demonstrated that the thoracic hairy skin in developing and adult rats receives abundant catecholaminergic sympathetic innervation. Botchkarev et al. (1999) have reported that the number of adrenergic nerve fibers and b2-adrenoreceptor expression of the hair follicle epithelium in murine skin increases during the early growth phase of the hair cycle (anagen). Subcutaneous injections of the noradrenaline-depleting agent guanethidine, or of the neurotoxin 6-hydroxydopamine (6-OHDA), induce a premature onset of anagen in the lower back skin of mice (Peters et al., 1999). Newborn mice were subcutaneously injected once with 6OHDA, and distinct areas of hairless and thinning of skin were observed surrounding the sites treated with 6OHDA (Asada-Kubota, 1995). Combining our SSL observations, we deduced that the sympathetic innervation might be the dominant factor to linear hair re-growth. Thus, the influence of the chemical sympathectomy on HLL re-growth was observed in our experiment. But no positive results could be observed. The sympathetic in the SSL must be active, and the arrector pili muscles along the SSL also must be active. Thus, the hair follicles along the SLL (or the HLL) must be stimulated continually. This mechanical stimulation and/or trophic functions are important factors to promote the hair growth and keep the hair follicles in an anagen phase. Thus, the tentative chemical sympathectomy cannot change the existent active growth state of the hair follicle nor disturb new hair-growth after shaving along the HLL. As reported, the rat hair growth was promoted by irradiation using Super Lizer (infrared ray). The mRNA expression levels of hepatocyte growth factor (HGF) and HGF activator measured by reverse transcriptase-polymerase chain reaction were high in the skin extracted from the irradiated rats (Miura et al., 1999). HGF as a mitogenic factor for dermal fibroblasts may be the factor in different hair lines (or zones) of the HLL and non-HLL. These may relate to the neurotransmitter with HGF in the hair growth. 867 When we considered whether hairs along the SSL or HLL grew actively during normal states, the dyeing method was selected. Our results indicate that the hairs along the hair-line do not grow in normal states, but grow significantly after being shaved. The unshaved hairs, however, did not grow at all. Of the shaved hairs, only those along the hair-line grew quickly. This finding indicates that the environmental influence on the shaved skin stimulated hair re-growth, or that the SSL or HLL is sensitive to a change of environment. We deduced, therefore, that the primary environmental factor to the shaved skin should be temperature. As a fact, the hair re-growth along the hair-line was significantly delayed when the room temperature was approximately 328C. We attempted to wrap the shaved area with thick cotton to preserve heat. But the results were difficult to judge. As a sum, the morphological differences of distribution between the HLL and non-HLL are genetically fundamental, and the abundant sympathetic innervations along the HLL are stimulators for the active growing phase of hair follicles. Temperature triggers fast regrowth after shaving. We may deduce that the HLL function to help the body adapt to environmental change. But how does the environment and/or temperature stimulate hair growth? How does HLL work in a normal body? And how do the HLLs develop in embryos? To make it clear, there is a long way to go. After all, here, indicated again, the skin is not an even sheet enclosing the body, but it includes a complex linear system that is divided into different parts/zones. We are sure that the hair-line is not just a superficial phenomenon of hair re-growth, but should involve some important physiological function of the body. Combining the HLL and the SSL, the experimental study on hair and skin sympathetic in rats should consider the difference between the HLL and the non-HLL. For example, when the test and control areas are arranged in the HLL and the non-HLL, respectively or alternatively, the results will be quite chaotic. Indeed, this is not a problem; just shaving an area of hairs and observing 1–3 days. ACKNOWLEDGMENT Thanks to Mr. Peter Walters for his English corrections. LITERATURE CITED Andreasen E. 1953. Cyclic changes in the skin of the mouse. Acta Pathol Microbiol Scand 32:157–163. Asada-Kubota M. 1995. Inhibition of hair growth by subcutaneous injection of a sympathetic neurotoxin, 6-hydroxydopamine in neonatal mice. Anat Embryol (Berl) 191:407–414. Borum K. 1954. Hair pattern and hair succession in the albino mouse. Acta Pathol Microbiol Scand 34:521–524. Botchkarev VA, Peters EM, Botchkareva NV, Maurer M, Paus R. 1999. Hair cycle-dependent changes in adrenergic skin innervation, and hair growth modulation by adrenergic drugs. J Invest Dermatol 113:878–887. Butcher EO. 1934. The hair cycles in the albino rat. Anat Rec 61:5– 19. De La Torre JC. 1980. Standardization of the sucrose potassium phosphate-glyoxylic acid histofluorescence method for tissue monoamines. Neurosci Lett 17:339–340. Dry FW. 1926. The coat of the mouse. J Genet 16:287–340. 868 LIU ET AL. Eaton GJ. 1976. Hair growth cycles and wave pattern in ‘nude’ mice. Transplantation 22:217–222. Johnson E. 1958. Quantitative studies of hair growth in the albino rat. I-III. J Endocrinol 16:337–350;351–359;360–368. Liu LY, Zhang H, Pan J, Pen A. 2005. The existence of a linear system consisted of sympathetic endings in the rat’s skin. Anat Embryol (Berl) 210:91–100. Militzer K. 2001. Hair growth pattern in nude mice. Cells Tissues Organs 168:285–294. Miura Y, Yamazaki M, Tsuboi R, Ogawa H. 1999. Promotion of rat hair growth by irradiation using Super Lizer. Jpn J Dermatol 109:2149–2152. Paus R, Peters EM, Eichmuller S, Botchkarev VA. 1997. Neural mechanisms of hair growth control. J Investig Dermatol Symp Proc 2:61–68. Peters EM, Maurer M, Botchkarev VA, Gordon DS, Paus R. 1999. Hair growth-modulation by adrenergic drugs. Exp Dermatol 8:274–281. Roersma ME, Veldhuis GJ. 2001. Proposal and evaluation of a Monte Carlo model for hair regrowth following plucking. Skin Res Technol 7:176–183. Schotzinger RJ, Landis SC. 1990. Postnatal development of autonomic and sensory innervation of thoracic hairy skin in the rat. A histochemical, immunocytochemical, and radioenzymatic study. Cell Tissue Res 260:575–587.