Amos Gilhar

Ellias Matanis

Hakim Munder

Ruth Miskin

Yehuda Ullmann

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99Acta Dermatovenerol Croat               2009;17(2):99-102                   MEDICAL RESEARCHCutaneous Morphometric Parameters of Young FVB/N Mice Sustained in Aged Mice and in Calorically  Restricted Transgenic αMUPA MiceEllias Matanis1, Hakim Munder1, Yehuda Ullmann1, Ruth Miskin2, Amos Gilhar11Skin Research Laboratory, B. Rappaport, Faculty of Medicine, Technion – Israel Institute of Technology, Haifa; 2Department of Biological Chemistry, Weizmann  Institute of Science, Rehovot, IsraelCorresponding author:Professor Amos GilharLaboratory for Skin Research, Rappaport BuildingB. Rappaport Faculty of MedicineTechnion – Israel Institute of TechnologyP.O.B. 9649, Bat-Galim, Haifa31096 Israel amos.gilhar@flim.health.gov.ilReceived: January 26, 2009Accepted: March 20, 2009Summary Caloric restriction (CR) extends the lifespan of diverse animal species and is currently the only therapeutic intervention known to attenuate aging and increase longevity in laboratory animals. The effect of CR on intrinsic skin aging is not well understood. To study this issue, we took advantage of transgenic αMUPA mice that spontaneously eat less (20-30%) when fed ad libitum and live longer compared to their wild type (WT) FVB/N control mice.  Herein we determined morphometric skin parameters in young (6-7 months) and aged (17-18 months) αMUPA and WT mice. In addition, we transplanted skin grafts excised from the aged or young αMUPA and WT mice into both types of young mice, to test whether the systemic environments of αMUPA and WT mice could affect the grafts differently. The results have shown that the mean epidermal thickness, number of hair follicles and number of dermal blood vessels were similar in all four groups regardless of age or mouse type. In addition, the post-graft specimens of all four groups exhibited increase in all parameters measured, in particular a remarkable 6-7 fold increase in epidermal thickness. However, no significant differences were detected in the post-graft samples between the four experimental groups. Our findings indicate that, at least in FVB/N mice, parameters measured in normal or grafted skin depend primarily on the intrinsic cutaneous capacity rather than on circulating factors as determined by age or reduced calories.Key wordS: skin aging, epidermal thickness, dermal blood vessels, hair follicles, caloric restrictionINTroduCTIoN Data on the aging of rodent skin are still scarce and somewhat inconsistent. The Charles River hairless mouse strain, a murine model of cutane-ous senescence, exhibited abnormalities in the epidermal barrier functions similar to those ob-served in humans (1). In addition, CBA mice have recently shown unique age-related histological modifications in the skin, including a prominent 100thinning of the epidermis as well as a reduced number of pilosebaceous units (2). However, epi-dermal stem cells of C57/B16 mice were resistant to cellular aging (3), and Fisher 344 rats exhibited an age-related increase in the epidermis, dermis and the fat layer (4), contrary to skin thinning that is characteristic of aged humans (5). We previously observed that grafting aged hu-man epidermis to immunodeficient mice resulted in a reversal of aging changes, including an in-creased proliferation index and the reappearance of the convoluted dermal/epidermal junction (5, 6). These findings suggest that systemic factors of young recipient mice could be involved in the beneficial influence detected in the old donor skin (7). The contribution of a young systemic environ-ment to the rejuvenation of aged tissues in mice was also detected after heterochronic parabiosis, in which sharing of circulatory systems between old and young mice led to rejuvenated Notch sig-naling, cell proliferation and tissue regeneration capacity of the aged muscle (8).   Caloric restriction (CR), usually 20-40% reduc-tion of ad libitum food consumption, extends the lifespan of diverse animal species and is currently the only therapeutic intervention known to attenu-ate aging and increase longevity in laboratory ani-mals. In rodents, CR prevents or delays the onset of major age-related diseases, such as cancer and type 2 diabetes, and leads to changes in cir-culating hormones and growth factors, including reduced levels of IGF-1 and insulin (9). CR also modifies the transcriptional response of tissues to-wards a state that reverses or resists age-related changes, and is thought to promote susceptibility to apoptotic and autophagic cell death in mitoti-cally competent tissues (10). Data on the effect of CR on rodent skin are still limited. In old Fisher 344 rats, CR delayed or prevented histomorpho-logical changes resulting from intrinsic skin aging (4). In young C57BL/6J mice, CR reduced cell pro-liferation rates in a variety of tissue types including epidermis (11).   Whether CR can affect intrinsic skin aging through systemic or local factors is not well under-stood. To study this issue, we took advantage of transgenic αMUPA mice that carry as a transgene the cDNA encoding the urokinase-type plasmino-gen activator (uPA) (12). αMUPA mice spontane-ously eat less (20-30%) when fed ad libitum and live longer compared to wild type (WT) FVB/N control mice (13). αMUPA mice exhibit additional similarities to calorically restricted mice, such as reduced body weight and lack of obesity, reduced levels of serum IGF-1 and glucose, enhanced capacity to conduct apoptosis in the liver, and reduced incidence of spontaneous tumors or car-cinogen-induced pre-neoplastic lesions (14, 15). αMUPA mice also maintain a youthful appearance throughout most of their lifetime.  We herein determined morphometric skin pa-rameters in young (6-7 months) and aged (17-18 months) αMUPA and WT mice. In addition, we performed skin transplantations of the young and aged αMUPA and WT mice into both types of young mice, to test whether the systemic environ-ment of αMUPA mice could affect the grafts differ-ently compared to that of the WT control mice.maTerIaL aNd meTHodS A total of 54 mice were included in this study, divided into the following four groups: 10 old WT FVB/N mice (mean body weight 38.9 ± 8.8 gram); 10 old αMUPA mice (mean body weight 26.8 ± 1.7 gram); 15 young WT mice (mean body weight 28.22 ± 0.3 gram); 19 young αMUPA mice (mean body weight 21.9 ± 1.54 gram). Unlike old WT mice, old αMUPA mice exhibited a young appear-ance of their fur. All of the mice were propagated and maintained at the Weizmann Institute Trans-genic Mouse Facilities. Animal care and research protocols were in accordance with institutional guidelines and were approved by the Institutional Committee on Animal Use. Skin specimens were obtained from the dorsal part of each mouse and were submitted for routine histological examination with hematoxylin and eo-sin. The histological assessment was performed by routine light microscope (6). Epidermal thick-ness was measured as described elsewhere (5, 6). Numbers of blood vessels were determined in the entire area of the dermis, and results were given as a mean number per mm2. Terminal hair count was performed in a similar manner. Dermal cells were enumerated without specific staining and were also given as the mean number per mm2. All measurements were conducted in a blind manner.reSuLTS The results showed that the mean epidermal thickness was quite similar in all four groups regard-less of age or mouse type, as follows: There was no difference between the epidermal thicknesses of old WT  (45.6 ± 8 μm) and young WT mice  (43 ± 6 μm) (Independent samples t-test, p=0.36). The mean epidermal thickness of old αMUPA mice (49 Matanis et al.      Acta Dermatovenerol CroatMorphometric parameters in FVB/N and αMUPA mice     2009;17(2):99-102ACTA DERMATOVENEROLOGICA CROATICA101± 4.5μm) was slightly however significantly higher than that of young αMUPA mice (43 ± 6 μm) (Inde-pendent samples t-test, p=0.01), but this 6-micron difference (95% confidence interval 1.56 - 10.44) is of no clinical implication. In addition, the mean numbers of hair follicles were similar in all four mouse groups: old WT mice (1.25± 0.36) versus young WT mice (1.35 ± 0.44) (Independent sam-ples t-test, p=0.56); old αMUPA mice (1.48 ± 0.25) versus young αMUPA mice (1.76 ±0.53) (Indepen-dent samples t-test, p=0.13). The mean numbers of blood vessels or dermal cells were also similar in all groups.  To compare the influence of αMUPA and WT mice while serving as graft recipients, skin speci-mens from old and young WT and αMUPA mice were transplanted onto young WT and αMUPA mice. Transplantation was performed as previ-ously described (6, 7), and the skin grafts were ex-cised after four weeks. The post-graft specimens from all four groups exhibited a significant increase in epidermal thickness, dermal cells and dermal blood vessels, in agreement with previous results (6.7).  For example, the mean epidermal thickness of old WT samples increased significantly from 42.8 ±5.9 μm before engraftment onto young WT mice to 280.7 ±141.6 μm after engraftment (Paired samples t-test, p=0.004) (Fig. 1). However, no sig-nificant difference was detected in the post grafts between the four experimental groups. of the old versus young WT group, and the ill ap-pearance of the old fur, did not interfere with the proliferative capacity of the old skin grafts.  Given the resistance of the old WT skin to ag-ing, it is not surprising that old αMUPA and old WT mice exhibited similar values for skin parameters. Furthermore, the findings that the old WT skin samples showed similar alterations irrespective of whether grafted onto αMUPA or WT mice, suggest that the systemic state induced by CR, at least in young αMUPA mice, did not interfere with the transplantation-imposed changes including the proliferative capacity. It is yet unknown, however, whether differences could be found after skin en-graftment onto old WT versus old αMUPA mice.  Together, our findings on the normal and grafted skin are consistent with the view, that skin parame-ters in mice are determined primarily by an intrinsic cutaneous capacity that is independent of circulat-ing factors as modified by age or reduced calories. references1.   Ghadially R, Brown BE, Sequeira-Martin SM, Feingold KR, Elias PM. The aged epidermal permeability barrier. Structural, functional, and lipid Biochemical abnormalities in humans and a senescent murine model. J Clin Invest 1995; 95 :2281-90.2.   Bhattacharyya TK, Thomas JR. Histomorpho-logic changes in aging skin: Observations in the CBA mouse model. Arch Facial Plast Surg 2004;  6:21-5.3.   Giangereco A, Qin M, Pintar JE, Watt FM.  Epi-dermal stem cells are retained in vivo throug-hout skin aging. Aging Cell 2008 ;7:250-259.4.   Bhattacharyya TK, Merz M, Thomas JR. Mo-dulation of cutaneous aging with calorie res-triction in Fischer 344 Rats: A histological stu-dy. Arch Facial Plast Surg 2007 ; 7:12-16. 5.   Gilhar A, Ullmann Y, Karry R, Shalaginov R, Assy B, Serafimovich S, et al. Aging of human epidermis: Reversal of aging changes corre-lates with reversal of keratinocyte fas expres-sion and apoptosis. J Gerontol A Biol Sci Med Sci  2004; 59:411-5.6.   Gilhar A, Pillar T, Etzioni A. Possible role of cytokines in cellular proliferation of the skin transplanted onto nude mice.  Arch Dermatol 1995; 131: 38-42.7.   Gilhar A, Ullmann Y, Karry R, Shalaginov R, Assy B, Serafimovich S, et al. Ageing of hu- dISCuSSIoN This study shows that old WT FVB/N mice did not differ from young WT mice with respect to all morphometric parameters measured, suggesting that the skin of the FVB/N mouse strain is resistant to intrinsic aging. This conclusion is further sup-ported by the findings showing that the post-graft samples of both old and young WT mice exhibited a similar impressive thickening compared to the normal skin samples. The latter result also indi-cates that the 35% increase in mean body weight Figure 1. Photomicrograph of epidermal samples. Samples were obtained from a young WT mouse (a), an old WT mouse. (b), and a post-graft of an old WT mouse transplanted onto a young WT mouse (c). Note a marked increase in epidermal thickness in the post-graft. Magnification was x360 in a and c, and x128 in b.Matanis et al.      Acta Dermatovenerol CroatMorphometric parameters in FVB/N and αMUPA mice     2009;17(2):99-102ACTA DERMATOVENEROLOGICA CROATICA102 ACTA DERMATOVENEROLOGICA CROATICAman epidermis: The role of apoptosis, Fas and telomerase. Br J Dermatol 2004; 150:56-63.8.   Conboy IM, Conboy MJ, Wagers AJ, Girma ER, Weissman IL, Rando TA. Rejuvenation of aged progenitor cells by exposure to a young systemic environment. Nature 2005; 433:760-4.9.   Masoro EJ. Overview of caloric restriction and ageing. Mech Ageing Dev 2005; 126:913-22. 10. Spindler SR, Dhahbi JM. Conserved and tis-sue-specific genic and physiologic responses to caloric restriction and altered IGFI signaling in mitotic and postmitotic tissues. Annu Rev Nutr 2007; 27: 193-217. 11. Varady KA, Roohk JD, McEvoy-Hein BK, Gay-linn BD, Thorner MO, Hellerstein  MK. Modi-fied alternate-day fasting regimens reduce cell proliferation rates to a similar extent as daily calorie restriction in mice. FASEB Journal 2008; 22:2090-2096.  12. Miskin R, Axelrod JH, Griep AE, Lee E, Belin D, Vassalli JD, et al. Human and murine uro-kinase cDNAs linked to the murine alpha A-crystallin promoter exhibit lens and non-lens expression in transgenic mice. Eur J Biochem 1990; 190: 31-38.13. Miskin R, Masos T. Transgenic mice over-ex-pressing urokinase-type plasminogen activa-tor in the brain exhibit reduced food consump-tion, body weight and size, and increased longevity. J Gerontol A Biol Sci Med Sci 1997; 52: B118-B124. 14. Tirosh O,  Aronis A, Zusman I, Kossoy G, Ya-hav S, Shinder D, et al. Mitochondrion-media-ted apoptosis is enhanced in long-lived αMUPA transgenic mice and calorically restricted wild-type mice. Exp Gerontol  2003; 38:955- 63.15. Tirosh O, Pardo M, Schwarz B, Miskin R. Long-lived αMUPA transgenic mice show re-duced SOD2 expression, enhanced apoptosis and reduced susceptibility to the carcinogen dimethylhydrazine. Mech Ageing Dev  2005; 126:1262-73Matanis et al.      Acta Dermatovenerol CroatMorphometric parameters in FVB/N and αMUPA mice     2009;17(2):99-102What are sunspots? To solve the problem - use Orizol cream - rice cream; year 1929.(from the collection of Mr. Zlatko Puntijar)

Cutaneous Morphometric Parameters of Young FVB/N Mice Sustained in Aged Mice and in Calorically Restricted Transgenic aMUPA Mice

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