Experimental Gerontology, Vol. 26, pp. 357-363, 1991 Printed in the USA. All rightsreserved.
THE
EFFECT
OF
SELECTIVE
0531-5565/91 $3.00 + .00 Copyright© 1991 PergamonPress plc
DESALIVATION
HEALING
ON WOUND
IN MICE
LIPA BODNER,1 AHUVA KNYSZYNSKI,2 SHIFRA ADLER-KuNIN2 and DAvm DANON3 lDepartment of Oral and Maxillofacial Surgery, Goldschleger School of Dental Medicine, Tel Aviv University, Tel Aviv, Israel, 2Department of Cell Biology, 3Department of Membrane Research, The Weizman Institute of Science, Rehovot, Israel
Abstract - - The effectiveness of wound licking in the acceleration of wound healing was evaluated in selectively desalivated mice. Rate of healing of experimentally induced cutaneous wounds was evaluated macroscopically by photograpgy at 0, 2, 4, and 6 days after wounding. Sialadenectomy of submandibular and sublingual glands significantly slowed down wound healing in animals caged together compared to sham-operated controls. Separate caging as compared to caging in groups slowed down healing in sham-operated animals at day 2 but not at day 4 and 6. No effect on the rate of healing in sialadenectomized mice was observed in separate caging compared to mice caged in groups. Ligation of the parotid duct had an insignificant effect. The rate of wound healing of sublingual sialadenectomized mice was slower than that of sham-operated controls, but not as slow as those of sublingual and submandibular sialadenectomized mice. The results suggest that the rate of healing of expermimentally induced cutaneous wounds of mice is slowed down when licking is prevented by separate caging which confLrms previous reports. Licking with submandibular saliva seems to be more effective than sublingual saliva. Parotid saliva or minor salivary glands secretions are the least effective. Key Words: skin, wound healing, salivary glands, Hcking, mice
INTRODUCTION HUMAN AND RODENT aging is accompanied by age-related structural and functional changes of the salivary glands. The glandular parenchymal volume is reduced. Parenchymal elements are replaced by fatty deposits and connective tissue (Waterhouse et al., 1973; Drummond & Chisholm, 1984; Sashima, 1986; Scott, 1986; Scott et al., 1986; Mintz & Mooradian, 1987). Age-related functional alteration of the salivary glands include a decrease in salivary flow rate (Ben-Aryeh et al., 1985; Bodner & Baum, 1985; Pedersen et al., 1985), a decline in
Correspondence to: Lipa Bodner, Department of Oral and Maxillofacial Surgery, The Maurice and Gabriela Goldschleger, School of Dental Medicine, Tel Aviv University, Ramat Aviv 69978, Tel Aviv, Israel. (Received 20 September 1990, Accepted 9 November 1990) 357
358
t. BODNER et
al
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'~
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2
4 TIME ( d a y s )
15
FIG. 1. The rate of wound healing in sialadenectomized mice and in sham-operated controls in which the submandibular (SM) and sublingual (SL) glands were removed. Mice were either caged separately or in groups. ©--C) SM and SL sialadenectomy, caged separately O--O SM and SL sialadenectomy, caged in groups A - - A Sham-operated, caged separately A - - A Sham-operated, caged in groups Note the marked difference in the rate of wound healing in favor of the caged groups on day 2 and the insignificant difference on days 4 and 6. Healing was slower in the sialadenectomized animals; there was no difference whether mice were caged alone or in a group. Values are mean -+ S.E.M. for 8-10 mice (-+ S.E.M. was drawn only where there was sufficient space without overlapping). protein synthesis and secretion (Kim et al., 1980; Kuyatt & Baum, 1981; Gresik et al., 1982), and a deterioration in electrolite transport (Gutman & Ben-Aryeh 1974; Bodner et al., 1983; Bodner & Baum, 1984). The salivary system in mammals include three pairs of major glands, namely the parotid, submandibular, and sublingual. These glands produce about 95% of the total volume of saliva. The remaining 5% of saliva originates from the minor salivary glands located throughout the oral cavity. The secretion of each gland is unique in its volume, composition and specific biologic activity (Young & Schneyer, 1981). Reduced salivary secretion leads to xerostomia or dryness of the mouth and alteration in all physiologic functions of saliva, such as licking, cleansing and lubricating the oral mucosa, digestion and antibacterial activity (Mandel & Watman 1976). Furthermore, sialadenectomy has an effect that indicates an important endocrine function of the salivary glands (Li et al., 1983). Wound licking plays a major role in the repair of cutaneous wounds in mice (Hutson et al., 1979; Li et al., 1980; Naill et al., 1982). Saliva contains several biologically active substances, such as growth promoting factors, immunoglobulins (Barka, 1980) and antibacterial substances (Young & Schneyer, 1981) that are delivered to the wound by licking. It is, however, unknown whether the decline in salivary gland function with aging (Bodner et al., 1983; Bodner & Baum, 1985) affects all salivary glands at the same rate.
SELECTIVE DESALIVAT1ON AND WOUND HEALING
359
100
'~
50
a
2
a
4 TIME (doys)
i
6
1=io. 2. The rate of wound healing in sialadenectomized mice and with sham-operated mice. In one group, the submandibular (SM) and sublingual (SL) glands were removed and parotid left intact (Q--Q). In a second group, the SM and SL were removed and parotid ducts were ligated (I--m). The third group was sham-operated (,t--A). All animals were caged in groups permitting communal licking, It can be seen that ligation of parotid gland did not cause any difference in the rate of wound healing in animals in which SM and SL glands were removed. Values are means ___ S.E.M. for 8-10 mice ( - S.E.M. was drawn only where there was sufficient space without overlapping). It is generally accepted that cutaneous wound healing is slower in the elderly than in the young. This has been reported in clinical studies (Goodson & Hunt, 1979; Grove, 1982) and demonstrated in experimental investigations on mice and rats (Roth et al., 1981; Cohen et al., Danon et al., 1989). The purpose of the present investigation was to study the effectiveness of saliva derived from individual glands in the promotion of wound healing by licking of experimental skin wound in mice. MATERIALS AND METHODS C B 6 F l (BALB/C x C57BL6) female mice, 6-weeks old, weighing approximately 22 g, were used. Mice were obtained from the animal colony of The Weizman Institute of Science and were fed standard pellets and water ad libitum. Anesthesia was performed by intraperitoneal injection of 0.6 mg per animal of sodium pentobarbiton (Ceva, Paris). Experimental mice were selectively sialadenectomized; control animals were sham-operated involving full exposure of the glands. The incisions of both groups were closed with 3/0 silk. Two circular experimental wounds in the back skin of all animals were made as previously described using an instrument for punching holes in leather belts (Danon et al., 1989).
Selective sialadenectomy Surgical sialadenectomy was performed through a midventral incison in the neck similar to the technique previously described for rats (Bowen et al., 1988). Three surgical proto-
360
L. BODNER et al,
100
I,i.I
,50 Z 0
I
I
2 4 TIME ( clays ) FIG. 3. The rate of wound healing in selectively sialadenectomized mice and sham-operated controis. All animals were caged in groups to allow communal licking. A--A Sham-operated animals (all glands are intact) O--O Submandibular (SM) and sublingual (SL) sialadenectomy (parotid intact) V--V SL sialadenectomy and parotid duct ligation (SM intact) I _ m SM and SL sialadenectomy and parotid duct ligation (only minor salivary glands intact) Values are means _+ S.E.M. for 8-10 mice (-+ S.E.M. was drawn only where there was sufficient space without overlapping). cols for selective sialadenectomy were followed: in protocol " A , " mice underwent bilateral sialadenctomy of submandibular (SM) and sublingual (SL) glands, leaving intact parotid and minor salivary glands (MSG); in protocol " B , " mice underwent sialadenctomy of the SL glands and ligation of the parotid ducts, leaving the SM and MSG; and in protocol " C , " the SM and SL glands were removed and the parotid ducts ligated, leaving only the MSG. For each surgical protocol, 20 animals were used. After surgery, 10 mice were kept for further study in two cages, five per cage, and the other 10 were caged separately, one mouse per cage. Skin wound
A pair of circular, full thickness cutaneous wounds were made by lifting the shaved back skin longitudinally and punching a hole with a leather belt punch (outer diameter 4.7 mm). This resulted in one wound on each side of the back left for secondary healing. Operations were performed under clean, but not aseptic conditions. Animals were caged in groups of five to allow communal licking of the wounds, or caged separately to prevent licking. The size of the wounds were recorded on day 0, 2, 4, and 6 after surgery using color slide photography at a fixed focal distance taken with a Nikon F-3 camera (24 × 36 mm film). Slides were projected at a given distance on computer paper of the same batch. Wound areas were traced, cut out, and weighed on a H-54 Mettler balance (Niall et al., 1982; Danon et al., 1989). Wound areas on each day were expressed as percentage of initial wound area. Data were analyzed by Student's t-test.
SELECTIVE DESALIVATION AND WOUND HEALING
361
RESULTS Surgery was without incidence in all animals, Food intake and body weight were not reduced and daily activity appeared normal throughout the experiment. Figure 1 presents the rate of wound healing as a function of time after surgery in protocol " A . " Sham-operated and sialadenectomized animals were compared. The rate of wound healing in mice caged separately was compared to the rate of healing in a caged group. A highly significant difference (p < 0.001) in rate of healing between sialadenectomized and sham-operated mice was found. The difference in the rate of healing between animals caged in groups or separately was insignificant for sialadenectomized mice. Sham-operated mice caged separately were significantly slower (p < 0.005) in wound healing compared to shamoperated mice kept in one cage on day 2, but had no significant difference on day 4 and 6. Figure 2 presents the rate of healing of protocol " B " in which the wounds of three groups of mice are described: sham-operated, SM and SL sialadenectomy, and SM and SL sialadenectomy as well as parotid ligation. Both experimental groups were significantly slower in wound healing (p < 0.001) compared to sham-operated animals. However, no significant differences were found between the SM and SL sialadenectomy group and the group with SM and SL removal and parotid duct ligation. Figure 3 presents the rate of wound healing in mice in which only the SL glands were removed and the parotid ducts ligated compared to sham-operated animals, or SM and SL removal with or without parotid ligation. There was a significant difference (p < 0.001) between sialadenectomized animals and sham-operated controls. With the removal of SM and SL glands, no significant difference was noted whether the parotid duct was ligated or not. When the SL gland was removed and the parotid duct was ligated, a situation was created in which the rate of wound healing was better than in totally sialadenectomized animals, but not as good as sham-operated (p < 0.03). DISCUSSION Slower wound healing in aged rats and mice has been attributed to several reasons (Goodson & Hunt, 1979). Deterioration of macrophage function is just one reason (Danon et al., 1989). Saliva is known to promote wound healing in young mice by wound licking (Hudson et al., 1979). The fact that salivary glands in mammals deteriorate with age may be another reason for the slower wound healing seen in advanced age. The deterioration of salivary glands may be indirectly implicated through an endocrine mechanism (Li et al., 1983). The present study confirmed previous reports on the acceleration of wound healing in mice caged in groups, allowing communal licking (Li et al., 1980; Niall et al., 1982). Separate caging of sham-operated mice slowed down healing on day 2 (Fig. 1) (p < 0.005) compared to sham-operated mice grouped in one cage. The fact that this difference leveled off on day 4 and day 6 may be attributed to the effect of licking on wound contraction followed by the wound healing process (Hutson et al., 1979). The experiment in which the SM and SL glands were removed (Fig. 1) demonstrates that there is no significant difference in the rate of healing between mice caged in groups or separately, indicating the importance of SM and SL saliva in the licking process. Furthermore, the fact that the rate of healing in SM and SL sialadenectomized mice was slower than in sham-operated mice cages separately may indicate a further indirect role of these glands on wound healing.
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Ligation o f the parotid gland had practically no effect on wound healing, indicating that licking per se is not sufficient and parotid saliva is not effective (Fig. 2). After removal of the SL gland and parotid ligation, w o u n d healing was slower than in sham-operated controls (p < 0.03) (Fig. 3). H o w e v e r , healing was e v e n s l o w e r when both S M and SL glands were r e m o v e d . This indicates that the relatively small SL gland as c o m p a r e d to the S M gland contains large amounts of active substances which affects wound healing. The results o f the present e x p e r i m e n t s h o w that the mechanical effect o f w o u n d licking is not sufficient to accelerate w o u n d healing. Licking with parotid saliva is not effective and, by contrast, licking with S M and SL saliva significantly p r o m o t e s w o u n d healing. The relative effect of the SL saliva on p r o m o t i o n o f w o u n d healing seems to be very important. The SL gland is about 10% o f the weight of the S M gland (Young & Schneyer, 1981), and saliva production is usually proportional to gland weight. Therefore, the r e m o v a l o f the SL causes a m a r k e d negative effect on w o u n d healing and may indicate that the SL gland secretes the most effective saliva for p r o m o t i o n of w o u n d healing. Acknowledgment -- This study was supported in part by the National Insurance Institute of Israel.
REFERENCES BARKA, T. Biologically active polypeptides in submandibular glands. J. Histochem. Cvtochem. 28, 836-859, 1980. BEN-ARYEH, H., MIRON, D., BERDICEVSKY, I., SZANGEL, R., and GUTMAN, D. Xerostomia in the elderly: Prevalence, diagnosis, complication and treatment. Gerodontol. 2, 77-82, 1985. BODNER, L., HOOPS, M.T., ITO, H., ROTH, G.S., and BAUM, B.J. Multiple transduction mechanisms are likely involved in calcium mediated exocrine secretory events in rat parotid cells. J. Biol. Chem. 258, 27742777, 1983. BODNER, L. and BAUM, B.J. Submandibular gland secretory function in young adult and aged rats. Comp. Biochem. Physiol. 77A, 235-238, 1984. BODNER, L. and BAUM, B.J. Characteristics of stimulated parotid gland secretion in the aging rat. Mech. Ageing Dev. 31, 337-342, 1985. BOWEN, W.H., PEARSON, S.K., and YOUNG, D.A. The effect of desalivation on coronal and root surface caries in rats. J. Dent. Res. 67, 21-23, 1988. COHEN, B.J., DANON, D., and ROTH, G.S. Wound repair in mice as influenced by age and antimacrophagh serum. J. Gerontol. 42, 295-301, 1987. DANON, D., KOWATCH, M.A., and ROTH, G.S. Promotion of wound repair in old mice by local injection of macrophages. Proc. Natl. Acad. Sci. U.S.A. 86, 2018-2020, 1989. DRUMMOND, J.R, and CHISHOLM, D.M. A qualitative and quantitative study of the ageing human labial salivary glands. Arch. Oral Biol. 29, 151-155, 1984. GOODSON, W.H. and HUNT, T.K. Wound healing and aging. J. Invest. Dermatol. 73, 88-91, 1979. GRESIK, E.W., BRENNAN, M., and AZMITIA, E. Age-related changes in EGF and protease in submandibular glands of C57BL/6J mice. GerodontoL 1, 81-84, 1982. GROVE, G.L. Age-related differences in healing of superficial skin wounds in humans. Arch. Dermatol. Res. 272, 381-385, 1982. GUTMAN, D. and BEN-ARYEH, H. The influence of age on salivary content and rate of flow. Int. J. Oral Surg. 3, 314-317, 1974. HUTSON, J.M., N1ALL, M., EVANS, D., and FOWLER, R. Effect of salivary glands on wound contraction in mice. Nature (London) 279, 793-795, 1979. KIM, S., WEINHOLD, P., HAN, S., and WANGER, D. Age-related decline in protein synthesis in the rat parotid gland. Exp. Gerontol. 15, 77-85, 1980. KUYATT, B.L. and BAUM, B.J. Characteristics of submandibular gland from young and aged rats. J. Dent. Res. 60, 936-941, 1981. LI, A.K.C., KOROLY, M.J., SCHATTENKERK, M.E., MALT, R.A., and YOUNG, M. Nerve growth factor: Acceleration of rate of wound healing in mice. Proc. Natl. Acad. Sci. U.S.A. 77, 4379-4381, 1980.
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LI, A.K.C., SCHATENKERK, M.E., DEVRIES, J.E., FORD, W.D.A., and MALT, R.A. Growth and metabolic alterations after sub-mandibular sialadenectomy in male mice. Am. J. Physiol. 244, R41-R44, 1983. MANDEL, I.D. and WATMAN, S. The salivary secretion in health and disease. Oral Sci. Rev. 8, 25-47, 1976. MINTZ, G.A. and MOORADIAN, A.D. Age-related changes in rat sublingual salivary gland morphology. Gerodontol. 6, 137-144, 1987. NIALL, M., GRAEME, B.R., and O'BRIEN, B. The effect of epidermal growth factor on wound healing in mice. J. Surg. Res. 33, 164-169, 1982. PEDERSEN, W., SCHUBERT, M., IZUTSU, K.T., MERSOL, T., and TRUELOVE, E. Age dependent decreases in human submandibular gland flow rate as measured under resting and post stimulation condition. J. Dent. Res. 64, 822-826, 1985. ROTH, G.S., HARMAN, M.H., and LAMBERG, S. I. Altered ovarian regulation of wound healing during aging. Proc. Soc. Exp. Biol. Med. 166, 17-23, 1981. SASHIMA, M. Age-related changes of rat submandibular gland: A morphometric and ultrastructural study. J. Oral Pathol. 15, 507-512, 1986. SCOTT, J. Structure and function in aging human salivary glands. Gerodontol. 5, 149-158, 1986. SCOTT, J., BODNER, L., and BAUM, B.J. Assessment of age related changes in the submandibular salivary glands of the rat using stereological analysis. Arch. Oral Biol. 31, 69-71, 1986. WATERHOUSE, J.P., CHISHOLM, D.M., WINTER, R.B., PATEL, M., and YALE, R.S. Replacement of functional parenchymal cells by rat and connective tissue in human submandibular salivary glands: An age-related change. J. Oral Pathol. 2, 16--27, 1973. YOUNG, J.A. and SCHNEYER, A.C. Composition of saliva in mammals. Aust. J. Exp. Biol. Med. Sci. 59, 1-53, 1981.
THE
EFFECT
OF
SELECTIVE
0531-5565/91 $3.00 + .00 Copyright© 1991 PergamonPress plc
DESALIVATION
HEALING
ON WOUND
IN MICE
LIPA BODNER,1 AHUVA KNYSZYNSKI,2 SHIFRA ADLER-KuNIN2 and DAvm DANON3 lDepartment of Oral and Maxillofacial Surgery, Goldschleger School of Dental Medicine, Tel Aviv University, Tel Aviv, Israel, 2Department of Cell Biology, 3Department of Membrane Research, The Weizman Institute of Science, Rehovot, Israel
Abstract - - The effectiveness of wound licking in the acceleration of wound healing was evaluated in selectively desalivated mice. Rate of healing of experimentally induced cutaneous wounds was evaluated macroscopically by photograpgy at 0, 2, 4, and 6 days after wounding. Sialadenectomy of submandibular and sublingual glands significantly slowed down wound healing in animals caged together compared to sham-operated controls. Separate caging as compared to caging in groups slowed down healing in sham-operated animals at day 2 but not at day 4 and 6. No effect on the rate of healing in sialadenectomized mice was observed in separate caging compared to mice caged in groups. Ligation of the parotid duct had an insignificant effect. The rate of wound healing of sublingual sialadenectomized mice was slower than that of sham-operated controls, but not as slow as those of sublingual and submandibular sialadenectomized mice. The results suggest that the rate of healing of expermimentally induced cutaneous wounds of mice is slowed down when licking is prevented by separate caging which confLrms previous reports. Licking with submandibular saliva seems to be more effective than sublingual saliva. Parotid saliva or minor salivary glands secretions are the least effective. Key Words: skin, wound healing, salivary glands, Hcking, mice
INTRODUCTION HUMAN AND RODENT aging is accompanied by age-related structural and functional changes of the salivary glands. The glandular parenchymal volume is reduced. Parenchymal elements are replaced by fatty deposits and connective tissue (Waterhouse et al., 1973; Drummond & Chisholm, 1984; Sashima, 1986; Scott, 1986; Scott et al., 1986; Mintz & Mooradian, 1987). Age-related functional alteration of the salivary glands include a decrease in salivary flow rate (Ben-Aryeh et al., 1985; Bodner & Baum, 1985; Pedersen et al., 1985), a decline in
Correspondence to: Lipa Bodner, Department of Oral and Maxillofacial Surgery, The Maurice and Gabriela Goldschleger, School of Dental Medicine, Tel Aviv University, Ramat Aviv 69978, Tel Aviv, Israel. (Received 20 September 1990, Accepted 9 November 1990) 357
358
t. BODNER et
al
100
'~
50
2
4 TIME ( d a y s )
15
FIG. 1. The rate of wound healing in sialadenectomized mice and in sham-operated controls in which the submandibular (SM) and sublingual (SL) glands were removed. Mice were either caged separately or in groups. ©--C) SM and SL sialadenectomy, caged separately O--O SM and SL sialadenectomy, caged in groups A - - A Sham-operated, caged separately A - - A Sham-operated, caged in groups Note the marked difference in the rate of wound healing in favor of the caged groups on day 2 and the insignificant difference on days 4 and 6. Healing was slower in the sialadenectomized animals; there was no difference whether mice were caged alone or in a group. Values are mean -+ S.E.M. for 8-10 mice (-+ S.E.M. was drawn only where there was sufficient space without overlapping). protein synthesis and secretion (Kim et al., 1980; Kuyatt & Baum, 1981; Gresik et al., 1982), and a deterioration in electrolite transport (Gutman & Ben-Aryeh 1974; Bodner et al., 1983; Bodner & Baum, 1984). The salivary system in mammals include three pairs of major glands, namely the parotid, submandibular, and sublingual. These glands produce about 95% of the total volume of saliva. The remaining 5% of saliva originates from the minor salivary glands located throughout the oral cavity. The secretion of each gland is unique in its volume, composition and specific biologic activity (Young & Schneyer, 1981). Reduced salivary secretion leads to xerostomia or dryness of the mouth and alteration in all physiologic functions of saliva, such as licking, cleansing and lubricating the oral mucosa, digestion and antibacterial activity (Mandel & Watman 1976). Furthermore, sialadenectomy has an effect that indicates an important endocrine function of the salivary glands (Li et al., 1983). Wound licking plays a major role in the repair of cutaneous wounds in mice (Hutson et al., 1979; Li et al., 1980; Naill et al., 1982). Saliva contains several biologically active substances, such as growth promoting factors, immunoglobulins (Barka, 1980) and antibacterial substances (Young & Schneyer, 1981) that are delivered to the wound by licking. It is, however, unknown whether the decline in salivary gland function with aging (Bodner et al., 1983; Bodner & Baum, 1985) affects all salivary glands at the same rate.
SELECTIVE DESALIVAT1ON AND WOUND HEALING
359
100
'~
50
a
2
a
4 TIME (doys)
i
6
1=io. 2. The rate of wound healing in sialadenectomized mice and with sham-operated mice. In one group, the submandibular (SM) and sublingual (SL) glands were removed and parotid left intact (Q--Q). In a second group, the SM and SL were removed and parotid ducts were ligated (I--m). The third group was sham-operated (,t--A). All animals were caged in groups permitting communal licking, It can be seen that ligation of parotid gland did not cause any difference in the rate of wound healing in animals in which SM and SL glands were removed. Values are means ___ S.E.M. for 8-10 mice ( - S.E.M. was drawn only where there was sufficient space without overlapping). It is generally accepted that cutaneous wound healing is slower in the elderly than in the young. This has been reported in clinical studies (Goodson & Hunt, 1979; Grove, 1982) and demonstrated in experimental investigations on mice and rats (Roth et al., 1981; Cohen et al., Danon et al., 1989). The purpose of the present investigation was to study the effectiveness of saliva derived from individual glands in the promotion of wound healing by licking of experimental skin wound in mice. MATERIALS AND METHODS C B 6 F l (BALB/C x C57BL6) female mice, 6-weeks old, weighing approximately 22 g, were used. Mice were obtained from the animal colony of The Weizman Institute of Science and were fed standard pellets and water ad libitum. Anesthesia was performed by intraperitoneal injection of 0.6 mg per animal of sodium pentobarbiton (Ceva, Paris). Experimental mice were selectively sialadenectomized; control animals were sham-operated involving full exposure of the glands. The incisions of both groups were closed with 3/0 silk. Two circular experimental wounds in the back skin of all animals were made as previously described using an instrument for punching holes in leather belts (Danon et al., 1989).
Selective sialadenectomy Surgical sialadenectomy was performed through a midventral incison in the neck similar to the technique previously described for rats (Bowen et al., 1988). Three surgical proto-
360
L. BODNER et al,
100
I,i.I
,50 Z 0
I
I
2 4 TIME ( clays ) FIG. 3. The rate of wound healing in selectively sialadenectomized mice and sham-operated controis. All animals were caged in groups to allow communal licking. A--A Sham-operated animals (all glands are intact) O--O Submandibular (SM) and sublingual (SL) sialadenectomy (parotid intact) V--V SL sialadenectomy and parotid duct ligation (SM intact) I _ m SM and SL sialadenectomy and parotid duct ligation (only minor salivary glands intact) Values are means _+ S.E.M. for 8-10 mice (-+ S.E.M. was drawn only where there was sufficient space without overlapping). cols for selective sialadenectomy were followed: in protocol " A , " mice underwent bilateral sialadenctomy of submandibular (SM) and sublingual (SL) glands, leaving intact parotid and minor salivary glands (MSG); in protocol " B , " mice underwent sialadenctomy of the SL glands and ligation of the parotid ducts, leaving the SM and MSG; and in protocol " C , " the SM and SL glands were removed and the parotid ducts ligated, leaving only the MSG. For each surgical protocol, 20 animals were used. After surgery, 10 mice were kept for further study in two cages, five per cage, and the other 10 were caged separately, one mouse per cage. Skin wound
A pair of circular, full thickness cutaneous wounds were made by lifting the shaved back skin longitudinally and punching a hole with a leather belt punch (outer diameter 4.7 mm). This resulted in one wound on each side of the back left for secondary healing. Operations were performed under clean, but not aseptic conditions. Animals were caged in groups of five to allow communal licking of the wounds, or caged separately to prevent licking. The size of the wounds were recorded on day 0, 2, 4, and 6 after surgery using color slide photography at a fixed focal distance taken with a Nikon F-3 camera (24 × 36 mm film). Slides were projected at a given distance on computer paper of the same batch. Wound areas were traced, cut out, and weighed on a H-54 Mettler balance (Niall et al., 1982; Danon et al., 1989). Wound areas on each day were expressed as percentage of initial wound area. Data were analyzed by Student's t-test.
SELECTIVE DESALIVATION AND WOUND HEALING
361
RESULTS Surgery was without incidence in all animals, Food intake and body weight were not reduced and daily activity appeared normal throughout the experiment. Figure 1 presents the rate of wound healing as a function of time after surgery in protocol " A . " Sham-operated and sialadenectomized animals were compared. The rate of wound healing in mice caged separately was compared to the rate of healing in a caged group. A highly significant difference (p < 0.001) in rate of healing between sialadenectomized and sham-operated mice was found. The difference in the rate of healing between animals caged in groups or separately was insignificant for sialadenectomized mice. Sham-operated mice caged separately were significantly slower (p < 0.005) in wound healing compared to shamoperated mice kept in one cage on day 2, but had no significant difference on day 4 and 6. Figure 2 presents the rate of healing of protocol " B " in which the wounds of three groups of mice are described: sham-operated, SM and SL sialadenectomy, and SM and SL sialadenectomy as well as parotid ligation. Both experimental groups were significantly slower in wound healing (p < 0.001) compared to sham-operated animals. However, no significant differences were found between the SM and SL sialadenectomy group and the group with SM and SL removal and parotid duct ligation. Figure 3 presents the rate of wound healing in mice in which only the SL glands were removed and the parotid ducts ligated compared to sham-operated animals, or SM and SL removal with or without parotid ligation. There was a significant difference (p < 0.001) between sialadenectomized animals and sham-operated controls. With the removal of SM and SL glands, no significant difference was noted whether the parotid duct was ligated or not. When the SL gland was removed and the parotid duct was ligated, a situation was created in which the rate of wound healing was better than in totally sialadenectomized animals, but not as good as sham-operated (p < 0.03). DISCUSSION Slower wound healing in aged rats and mice has been attributed to several reasons (Goodson & Hunt, 1979). Deterioration of macrophage function is just one reason (Danon et al., 1989). Saliva is known to promote wound healing in young mice by wound licking (Hudson et al., 1979). The fact that salivary glands in mammals deteriorate with age may be another reason for the slower wound healing seen in advanced age. The deterioration of salivary glands may be indirectly implicated through an endocrine mechanism (Li et al., 1983). The present study confirmed previous reports on the acceleration of wound healing in mice caged in groups, allowing communal licking (Li et al., 1980; Niall et al., 1982). Separate caging of sham-operated mice slowed down healing on day 2 (Fig. 1) (p < 0.005) compared to sham-operated mice grouped in one cage. The fact that this difference leveled off on day 4 and day 6 may be attributed to the effect of licking on wound contraction followed by the wound healing process (Hutson et al., 1979). The experiment in which the SM and SL glands were removed (Fig. 1) demonstrates that there is no significant difference in the rate of healing between mice caged in groups or separately, indicating the importance of SM and SL saliva in the licking process. Furthermore, the fact that the rate of healing in SM and SL sialadenectomized mice was slower than in sham-operated mice cages separately may indicate a further indirect role of these glands on wound healing.
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Ligation o f the parotid gland had practically no effect on wound healing, indicating that licking per se is not sufficient and parotid saliva is not effective (Fig. 2). After removal of the SL gland and parotid ligation, w o u n d healing was slower than in sham-operated controls (p < 0.03) (Fig. 3). H o w e v e r , healing was e v e n s l o w e r when both S M and SL glands were r e m o v e d . This indicates that the relatively small SL gland as c o m p a r e d to the S M gland contains large amounts of active substances which affects wound healing. The results o f the present e x p e r i m e n t s h o w that the mechanical effect o f w o u n d licking is not sufficient to accelerate w o u n d healing. Licking with parotid saliva is not effective and, by contrast, licking with S M and SL saliva significantly p r o m o t e s w o u n d healing. The relative effect of the SL saliva on p r o m o t i o n o f w o u n d healing seems to be very important. The SL gland is about 10% o f the weight of the S M gland (Young & Schneyer, 1981), and saliva production is usually proportional to gland weight. Therefore, the r e m o v a l o f the SL causes a m a r k e d negative effect on w o u n d healing and may indicate that the SL gland secretes the most effective saliva for p r o m o t i o n of w o u n d healing. Acknowledgment -- This study was supported in part by the National Insurance Institute of Israel.
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