|
FIGURE 1 Leptin is expressed in nerves and blood vessels in the dorsal skin of Xenopus laevis juveniles. (A) Leptin protein is expressed in blood vessels (BV) and nerves (N) in the dermis and hypodermis. Only background fluorescence from granular glands (G) is present in tissue stained with antibody that was incubated with recombinant Xenopus leptin protein (4 μg) prior to staining (pre-absorbed). Scale bars, 100 µm. (B) Leptin is expressed in the endothelial cells of a large blood vessel (BV) in the hypodermis and unknown scattered cell bodies (solid arrows). Leptin also co-localizes with acetylated alpha-tubulin (AαT, yellow), a neural marker in axons and some nerve cell bodies (hollow arrows). Granular glands are autofluorescent after bleaching and clearing at 488 nm (as seen in the AαT panel). Scale bars, 100 µm. (C) Leptin protein co-localizes with axons innervating a granular gland. (D) Cross section showing leptin in the endothelial cells of a blood vessel in the hypodermis, up against the stratum corneum (SC) of the dermis. Scale bar, 25 µm.
|
|
FIGURE 2 Leptin stimulates JAK/STAT signaling in skin. Leptin-induced phosphorylated STAT3-ir (pSTAT3-ir, yellow) in 16-µm sections of juvenile Xenopus laevis dorsal skin; skin was fixed 6 h after intraperitoneal injection with (A) saline or (B) leptin. Leptin stimulates pSTAT3-ir strongly in the epidermis and at the apical end of the granular glands and hypodermis (representative image from n = 3). Scale bar, 100 µm (n = 3). Epidermis (E), dermis (D), stratum corneum of the dermis (SC), and thin hypodermis (H). Scale bar, 50 µm.
|
|
FIGURE 3 Leptin protein is rapidly localized to injured skin. (A) Whole mount of leptin protein (aqua) and acetylated α-tubulin (AαT, yellow) at the time of injury in juvenile Xenopus laevis dorsal skin. Dotted line indicates injury at the center of the donut-shaped explant. Leptin is highly expressed in blood vessels and nerve tissue (indicated by AαT) associated with the injury site. Granular glands are autofluorescent after bleaching and clearing at 488 nm (as seen in the AαT panel). Scale bar, 100 µm. (B) Leptin protein expression in 16-µm cross sections of juvenile X. laevis dorsal skin after injury. Leptin expression (aqua) is concentrated in the injured tissue at 0 and 24 hpi and nearly depleted by 48 hpi in vitro except for some localized in the epidermis (DAPI, magenta). Epidermis (E), dermis (D), stratum corneum of the dermis (SC), and thin hypodermis (H). Arrow in 24 h panel indicates blood vessel. Scale bars, 100 µm. (C) Leptin is most highly expressed in the central dermis after injury, but expression within 300 µm of the injury site in all skin layers is depleted by 48 hpi in culture (standard least squares ANOVA, time point, p < 0.0001).
|
|
FIGURE 4 pSTAT3-ir increases after full thickness injury of dorsal skin. (A) The highest pSTAT3 activation was associated directly with the injured edge of the explant (integrated density: 589,578 ± 201,764 ADU, n = 3). (B) By contrast, the end of tissue explant that was non-injured and cut after fixation had less pSTAT3-ir activation (integrated density: 267,042 ± 45,092 ADU).
|
|
FIGURE 5 Leptin treatment (0, 10, and 100 ng/ml) in culture media significantly improved wound healing in juvenile Xenopus laevis dorsal skin explants at 48 h of culture. (A) At 24 h, treatments at both 10 and 100 ng/ml significantly increased wound closure compared to the control, and by 48 h, the 100 ng/ml treatment continued to improve wound healing compared to the control (repeated-measures MANOVA, treatment = 0.0034, Tukey’s HSD, p < 0.05, n = 12/treatment). (B) Representative samples of dorsal skin explants in culture showing the interior wound of the “punch-in-a-punch” donut-shaped biopsy. Wound closure measured as the total area reduction of the central wound compared to 0 hpi. Scale bars, 0.5 mm.
|
|
FIGURE 6 Pretreatment with leptin rescues effect of food restriction on wound closure in vitro. Effect of leptin on in vitro wound healing of juvenile Xenopus laevis dorsal skin explants where animals were fed (A) once a day or (B) food restricted for 31 days and then injected in the peritoneum (IP) with leptin (200 ng/g body weight) or saline every other day for 7 days. Dorsal skin donut explant wounds were then treated with (black) or without leptin (red) in culture (100 ng/ml) for 48 h. Pretreatment with IP leptin injection significantly increased the rate of wound closure in food-restricted animals over those with pretreatment injections of saline by 48 h, but there was no effect of pretreatment injection in fed animals (ANOVA, fed status * injection, p = 0.0253); asterisk indicates significant increase in wound healing in leptin pretreated vs. saline pretreated explants (Tukey’s HSD, p < 0.05, n = 8/treatment). There was no significant effect of leptin in the culture media (black vs. red) in this model (p = 0.1809).
|
|
FIGURE 7 Leptin suppresses microbial growth in culture media of skin explants. Proportion of culture wells visually turbid with bacterial infection for juvenile Xenopus laevis dorsal skin wound explant cultured in increasing leptin concentration at 48 hpi. Increasing leptin concentration (0 ng/ml, n = 70; 10 ng/ml, n = 38; 100 ng/ml, n = 70) significantly decreased the number of infected wells (likelihood ratio test, p = 0.0202).
|
|
Figure 1. Leptin is expressed in nerves and blood vessels in the dorsal skin of Xenopus laevis juveniles. (A) Leptin protein is expressed in blood vessels (BV) and nerves (N) in the dermis and hypodermis. Only background fluorescence from granular glands (G) is present in tissue stained with antibody that was incubated with recombinant Xenopus leptin protein (4 μg) prior to staining (pre-absorbed). Scale bars, 100 µm. (B) Leptin is expressed in the endothelial cells of a large blood vessel (BV) in the hypodermis and unknown scattered cell bodies (solid arrows). Leptin also co-localizes with acetylated alpha-tubulin (AαT, yellow), a neural marker in axons and some nerve cell bodies (hollow arrows). Granular glands are autofluorescent after bleaching and clearing at 488 nm (as seen in the AαT panel). Scale bars, 100 µm. (C) Leptin protein co-localizes with axons innervating a granular gland. (D) Cross section showing leptin in the endothelial cells of a blood vessel in the hypodermis, up against the stratum corneum (SC) of the dermis. Scale bar, 25 µm.
|
|
Figure 2. Leptin stimulates JAK/STAT signaling in skin. Leptin-induced phosphorylated STAT3-ir (pSTAT3-ir, yellow) in 16-µm sections of juvenile Xenopus laevis dorsal skin; skin was fixed 6 h after intraperitoneal injection with (A) saline or (B) leptin. Leptin stimulates pSTAT3-ir strongly in the epidermis and at the apical end of the granular glands and hypodermis (representative image from n = 3). Scale bar, 100 µm (n = 3). Epidermis (E), dermis (D), stratum corneum of the dermis (SC), and thin hypodermis (H). Scale bar, 50 µm.
|
|
Figure 3. Leptin protein is rapidly localized to injured skin. (A) Whole mount of leptin protein (aqua) and acetylated α-tubulin (AαT, yellow) at the time of injury in juvenile Xenopus laevis dorsal skin. Dotted line indicates injury at the center of the donut-shaped explant. Leptin is highly expressed in blood vessels and nerve tissue (indicated by AαT) associated with the injury site. Granular glands are autofluorescent after bleaching and clearing at 488 nm (as seen in the AαT panel). Scale bar, 100 µm. (B) Leptin protein expression in 16-µm cross sections of juvenile X. laevis dorsal skin after injury. Leptin expression (aqua) is concentrated in the injured tissue at 0 and 24 hpi and nearly depleted by 48 hpi in vitro except for some localized in the epidermis (DAPI, magenta). Epidermis (E), dermis (D), stratum corneum of the dermis (SC), and thin hypodermis (H). Arrow in 24 h panel indicates blood vessel. Scale bars, 100 µm. (C) Leptin is most highly expressed in the central dermis after injury, but expression within 300 µm of the injury site in all skin layers is depleted by 48 hpi in culture (standard least squares ANOVA, time point, p < 0.0001).
|
|
Figure 4. pSTAT3-ir increases after full thickness injury of dorsal skin. (A) The highest pSTAT3 activation was associated directly with the injured edge of the explant (integrated density: 589,578 ± 201,764 ADU, n = 3). (B) By contrast, the end of tissue explant that was non-injured and cut after fixation had less pSTAT3-ir activation (integrated density: 267,042 ± 45,092 ADU).
|
|
Figure 5. Leptin treatment (0, 10, and 100 ng/ml) in culture media significantly improved wound healing in juvenile Xenopus laevis dorsal skin explants at 48 h of culture. (A) At 24 h, treatments at both 10 and 100 ng/ml significantly increased wound closure compared to the control, and by 48 h, the 100 ng/ml treatment continued to improve wound healing compared to the control (repeated-measures MANOVA, treatment = 0.0034, Tukey’s HSD, p < 0.05, n = 12/treatment). (B) Representative samples of dorsal skin explants in culture showing the interior wound of the “punch-in-a-punch” donut-shaped biopsy. Wound closure measured as the total area reduction of the central wound compared to 0 hpi. Scale bars, 0.5 mm.
|
|
Figure 6. Pretreatment with leptin rescues effect of food restriction on wound closure in vitro. Effect of leptin on in vitro wound healing of juvenile Xenopus laevis dorsal skin explants where animals were fed (A) once a day or (B) food restricted for 31 days and then injected in the peritoneum (IP) with leptin (200 ng/g body weight) or saline every other day for 7 days. Dorsal skin donut explant wounds were then treated with (black) or without leptin (red) in culture (100 ng/ml) for 48 h. Pretreatment with IP leptin injection significantly increased the rate of wound closure in food-restricted animals over those with pretreatment injections of saline by 48 h, but there was no effect of pretreatment injection in fed animals (ANOVA, fed status * injection, p = 0.0253); asterisk indicates significant increase in wound healing in leptin pretreated vs. saline pretreated explants (Tukey’s HSD, p < 0.05, n = 8/treatment). There was no significant effect of leptin in the culture media (black vs. red) in this model (p = 0.1809).
|
|
Figure 7. Leptin suppresses microbial growth in culture media of skin explants. Proportion of culture wells visually turbid with bacterial infection for juvenile Xenopus laevis dorsal skin wound explant cultured in increasing leptin concentration at 48 hpi. Increasing leptin concentration (0 ng/ml, n = 70; 10 ng/ml, n = 38; 100 ng/ml, n = 70) significantly decreased the number of infected wells (likelihood ratio test, p = 0.0202).
|
