Unwounded Mouse Research Articles

Dermal injury drives a skin to gut axis that disrupts the intestinal microbiome and intestinal immune homeostasis in mice

The composition of the microbial community in the intestine may influence the functions of distant organs such as the brain, lung, and skin. These microbes can promote disease or have beneficial functions, leading to the hypothesis that microbes in the gut explain the co-occurrence of intestinal and skin diseases. Here, we show that the reverse can occur, and that skin directly alters the gut microbiome. Disruption of the dermis by skin wounding or the digestion of dermal hyaluronan results in increased expression in the colon of the host defense genes Reg3 and Muc2, and skin wounding changes the composition and behavior of intestinal bacteria. Enhanced expression Reg3 and Muc2 is induced in vitro by exposure to hyaluronan released by these skin interventions. The change in the colon microbiome after skin wounding is functionally important as these bacteria penetrate the intestinal epithelium and enhance colitis from dextran sodium sulfate (DSS) as seen by the ability to rescue skin associated DSS colitis with oral antibiotics, in germ-free mice, and fecal microbiome transplantation to unwounded mice from mice with skin wounds. These observations provide direct evidence of a skin-gut axis by demonstrating that damage to the skin disrupts homeostasis in intestinal host defense and alters the gut microbiome.

Prrx1 Fibroblasts Represent a Pro-fibrotic Lineage in the Mouse Ventral Dermis

SUMMARYFibroblast heterogeneity has been shown within the unwounded mouse dorsal dermis, with fibroblast subpopulations being identified according to anatomical location and embryonic lineage. Using lineage tracing, we demonstrate that paired related homeobox 1 (Prrx1)-expressing fibroblasts are responsible for acute and chronic fibroses in the ventral dermis. Single-cell transcriptomics further corroborated the inherent fibrotic characteristics of Prrx1 fibroblasts during wound repair. In summary, we identify and characterize a fibroblast subpopulation in the mouse ventral dermis with intrinsic scar-forming potential.

Effects of Vitamin D Receptor Knockout and Vitamin D Deficiency on Corneal Epithelial Wound Healing and Nerve Density in Diabetic Mice.

Diabetic keratopathy occurs in ∼70% of all people with diabetes. This study was designed to examine the effects of vitamin D receptor knockout (VDR-/-) and vitamin D deficiency (VDD) on corneal epithelial wound healing and nerve density in diabetic mice. Diabetes was induced using the low-dose streptozotocin method. Corneal epithelial wounds were created using an Algerbrush, and wound healing was monitored over time. Corneal nerve density was measured in unwounded mice. VDR-/- and VDD diabetic mice (diabetic for 8 and 20 weeks, respectively) had slower healing ratios than wild-type diabetic mice. VDR-/- and VDD diabetic mice also showed significantly decreased nerve density. Reduced wound healing ratios and nerve densities were not fully rescued by a supplemental diet rich in calcium, lactose, and phosphate. We conclude that VDR-/- and VDD significantly reduce both corneal epithelial wound healing and nerve density in diabetic mice. Because the supplemental diet did not rescue wound healing or nerve density, these effects are likely not specifically related to hypocalcemia. This work supports the hypothesis that low vitamin D levels can exacerbate preexisting ophthalmic conditions, such as diabetes.

Requirement for the collagen receptor Endo180 in collagen gel contraction mediated by corneal fibroblasts

The interaction of keratocytes with extracellular matrix components plays an important role in the maintenance of corneal transparency and shape as well as in the healing of corneal wounds. In particular, the interaction of these cells with collagen and cell-mediated collagen contraction contribute to wound closure. Endo180 is a receptor for collagen that mediates its cellular internalization. We have now examined the role of Endo180 in collagen contraction mediated by corneal fibroblasts (activated keratocytes). Antibodies to Endo180 inhibited the contractile activity of mouse corneal fibroblasts embedded in a three-dimensional collagen gel and cultured in the presence of serum, with this effect being both concentration and time dependent and essentially complete at an antibody concentration of 0.2 μg/ml. Whereas corneal fibroblasts cultured in a collagen gel manifested a flattened morphology with prominent stress fibers under control conditions, they showed a spindlelike shape with few stress fibers in the presence of antibodies to Endo180. Antibodies to Endo180 had no effect on the expression of α–smooth muscle actin or the extent of collagen degradation in collagen gel cultures of corneal fibroblasts. Immunohistofluorescence analysis did not detect the expression of Endo180 in the unwounded mouse cornea. However, Endo180 expression was detected in keratocytes migrating into the wound area at 3 days after a corneal incisional injury. Together, our results suggest that Endo180 is required for the contraction of collagen matrix mediated by corneal fibroblasts and that its expression in these cells may contribute to the healing of corneal stromal wounds.

Topical Application of a Mast Cell Stabilizer Improves Impaired Diabetic Wound Healing

Impaired wound healing in the diabetic foot is a major problem often leading to amputation. Mast cells have been shown to regulate wound healing in diabetes. We developed an indole-carboxamide type mast cell stabilizer, MCS-01, which proved to be an effective mast cell degranulation inhibitor invitro and can be delivered topically for prolonged periods through controlled release by specifically designed alginate bandages. In diabetic mice, both pre- and post-wounding, topical MCS-01 application accelerated wound healing comparable to that achieved with systemic mast cell stabilization. Moreover, MCS-01 altered the macrophage phenotype, promoting classically activated polarization. Bulk transcriptome analysis from wounds treated with MCS-01 or placebo showed that MCS-01 significantly modulated the mRNA and microRNA profile of diabetic wounds, stimulated upregulation of pathways linked to acute inflammation and immune cell migration, and activated the NF-κB complex along with other master regulators of inflammation. Single-cell RNA sequencing analysis of 6,154 cells from wounded and unwounded mouse skin revealed that MCS-01 primarily altered the gene expression of mast cells, monocytes, and keratinocytes. Taken together, these findings offer insights into the process of diabetic wound healing and suggest topical mast cell stabilization as a potentially successful treatment for diabetic foot ulceration.

Breaking up is hard to do: Does splitting cages of mice reduce aggression?

Injurious aggression in group housed male laboratory mice is a common welfare issue that can also negatively affect study outcomes. Often, one mouse in the cage appears unwounded, and the current standard practice is to remove this presumed aggressor. This procedure is not based on empirical evidence and may impede welfare by singly housing animals. We experimentally tested the hypothesis that the apparently uninjured mouse is indeed the aggressor, and that aggression is reduced in his absence. We separated cages of four or five male mice, reported for fight wounds to our university’s veterinary service, into cages of two or three mice containing either only wounded mice (“wounded” treatment) or both wounded and unwounded mice (“mixed” treatment). We recorded aggressive behavior for 30 min immediately pre- and post-separation, and scored wound severity at separation and over two weeks after. We predicted that if unwounded mice are aggressors: mice in the wounded treatment would show less escalated aggression (involving biting) than mice in the mixed treatment, and would be wounded less and/or heal faster during the two weeks following separation. Wound scores decreased significantly after separation in both treatments (wounded: p < 0.0001; mixed: p = 0.011), but mice in the wounded treatment healed faster than those in the mixed treatment (p = 0.006). There was no significant effect of treatment on duration of escalated aggression in the 30 min following separation (p = 0.240), nor did treatment predict which cages would be re-separated due to continued aggression (p = 0.104). Our results support the hypothesis that the unwounded mouse is the aggressor, as mice in cages with an unwounded mouse healed more slowly than those without. Both types of groups healed significantly over time, suggesting that separation into groups of two or three is a possible management alternative to social isolation of the presumed aggressor. By identifying spontaneous cases of severe aggression in an existing colony, we obtained a heterogeneous and representative sample of clinical cases, bolstering the generalizability of our conclusions.

Ionic Components of Wound Current at Mouse Skin Incisional Wounds

It has been assumed that skin wound currents are produced by passive ion leakage from wounded skin. However, what ions contribute to the skin wound current? Thirty male C57BL/6 mice were used in this study. A lancet wound (about 5mm in length) was made by cutting into the full thickness skin. We measured the dynamic time courses of individual ion flux with ion-selective probes at skin wound. The probe is aligned perpendicular to the surface of skin wound. All ions (Na+, Cl-, K+, Ca2+ and H+) showed a small steady efflux at unwounded mouse skin. However, there were large efflux of Na+, Cl-, K+, Ca2+ and H+ 15 minutes after wounding. These values maintained for 534 seconds, which were significantly higher than the ions flux of intact skin (P< 0.05). Our results suggest that Na+, Cl-, K+, Ca2+ and H+ contribute to the wound currents at skin wounds. A most significant observation is that the skin wound currents are carried mainly by the largest sodium and chloride efflux, then potassium, while the calcium and hydrogen are the least.

Vimentin knockdown decreases corneal opacity.

Wound induced corneal fibrosis can lead to permanent visual impairment. Keratocyte activation and differentiation play a key role in fibrosis, and vimentin, a major structural type III intermediate filament, is a required component of this process. The purpose of our study was to develop a nonviral therapeutic strategy for treating corneal fibrosis in which we targeted the knockdown of vimentin. To determine the duration of plasmid expression in corneal keratocytes, we injected a naked plasmid expressing green fluorescent protein (GFP; pCMV-GFP) into an unwounded mouse corneal stroma. We then injected pCMV-GFP or plasmids expressing small hairpin RNA in the corneal wound injury model (full-thickness corneal incision) to evaluate opacification. GFP expression peaked between days 1 and 3 and had prominent expression for 15 days. In the corneal wound injury model, we found that the GFP-positive cells demonstrated extensive dendritic-like processes that extended to adjacent cells, whereas the vimentin knockdown model showed significantly reduced corneal opacity. These findings suggest that a nonviral gene therapeutic approach has potential for treating corneal fibrosis and ultimately reducing scarring.

Corneal Goblet Cells and Their Niche: Implications for Corneal Stem Cell Deficiency

Goblet cells are terminally differentiated cells secreting mucins and antibacterial peptides that play an important role in maintaining the health of the cornea. In corneal stem cell deficiency, the progenitor cells giving rise to goblet cells on the cornea are presumed to arise from differentiation of cells that migrate onto the cornea from the neighboring conjunctiva. This occurs in response to the inability of corneal epithelial progenitor cells at the limbus to maintain an intact corneal epithelium. This study characterizes clusters of cells we refer to as compound niches at the limbal:corneal border in the unwounded mouse. Compound niches are identified by high expression of simple epithelial keratin 8 (K8) and 19 (K19). They contain variable numbers of cells in one of several differentiation states: slow-cycling corneal progenitor cells, proliferating cells, nonproliferating cells, and postmitotic differentiated K12+Muc5ac+ goblet cells. Expression of K12 differentiates these goblet cells from those in the conjunctival epithelium and suggests that corneal epithelial progenitor cells give rise to both corneal epithelial and goblet cells. After wounds that remove corneal epithelial cells near the limbus, compound niches migrate from the limbal:corneal border onto the cornea where K8+ cells proliferate and goblet cells increase in number. By contrast, no migration of goblet cells from the bulbar conjunctiva onto the cornea is observed. This study is the first description of compound niches and corneal goblet cells and demonstration of a role for these cells in the pathology typically associated with corneal stem cell deficiency.

IL-6 modulates TGF-β expression in skin from IL-6 deficient mice (97.11)

Abstract IL-6 and Tgf-β have been shown to play a role in skin development and maintenance. However, a link between these two cytokines has yet to be identified therefore; therefore, in this study we investigated the modulation of Tgf-β1 and Tgf-βR2 by IL-6 in skin. Recombinant IL-6 treatment of IL-6KO lattices and intradermal injections of IL-6 into unwounded mice showed a significant induction of Tgf-β1 mRNA and protein, where Tgf-β1 expression localized in the dermis, while Tgf-βR2 expression resided primarily in the epidermis in IL-6KO mice. IL-6 also increased Tgf-βR2 mRNA and protein expression, and potentiates TGFb mediated decreases in proliferation in cultured keratinocytes. Wounded IL-6KO and C57BL/6 control mice showed Tgf-β1 and βR2 mRNA expression was significantly greater in unwounded and seven day-old wounds from WT mice, however; protein expression was not different. Use of inhibitors indicated that IL-6 modulates Tgf-β through a Mapk/Erk dependent mechanism. These studies indicate that IL-6 has the ability to modulate the expression of Tgf-β and Tgf-βR2 mRNA in dermal fibroblasts and unwounded skin through a Mapk/Erk dependent mechanism. Protein expression levels indicated additional regulation by factors other than IL-6 within the wound. IL-6 treatment also potentiates the differentiative effects of Tgf-β on cultured keratinocytes. These results may provide a possible mechanism defining the role of IL-6 in skin maintenance and may indicate a new association of IL-6 with Tgf-β in pathologies associated fibrosis. This work was funded by NIH/NIGMS grant R01 GM067745.

Alcohol extract of Echinacea pallida reverses stress-delayed wound healing in mice

Healing of open skin wounds begins with an inflammatory response. Restraint stress has been well documented to delay wound closure, partially via glucocorticoid (GC)-mediated immunosuppression of inflammation. Echinacea, a popular herbal immunomodulator, is purported to be beneficial for wound healing. To test the hypothesis, an alcohol extract of E. pallida was administrated orally to mice for 3 days prior to, and 4 days post wounding with a dermal biopsy on the dorsum. Concomitantly, mice were exposed to 3 cycles of daily restraint stress prior to, and 4 cycles post wounding. Echinacea accelerated wound closure in the stressed mice, but had no apparent wound healing effect for the non-stressed mice when compared to their respective controls. To test if the positive healing effect is through modulation of GC release, plasma corticosterone concentrations were measured in unwounded mice treated with restraint stress and the herbal extract for 4 days. Plasma GC in restraint stressed mice gavaged with Echinacea was not different from mice treated with restraint only, but was increased compared to the vehicle control. This data suggests that the improved wound healing effect of Echinacea in stressed mice is not mediated through modulation of GC signaling.

Connective tissue growth factor promoter activity in normal and wounded skin

In skin, connective tissue growth factor (CTGF/CCN2) is induced during tissue repair. However, what the exact cell types are that express CTGF in normal and wounded skin remain controversial. In this report, we use transgenic knock-in mice in which the Pacific jellyfish Aequorea victoria enhanced green fluorescent protein (E-GFP) gene has been inserted between the endogenous CTGF promoter and gene. Unwounded (day 0) and wounded (days 3 and 7) skin was examined for GFP to detect cells in which the CTGF promoter was active, α-smooth muscle actin (α-SMA) to detect myofibroblasts, and NG2 expression to detect pericytes. In unwounded mice, CTGF expression was absent in epidermis and was present in a few cells in the dermis. Upon wounding, CTGF expression was induced in the dermis. Double immunolabeling revealed that CTGF-expressing cells also expressed α-SMA, indicating the CTGF was expressed in myofibroblasts. A subset (~30%) of myofibroblasts were also NG2 positive, indicating that pericytes significantly contributed to the number of myofibroblasts in the wound. Pericytes also expressed CTGF. Collectively, these results indicate that CTGF expression in skin correlates with myofibroblast induction, and that CTGF-expressing pericytes are significant contributors to myofibroblast activity during cutaneous tissue repair.

RANK, RANKL, OPG, and M-CSF expression in stromal cells during corneal wound healing.

To examine the influx of monocytes into the cornea after epithelial scrape injury and the expression of chemokines that potentially regulate monocyte phenotype in cultured corneal fibroblasts and keratocytes in situ. Monocytes were detected by immunocytochemistry for the monocyte-specific antigen CD11b, in unwounded and epithelial scrape-wounded mouse corneas. The receptor activator of NF-kappa B ligand (RANKL), osteoprotegerin (OPG), and monocyte chemotactic and stimulating factor (M-CSF) mRNAs were detected in cultured mouse stromal fibroblasts by RT-PCR and RNase protection assay. RANKL, OPG, and M-CSF proteins were detected in cultured mouse stromal fibroblasts by immunoprecipitation and Western blot analysis. RANKL, RANK, M-CSF, and OPG proteins were detected in unwounded and wounded mouse corneas by immunocytochemistry. Chimeric mice with green fluorescent protein-labeled bone marrow-derived cells underwent corneal scrape injury and were monitored by fluorescence microscopy and immunocytochemistry. A small number of cells expressing the monocyte-specific CD11b antigen were detected in the stromas of unwounded mouse corneas. A larger number of CD11b-positive cells was detected in the stroma at 24 or 48 hours after epithelial scraping injury. Experiments with chimeric mice with fluorescent green protein-labeled, bone marrow-derived cells demonstrated conclusively the origin of these CD11b(+) cells. RANKL, OPG, and M-CSF mRNAs and proteins were detected in cultured mouse stromal fibroblasts. RANKL, M-CSF, and OPG proteins were detected in unwounded corneas, but were expressed at higher levels in stromal cells during the 24- to 48-hour interval after epithelial scrape injury. RANK was detected in stromal cells presumed to be monocytes at 24 and 48 hours after epithelial injury. Cells expressing the CD11b monocyte-specific antigen appear in the corneal stroma in high numbers by 24 hours after epithelial injury and persist beyond 10 days after wounding. Cultured corneal fibroblasts and keratocytes in situ express RANKL, OPG, and M-CSF cytokines involved in regulating osteoclast differentiation from monocytes in bone. Cells expressing RANK were detected in the stroma at 24 and 48 hours after epithelial injury. The cytokine systems that regulate monocyte transition to osteoclast in bone are upregulated in the cornea in response to epithelial injury and may participate in regulating monocyte phenotype during corneal stromal wound healing.

Expression of type XVIII collagen during healing of corneal incisions and keratectomy wounds.

To determine the distribution of type XVIII collagen in mouse ocular tissues and to investigate the expression of type XVIII collagen during healing of corneal incisions and keratectomy wounds. Immunohistochemical analysis of type XVIII collagen was performed in mouse ocular tissue, with polyclonal antibodies to the hinge domain. For wound-healing experiments, excimer laser keratectomy and single linear incisions were performed on mouse corneas. The corneas were harvested at various time points after wounding and processed for immunohistochemistry, in situ hybridization, competitive reverse transcription-polymerase chain reaction (RT-PCR), and Western blot analysis. In the unwounded mouse cornea, type XVIII collagen was expressed by the corneal epithelial cells. Type XVIII collagen was immunolocalized to the mouse corneal epithelium, epithelial basement membrane, Descemet's membrane, ciliary epithelium, lens capsule, retinal inner limiting membrane, and Bruch's membrane. In the early stages of wound healing after excimer laser keratectomy (days 3 and 7), type XVIII collagen staining of the epithelial basement membrane was absent, whereas its localization to Descemet's membrane was unchanged. After linear corneal incisions, however, type XVIII collagen was clearly seen in the stroma and in the epithelial basement membrane. Type XVIII collagen immunolocalization to the subepithelial stromal wound region peaked at 1 week after wounding, and its mRNA showed a corresponding temporal increase in expression within the same region after linear corneal incisions. The results suggest that type XVIII collagen is broadly expressed in ocular tissues and that it may have a role in wound healing, especially after incisional corneal wounds.

Epithelial Injury Induces Keratocyte Apoptosis: Hypothesized Role for the Interleukin-1 System in the Modulation of Corneal Tissue Organization and Wound Healing

The purpose of this study was to determine the role of programmed cell death (apoptosis) in the disappearance of keratocytes beneath an epithelial debridement wound in the cornea and to investigate a potential role of interleukin-1 (IL-1) in induction of apoptosis in stromal fibroblasts in vitro and keratocytes in vivo. Keratocyte and stromal fibroblast cell morphology was examined in wounded and unwounded mouse corneas using transmission electron microscopy. Nuclear DNA fragmentation was detected with the TUNEL assay for 3′-hydroxyl DNA ends. The effect of IL-1 on keratocytes in vivo was determined by microinjection of IL-1 alpha into the central corneal stroma via a limbal entry site. The in vitro effects of interleukin-1 alpha (IL-1 alpha) and interleukin-1 beta (IL-1 beta) were determined with primary cultures of human corneal stromal and dermal fibroblasts. Cell shrinkage, blebbing with formation of membrane bound bodies, condensation and fragmentation of the chromatin, and DNA fragmentation, consistent with apoptosis were detected in anterior stromal keratocytes after epithelial scrape wounds. Thus, disappearance of keratocytes from the underlying stroma following epithelial debridement is mediated by apoptosis. Microinjection of IL-1 alpha into the central stroma of the mouse cornea caused a redistribution of keratocytes in the stroma via apoptosis and, possibly, negative chemotaxis. IL-1 alpha and IL-1 beta induced apoptosis in corneal stromal and dermal fibroblasts in vitro. The epithelial/endothelial-stromal IL-1 system may mediate corneal tissue organization and responses to mechanical- and pathogen-induced injury through induction of keratocyte apoptosis. Keratocyte apoptosis is likely an initiating event in wound healing following corneal surgery. We hypothesize that derangement's in this system may have a role in the pathogenesis of keratoconus and other diseases of the cornea.