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J Appl Physiol 97: 641-647, 2004. First published April 2, 2004; doi:10.1152/japplphysiol.00200.2004
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Hindlimb unloading depresses corneal epithelial wound healing in mice

Zhijie Li, Chantal A. Rivera, Alan R. Burns, and C. Wayne Smith

Section of Leukocyte Biology, Departments of Pediatrics, Medicine, and Immunology, Baylor College of Medicine, Houston, Texas 77030

Submitted 23 February 2004 ; accepted in final form 31 March 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
C57BL/6 mice were subjected to hindlimb unloading (HU) for a period of 3 wk to determine the possible effects on epithelial wound healing. A standardized corneal epithelial wound was performed, and parameters of the inflammatory response and reepithelialization were analyzed over an observation period of 96 h. Wound closure was significantly retarded in mice during HU with reepithelialization being delayed by ~12 h. Both epithelial migration and cell division were significantly depressed and delayed. The inflammatory response to epithelial wounding was also significantly altered during HU. Neutrophils, as detected by the Gr-1 marker, were initially elevated above normal levels before wounding and during the first few hours afterward, but there was a significant reduction in neutrophil response to wounding at times where neutrophil influx and migration in controls were vigorous. A similar pattern was seen with CD11b+CD11c+ cells (monocyte lineage). Langerhans cells are normally resident within the peripheral corneal epithelium. They respond to injury by initially leaving the epithelial site within 6 h and returning to normal levels by 96 h, 2 days after reepithelialization is complete. During HU, this pattern is distinctly different, with Langerhans cell numbers slowly diminishing, reaching a nadir at 96 h, which is significantly below normal. Evidence for systemic effects of HU is provided by findings that collagen deposition within subcutaneous sponges was significantly reduced during HU. In conclusion, HU, a ground-based model simulating some physiological aspects of spaceflight, impairs wound repair of corneas. Multiple factors, both local and systemic, likely contribute to this delayed wound healing.

leukocyte; Langerhans cell; dendritic cell; proliferation; cornea; epithelium

WITH THE EXPECTED INCREASE in extended-length space-exploration missions and the establishment of international space stations, there is an increased chance of injury occurring in astronauts due to trauma or unexpected emergency surgery. Little is known about wound healing under conditions of microgravity. Published studies of wound healing in microgravity or ground-based simulations have been confined to repair of bone, muscle, and connective tissues (8, 25, 28, 30, 40). Hindlimb unloading (HU) has been used as a ground-based model (49, 50) that simulates several physiological changes associated with prolonged exposure to microgravity. Our laboratory recently published studies (41), using this model with mice and rats, showing that there is a low-grade portal endotoxemia with an associated acute-phase response and hepatocyte injury after 3–4 wk of unloading. Given recent evidence that some acute-phase proteins may influence inflammatory processes and healing (7, 20–22, 46), we began a study to determine whether wound healing is altered in animals exposed to HU. We chose to investigate a defined injury to corneal epithelium and determine the effect of HU on the inflammatory response and corneal epithelial basal cell migration and proliferation. Our results indicate that inflammation and epithelial healing are significantly altered.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals.   All animal protocols used in this study were reviewed and approved by the Baylor College of Medicine Animal Care and Use Committee. Male C57BL/6 mice 6–8 wk of age and weighting 22–26 g were acclimatized for 1 wk before use in an approved animal care facility with a 12:12-h light-dark cycle.

Animal suspension technique.   The HU model described previously was used (41). Briefly, animals were anesthetized by use of Nembutal sodium solution, and a cast-like apparatus was applied to the tail. To facilitate free movement about the cage, the cast was attached to a swivel anchored to the cage top, which allowed a 360° range of movement. Animals were suspended in a 30° head-down-tilt position. Nonsuspended (control) animals housed individually were used as controls during the suspension periods.

Corneal epithelial wounds.   Mice were anesthetized with intraperitoneal injection of Nembutal. Central circular (2-mm diameter) corneal epithelial wounds were made through the whole epithelium with a trephine. The epithelium within the lesion, with the basement membrane intact, was removed with a diamond blade for refractive surgery (Accutome) under a dissecting microscope. Care was taken not to injure the corneal stroma.

Quantification of reepithelialization rates.   The injured corneas (16 corneas from 4 control and 4 HU mice) were stained with fluorescein and photographed with a digital camera every 6 h, beginning immediately after wounding, to evaluate the reepithelialization and to detect any sign of infection. The images were analyzed with Optimus 6.2 software (Media Cybernetics) using the visible grid as a standard. Parameters measured included area, perimeter, shape, major axis, and minor axis.

Whole-mount immunohistology for the corneas.   Corneas were excised and then fixed for 30 min in 1% paraformaldehyde-PBS, washed three times for 5 min each wash with PBS, permeabilized for 20 min with 0.1% Triton X-100 in PBS, blocked for 20 min with 1% BSA in PBS, and incubated with different diluted FITC and phycoerythrin-conjugated monoclonal antibodies to label different inflammatory cells. Anti-CD11b-FITC (clone M1/70), anti-CD11c-phycoerythrin (clone HL3), and Gr-1-FITC were obtained from PharMingen. Radial cuts were made in the cornea (Fig. 1) so that it could be flattened by a coverslip, and it was mounted in Airvol (Air Products and Chemicals) containing 1 µM 4',6-diamidino-2-phenylindole (Sigma Chemical, St. Louis, MO) to assess nuclear morphology and cell division. In this study, CD11c-positive cells with classic dendritic cell (DC) morphology in the epithelium were considered Langerhans cells, Gr-1-positive cells with nuclear lobulation were recognized as neutrophils, and CD11b and CD11c double-positive cells in the stroma were considered to be of the monocyte lineage. Different sets of mice (control and HU, each n = 4) were used for each cell type examined. Epithelial and stroma sheets were examined at 400-fold magnifications using a x10 eyepiece and a x40 objective lens. To compare the relative level of inflammatory cells in the different areas, limbal, peripheral, paracentral, and central fields for each cornea were assessed and counted separately, as illustrated in Fig. 1. At least four corneas were examined for immunohistology, and four quadrants were analyzed for each cornea to obtain the average numbers for each field. The limbus was defined as the intervening zone between the cornea and conjunctiva as the most peripheral field illustrated in Fig. 1. The peripheral field was defined as the field immediately central to the limbus. The paracentral field was defined as the third field. The central field was defined as the fourth field, as shown including an additional central field of view. Using deconvolution restoration microscopic analysis (DeltaVision), we obtained multiple z-plane sections to determine the position of the stained cells within the corneal whole mounts.



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  		Fig. 1. Diagram showing microscopic fields examined during analysis of the cornea. Analysis was performed by counting specific parameters (e.g., number of neutrophils) in 4 microscopic fields (magnification, x40) as indicated in each of 4 regions of the cornea.

 
Subcutaneous implantation of sponges.   Mice were either subjected to HU or housed individually as described above for 7 days. All were then prepared for surgery, anesthetized with Nembutol (50 mg/kg ip), and restrained in a prone position, and the backs were shaved and disinfected with 70% alcohol and providone iodine. A full-thickness longitudinal cutaneous incision 1 cm long was made on the midline near the base of the neck. Lateral subcutaneous pouches were dissected over the scapular regions, and a single PVA sponge (10-mm diameter, 1 mm thick; Xomed, Jacksonville, FL) in each pouch. The incisions were closed with two interrupted stainless-steel staples, and the mice were returned to their individual cages (either for HU or control conditions) for either 7 or 10 days. The mice were then killed by cervical dislocation after isofluorane anesthesia, and the sponges were removed and extracted for chemical analysis of hydroxyproline (48) as a surrogate measure of collagen (6).

Statistical analysis.   Data analysis was preformed using a two-way repeated-measures ANOVA with pairwise multiple comparisons using Tukey's test. A P value of <0.05 was considered significant. Data are expressed as means ± SE.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Delayed wound healing in the HU mice.   A significant difference between HU and control mice was observed in the gross appearance of the wounds at 6, 12, 18, and 24 h (Fig. 2). Reepithelization was typically complete in controls by 24 h but not until 30–36 h in HU mice. The main effects of HU appeared to be a delay in the onset of wound closure by >6 h. There may have been some reduction in the rate of closure after this time so that epithelial coverage was delayed by up to 12 h in some mice. The epithelial basal cell density was assessed across the diameter of the cornea. By 12 h, epithelial basal cells had moved into the wounded area in control corneas. This was contrasted to HU mice where the wounded area was still devoid of epithelial cells (Fig. 3). By 96 h after wounding, the density in the previously wounded area in control mice was the same as the peripheral density. In contrast, the basal cell density was significantly lower in the central and paracentral fields of corneas from HU mice (Fig. 3).



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  		Fig. 2. Corneal epithelial wound healing of hindlimb-unloaded (HU) mice. A: example of the healing process in HU and control mice. Wounds were photographed at the times indicated. B: wound closure kinetics in HU mice ({blacktriangleup}) and control mice ({blacksquare}). **P < 0.005 and *P < 0.05 vs. control conditions.

 


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  		Fig. 3. Epithelial basal cell density after wounding. At 12 and 96 h after wounding, basal cell density was determined across the diameter of corneas from limbus to limbus in HU and control mice. *P < 0.01 and **P < 0.005 vs. HU conditions.

 
Corneal epithelial basal cell proliferation.   To evaluate the proliferative phase of wound healing, the number of dividing cells in the corneal basal cell layer was determined. Dividing cells were mainly located in the limbal and peripheral regions in both groups during wound healing (Fig. 4). Compared with the control group, the peak level for dividing cells across the cornea in HU mice was significantly reduced and delayed by ~6 h (Fig. 4, B and C). HU mice also showed significantly reduced and delayed dividing numbers at 18 h after wounding (P < 0.005). By 96 h, mitotic cells were rare in control corneas but significantly (P < 0.01) elevated in HU corneas. Thus the proliferative aspects of corneal epithelial wound healing were significantly inhibited and delayed at both early and late stages in mice subjected to HU.



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  		Fig. 4. Dividing cell dynamics during corneal epithelial wound healing in HU mice. A: control mice. B: HU mice. C: comparison of total dividing cells in control and HU mice. Mitotic cells in the corneal epithelium were analyzed and quantitated by in situ 4',6-diamidino-2-phenylindole (DAPI) staining of whole-mount corneas. The mean numbers of dividing cells in different areas (limbus, {blacksquare}; paralimbus, {blacklozenge}; paracentral, {triangleup}; central, x) across the corneas from limbus to limbus in HU mice and control mice are shown. *P < 0.05 vs. control. D: photographs of the dividing cells in the limbus at 18 h after wounding: White arrows indicate the dividing cell with paired nuclei (DAPI staining; original magnification, x400).

 
Neutrophil infiltration to wounded corneas.   Neutrophil infiltration was measured by counting the number of GR-1-positive cells in the different areas from limbus to limbus through the central wound area and at various time points (0, 6, 12, 24, 48, 96, 264 h) postinjury. In corneas from control mice collected without wounding, neutrophils were very rare and occasionally found in the limbal region. In HU mice, however, there was a significantly greater number of neutrophils in the limbus of uninjured corneas after 3 wk of suspension (P < 0.005) (Fig. 5). In wounded corneas of both HU and control mice, neutrophil infiltration was observed as early as 6 h postinjury, peaked at hour 12 postinjury, and decreased after 18 h (Fig. 5A). Analyses of the different areas of wounded corneas show that, in control mice, neutrophils increased in the limbus, peripheral, and central cornea, peaked at 12 h, and then decreased markedly after 24 h, coincident with completion of reepithelialization. With HU, however, the pattern of neutrophil infiltration was altered considerably. Accumulation in the limbus exceeded that of control mice (Fig. 5B) at 6 h postwounding, but by 12 h accumulation in the limbus was significantly below that of control mice, and migration of neutrophils into regions of the stroma beneath the wounded area was substantially reduced (Fig. 5, C and D). The rate of migration was markedly slower in the HU corneas (not reaching the central regions until 12–18 h after wounding), and the magnitude of the response was significantly less.



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  		Fig. 5. Dynamics of polymorphonuclear neutrophils (PMN) in whole cornea (A) and in different fields of the corneas (B–D) during wound healing. Neutrophil infiltration was analyzed by immunohistochemical staining of wounded corneas and quantitation of Gr-1-immunopositive cells. The mean numbers of neutrophils across the corneas from limbus to limbus in the corneas of HU mice ({lozenge}) and control mice ({blacksquare}) are shown. **P < 0.005 and *P < 0.05 vs. control. Right: photographs of anti-GR-1 positive cells of central area stroma at 12 h postwounding (original magnification, x400).

 
CD11b+CD11c+ cell infiltration into wound area.   Compared with control mice, the limbus of suspended mice contained higher CD11b+CD11c+ double-positive cell numbers (P < 0.005; Fig. 6B) before wounding. In wounded corneas of both HU and control mice, CD11b+CD11c+ cell infiltration was observed as early as 6 h postinjury. Cell infiltration peaked at 12 and 24 h postinjury in control mice and HU mice, respectively, and began to decrease after 12 h in controls and 48 h in HU mice (Fig. 6A). Analyses of the different areas of wounded corneas show that, in control mice, CD11b+CD11c+ cells increased gradually in the limbus, peripheral, and central cornea fields, peaked after 12 h, and then decreased after 18 h. In suspended mice, peripheral fields followed a similar pattern with significantly lower cell numbers, whereas CD11b+CD11c+ cell migration into the central fields peaked at 18 h (Fig. 6D). Taken together, these data suggest that CD11b+CD11c+ cell recruitment was significantly inhibited by HU.



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  		Fig. 6. Dynamics of dendritic (CD11b+CD11c+) cell migration in whole cornea (A) and in different fields of the cornea (B–D) during wound healing. Cell infiltration was analyzed by immunohistochemical staining of the wounded cornea and quantitation of CD11b and CD11c double immunopositive cells. The mean numbers of cells across the cornea from limbus to limbus in the corneas of HU mice ({lozenge}) and control mice ({blacksquare}) are shown. **P < 0.005 and *P < 0.05 vs. control.

 
Effects of HU on Langerhans cell migration to wound area.   Langerhans cell infiltration was assessed using CD11c antibody staining and dendritic morphology. At baseline, Langerhans cells (CD11c+ cells) were mainly located at the limbus as described previously (17) (Fig. 7). The density of these cells gradually decreased from the limbus toward the center. No significant difference was found between HU mice and control mice before wounding (Fig. 8). Within 6 h after wounding, Langerhans cell numbers in corneal epithelium of control mice decreased markedly and then returned to basal levels within 96 h. In contrast, there was no change in Langerhans cells within 12 h, but a gradual decrease to a significantly lower level occurred at 96 h. Recovery to normal levels was seen at 11 days (data not shown). These data suggest that the HU condition significantly altered Langerhans cell migration during wound healing.



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  		Fig. 7. Morphology and distribution of Langerhans cells in control and HU corneas before wounding. A: typical dendritic CD11c+ cells (red) in the corneal limbus (magnification, x400). Epithelial cell nuclei are blue. B: similar distribution of Langerhans cells in control and HU mouse corneas.

 


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  		Fig. 8. Langerhans cell migration during wound healing in HU mice. Langerhans cell migration was analyzed by immunohisochemical staining of wounded cornea epithelium and quantitation of CD11c immunopositive cells. The mean numbers of total Langerhans cells in the corneas of HU mice ({lozenge}) and control mice ({blacksquare}) are shown. **P < 0.005 and *P < 0.05 vs. control.

 
Evidence for systemic alterations in wound healing.   To determine whether the abnormalities in corneal wound healing were possibly manifestations of a systemic influence of HU on wound healing, we chose a simple measure of collagen deposition in subcutaneous wounds. At both 7 and 10 days after subcutaneous implantation of PVA sponges, the hydroxyproline content was significantly lower in mice subjected to HU than in controls (Fig. 9).



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  		Fig. 9. Hydroxyproline content of subcutaneous sponges (n = 12 for 7-day experiment and n = 8 for 10-day experiment). Mice were subjected to HU or control conditions for 7 days before implantation of PVA sponges. After an additional 7 or 10 days of HU or control conditions, sponges were extracted for hydroxyproline as a surrogate measure for collagen deposition. *P < 0.05 vs. control.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Although adequate documentation exists on the adverse effects of space travel on various human organ systems and immune systems, there are no reported studies on wound healing in space. Experiments on tissue repair processes are of particular interest. Studies performed in rat skeletal muscle and bone revealed that microgravity (flown on Cosmos-2044 and STS-29) and tail suspension inhibited the repair processes (25). More recent studies have shown that both spaceflight and HU result in reduced connective tissue repair, including collagen accumulation, cellular influx, and response to growth factor stimulation (8, 40). These studies, like the present one, support the concept that microgravity and suspension can profoundly influence the tissue-repair process.

Inflammation and wound healing.   A number of potential mechanisms may play a role in delayed wound healing. Effective wound healing appears to be dependent on circulating leukocytes (12). Preventing leukocyte migration to corneal wounds results in impaired healing, including decreased proliferation of corneal epithelial cells and corneal fibroblasts (13, 14). Neutrophils are the first inflammatory cells to reach the wound. The primary function of neutrophils in this condition is microbial clearance. In addition, neutrophils perform wound healing-enhancing abilities by producing angiogenic factor vascular endothelial growth factor, tumor necrosis factor-{alpha}, and interleukin-1. Neutrophils also express proteinases that are involved in degrading the matrix of the wound bed, enabling further cellular infiltration by macrophages, DCs, and fibroblasts. Several studies carried out in orbiting spacecraft during the past decade on rodents and humans also showed significant changes in both the number and functional capacity of peripheral blood leukocytes (1, 24). In addition, a 50% reduction in neutrophil influx in the wounded corneas was seen in our suspended animals compared with control animals. Effects on leukocyte kinetics are obvious targets of future studies (35, 45), as are hormonal changes that may alter innate immune responses (33, 35, 44).

Another area of interest is the potential effects of acute-phase proteins on inflammation and wound healing. This is of interest in light of our recent finding that HU induces an acute-phase response in livers of suspended animals that appears to be linked to a portal endotoxemia (41). It is well known that endotoxin will induce a systemic inflammatory response, but our laboratory's recent work indicates that, in HU, elevations in endotoxin are found only in the portal venous supply, suggesting that the liver is efficient in clearing the endotoxin. However, there is a significant response of the liver that appears to be dependent on this endotoxin. Specifically, hepatic injury is indicated by systemic plasma elevations in liver enzymes and a distinct acute-phase response as evidenced by upregulation of serum amyloid A. Recent studies have shown that serum amyloid A can prime neutrophils for enhanced function (20, 21), may alter platelet functions (47), and may participate in other physiological and pathological processes (46). In addition, another acute-phase protein {alpha}1-acid glycoprotein appears to have modulatory activity in the inflammatory process (22). These examples raise the question of whether the liver response to HU could have systemic effects sufficient to alter healing and inflammation in the cornea. Our evidence that collagen deposition in subcutaneous wounds was reduced in HU mice supports the hypothesis of a systemic depression in wound healing. Further support comes from a study of collagen deposition in implanted sponges in rats during actual spaceflight where both sponge cellularity and collagen were significantly diminished (8). Future studies will be directed at investigating the possibility that systemic effects of HU involve the acute-phase proteins of the liver in response to portal endotoxemia.

DCs and wound healing.   DCs are specialized innate immunity cells with critical functions in presenting antigen to lymphocytes (2, 3). The altered behavior of these cells [Langerhans cells (17)] after epithelial injury in the HU mice raises the important question of possible alterations in subsequent immune functions. After trauma or injury, immature DCs with high phagocytic capacity, such as Langerhans cells in the peripheral cornea, capture injury-induced antigens (such as apoptotic and necrotic cell fragments and invading pathogens). The antigen/pathogen induces the immature DC to undergo phenotypic and functional changes that culminate in the complete transition from Ag-capturing cell to matured antigen presentation cells. Matured DCs will migrate from the peripheral tissue to the draining lymphoid organs and then bind and stimulate T cells in the T cell areas of lymphoid tissues. A recent study (26) shows that specialized T lymphocytes, epidermal {gamma}{delta} T cells, exist in the mouse skin. These cells can be activated after recognizing an injury-induced antigen and other signals and accelerate wound healing by secreting growth factors. Similarly, in the central nervous system of mice, autoreactive T cells can promote the revascularization and posttraumatic healing of brain in an antigen-specific fashion (23). In addition, some evidence demonstrates that blocking or deleting lymphocytes indirectly delays wound healing (4, 11). T and B lymphocytes are present in the early stages of rat corneal wound healing (39), and further studies will be necessary to determine whether this is true in mice and other species. DCs may also contribute, by production of inflammatory cytokines such as interleukin-1 and nitric oxide, factors that play an important role in wound healing (42, 51).

Recent data suggest that normal corneal stroma has a significant number of resident DC, and inflammation and trauma may induce DC migration into diseased corneas (18, 19). DC might also be involved in the corneal wound-healing process. The present study found that the CD11b+CD11c+ cell migration pattern observed during corneal wound healing paralleled that for neutrophil migration. At this point, we have not distinguished which subpopulations of these cells exhibit the behavior found in HU mice.

Microgravity and cell differentiation, proliferation, and cell cycle.   It appears that microgravity and HU have significant effects on cell proliferation, differentiation, and cycles. The tissues mainly studied included muscles (29, 37, 43), bone and cartilage (5, 10, 31, 34, 52), skin (9), retina (15), testis (27), and even lower vertebrae tissue (16, 36). The present study for the first time showed that HU might also significantly inhibit and delay the proliferation of the corneal epithelium during wound healing. Whether this is linked to alterations in innate immune responses, functional changes of lacrimal gland and tear functions (32), or corneal nerve functions (38) remains to be determined.

In conclusion, our experiments for the first time indicate that there may be intrinsic defects in the capacity of wound repair to corneas in the space environment. Multiple factors, both local and systemic, likely contribute to delayed wound healing. Because it is highly possible that trauma will take place during space exploration, we think these studies underscore the need to understand the basic mechanisms of altered wound healing.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This project is supported by National Aeronautics and Space Administration Grant NAG 9-1253 and National Institutes of Health Grants HL-070537 and AI-46773.


   FOOTNOTES
 

Address for reprint requests and other correspondence: C. Wayne Smith. Leukocyte Biology Section, Children's Nutrition Research Center, 1100 Bates, Rm. 6014, Baylor College of Medicine, Houston, TX 77030-2600 (E-mail: cwsmith{at}bcm.tmc.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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