Vanadate ingestion increases the gain in wound breaking strength and leads to better organized collagen fibers in rats during healing.

Repair of incision wounds closed by suturing is evaluated by the progressive gain in wound breaking strength. Previously the closure of open wounds in rats ingesting vanadate, an inhibitor of tyrosine phosphate phosphatases, was shown to occur with deposition of more uniformly organized collagen fiber bundles. The hypothesis of this study was that deposition of more uniformly organized collagen fibers would enhance the gain in wound breaking strength of incisional wounds. Six adult rats received vanadate-supplemented saline drinking water for 1 week before placement of two 6-cm, parallel, suture-closed wounds on their backs. Six control rats received identical wounds and were given saline drinking water. The drinking water regimen was continued for 1 week after wounding, and then wound strength was tested with a tensiometer and tissue samples were obtained for histologic evaluation. Wound breaking strength doubled in vanadate-treated rats compared with controls. Bright-field and polarized light microscopy showed that the connective tissue matrix of granulation tissue from control rats was oriented perpendicular to the surface of the skin. In contrast, the connective tissue matrix of granulation tissue from vanadate-treated rats was oriented parallel to the skin surface. The gap in granulation tissue between the edges of the wounds in the vanadate-treated rats was greater than that in controls. Electron microscopy showed that wounds in the vanadate-treated contained uniform collagen fibers that were 20 percent greater in diameter and more evenly spaced than they were in controls. It is proposed that these changes in the organization of collagen fibers within incisional wounds were responsible for the increased wound breaking strength observed in rats ingesting vanadate.

Vanadate and the absence of myofibroblasts in wound contraction.

HYPOTHESIS: Fibroblasts, not myofibroblasts, are responsible for wound contraction. Only myofibroblasts express a smooth muscle actin for which vanadate blocks its expression. Wound contraction in vanadate-treated rats will proceed normally in the absence of myofibroblasts. DESIGN: Laboratory study using rats. METHODS: Wound healing in rats receiving vanadate parenterally, an inhibitor of tyrosine phosphate phosphatases, was investigated. For 21 days, treated rats received drinking water containing vanadate, 0.2 mg/mL, in isotonic sodium chloride solution, and the control rats received isotonic sodium chloride solution alone. On day 7, 4 square, full-excision wounds were made dorsally and measured, then 2 polyvinyl alcohol sponges were placed ventrally in subcutaneous pockets. RESULTS: After 2 weeks, the wound area in the rats receiving vanadate measured 7.1 +/- 1.8 U (mean+/-SD), and the wound area in the control rats measured 7.2 +/- 2.2 U. The control rats' granulation tissue (GT) had myofibroblasts, or alpha-smooth muscle (alpha-SM) actin-positive fibroblasts, whereas the vanadate-treated group's fibroblasts were devoid of alpha-SM actin. By Western blot analysis, GT homogenates in the vanadate-treated group contained less alpha-SM actin. By electron microscopy, control rats' GT showed classic myofibroblast populations, and the collagen fiber bundles were randomly organized. In contrast, the wounds in the vanadate-treated group showed unencumbered fibroblast populations and neatly ordered, parallel collagen fiber bundles. By polarized light microscopy, the GT of the vanadate-treated group displayed orderly collagen fiber bundles. CONCLUSIONS: The differentiation of fibroblasts into myofibroblasts requires the dephosphorylation of selected tyrosine phosphate residues. In the absence of myofibroblasts, the rate of rat wound contraction is normal, and collagen fiber bundles have a more orderly arrangement. Myofibroblasts are not required for wound contraction.

Repeated additions of hyaluronan alters granulation tissue deposition in sponge implants in mice.

The role for the metabolism of hyaluronic acid in the repair process is uncertain. Fetal dermal wounds do not heal by scarring and have sustained high levels of hyaluronic acid. In contrast, adult dermis is repaired by scarring and has less hyaluronic acid. Initially after injury, hyaluronic acid is elevated in both adult and fetal wounds, and although it remains elevated in fetal repair, it is rapidly degraded in adult wounds. The chronic addition of hyaluronic acid or hyaluronidase to polyvinyl alcohol sponge implants in adult mice was investigated in this study. Polyvinyl alcohol sponge implants containing a central reservoir were placed subcutaneously in the dorsum of adult male CD-1 mice. Mice were divided into three groups: a phosphate-buffered saline control, a 20 microgram hyaluronic acid treatment group, and a 10 U hyaluronidase treatment group. The central reservoir of each sponge implant received appropriate compound every 3 days for 2 weeks via transdermal injection and were then evaluated histologically. At 2 weeks, the cellular density and the quantity of granulation tissue deposition were the greatest in the hyaluronidase group and were lowest in the hyaluronic acid group. In addition, the organization of collagen fiber bundles was the most dense in the hyaluronidase group and least in the hyaluronic acid group. In a second experiment, polyvinyl alcohol sponge implants in mice received either phosphate-buffered saline solution or 20 microgram hyaluronic acid every 3 days for 1 week. On day 5, an aliquot of fluorescently tagged native collagen was injected into the sponges. Sponges were harvested at day 7, cryosections made, and the presence of autofluorescent collagen fibers assessed. The autofluorescent collagen fiber bundles in the phosphate-buffered saline solution group were organized in thick parallel bundles, whereas the collagen bundles from hyaluronic acid-treated implants were organized in fine lacelike structures. Chronic addition of hyaluronic acid appears to mimic the fetal dermal connective tissue matrix in which repair proceeds with diminished collagen deposition, organized in finer collagen fiber bundles in granulation tissue. On the other hand, the removal of hyaluronic acid by the chronic administration of hyaluronidase increases the amount of granulation tissue. Elevated levels of hyaluronic acid in granulation tissue appear to modulate the ability of resident fibroblasts to organize collagen fiber bundles.

Hyaluronan induces scarless repair in mouse limb organ culture.

BACKGROUND/PURPOSE: Wounded fetal mouse limbs harvested from two distinct time points in gestation heal differently in organ culture. The healing of a gestational day 14 limb is by scarless repair, whereas gestational day 18 (gd 18) limbs heal by scarring. The persistence of elevated levels of hyaluronic acid (HA) is a major difference in the extracellular matrix of scarless repair. The purpose of this study was to demonstrate that chronic additions of HA to incisional wounds of gd 18 limbs induces scarless repair. METHODS: Time-dated pregnant CD-1 mice (term, 20 days) were killed on gestational day 18 and fetuses were harvested via laparotomy. A through and through stab wound was made in each forelimb with a 1-mm microscapel, and the wound was closed with a single 10-0 nylon suture. The forelimbs were amputated at the level of the shoulder and placed in organ culture. Daily medium changes with 1 mL of BGJb (devoid of serum) were made. Half the cultures received 10 microL of HA (4 mg/mL) directly to the wound site with each medium change. The other half of the cultures received 10 microL of phosphate-buffered saline (PBS-control). At day 7, the limbs were harvested, fixed in methyl Carnoys solution, paraffin embedded, and 5-microm serial sections cut. The sections were stained with H&E or Sirius red/fast green. The sections were viewed in a blinded fashion by two observers. Suture defined the wound site, and the sections were graded for healing by scarring. RESULTS: Minimal limb growth occurred in both control and HA-treated limbs. Grossly, both control and treated limbs healed incisional wounds by 7 days in culture. Limbs from both treatment and control groups showed viability by microscopic analysis. The limbs treated with HA had no appreciable scar morphologically in sections in which epithelial dimpling and suture were evident. The orientation of the collagen fiber bundles in the control wounds were in parallel arrays perpendicular to the incision. The orientation of the collagen fiber bundles in the HA-treated limbs had a basket weave pattern that was indistinguishable from unwounded dermis. The direct repeated additions of HA to healing organ cultured limb explants of gestational day 18 fetal mice promoted scarless repair. CONCLUSIONS: This result demonstrates that chronic elevation of HA in the microenvironment of a wound affects healing by promoting the deposition of a more dermal-like connective tissue matrix in the wound site. The maintenance of elevated levels of HA could have utility in the clinical setting to improve the organization of connective tissue, leading to the reduction of scar complications.

Dexamethasone abrogates the fibrogenic effect of transforming growth factor-beta in rat granuloma and granulation tissue fibroblasts.

Administration of TGF-beta, a fibrogenic inflammatory growth factor, promotes fibrosis and scarring. Dexamethasone, an anti-inflammatory steroid, inhibits wound healing and reduces fibrosis. The current studies were initiated to determine whether the co-administration of dexamethasone was able to abrogate the fibrogenic effect of TGF-beta. Polyvinyl alcohol sponges were implanted subcutaneously on the abdominal area of rats and directly injected with vehicle, dexamethasone, TGF-beta, or dexamethasone plus TGF-beta. Dexamethasone was able to block the fibrogenic effect of TGF-beta. Collagen and noncollagen protein synthesis was measured as a function of TGF-beta or dexamethasone concentrations in fibroblasts isolated from granulation tissue. Addition of dexamethasone to cultures treated simultaneously with TGF-beta blocked the fibrogenic response of TGF-beta. To study the molecular regulation of collagen gene expression by TGF-beta or dexamethasone, fibroblasts derived from granulation tissue were stably transfected with the ColCat 3.6 plasmid, which contains the rat pro alpha1(I) collagen promoter linked to the chloramphenicol acetyltransferase (CAT) gene. Dexamethasone decreased CAT activity whereas TGF-beta increased the activity of this reporter gene. The increase in CAT activity observed with TGF-beta treatment was significantly decreased when dexamethasone was added to the cultures, although CAT activity did not return to control level. Since collagen synthesis in fibroblasts treated simultaneously with dexamethasone and TGF-beta1 was found to be the same as that of untreated samples, the data indicate that there is a dexamethasone-mediated posttranscriptional regulation of pro alpha1(I) collagen mRNA. These studies demonstrate that at the in vivo level, the cellular level, and the molecular level, dexamethasone is able to block the fibrogenic effect of TGF-beta.

A simple method to assess the relative amount of collagen deposition in wounded fetal mouse limbs.

A modification of the Sirius red and fast green dye staining technique which binds selectively to collagen and noncollagenous proteins, respectively, has been used to quantify the amount of collagen deposition occurring in wounded fetal mouse limbs. Wounded day 14 and 18 fetal mouse limbs were grown in serum-free organ culture for 1 to 7 days, fixed in formalin, embedded in paraffin, and sectioned. The sections were stained with Sirius red and fast green dyes, and those sections obtained from either wounded or unwounded tissue were identified microscopically. The sections were then scraped off microscopic slides. Dye bound to the tissue sections was then eluted with a mixture of sodium hydroxide and methanol, and the optical densities were determined spectrophotometrically. There was a 98.5% correlation between the absorbance of Sirius red dye (collagen) and fast green dye (noncollagenous proteins) of eluted stain and hydroxyproline and leucine content, respectively, as determined by high-performance liquid chromatography. In addition, there was a greater collagen/protein ratio in wounded compared with unwounded sections of day 18 limbs at 7 days after wounding (p = 0.005). However, no difference in the collagen/protein ratio was detected between wounded and unwounded regions of day 14 limbs at either day 1 or 7 after wounding. These results are consistent with previous histologic observations indicating greater collagen deposition in wounded regions of day 18 compared with day 14 limbs at 7 days after wounding. With the use of this technique, it is now possible to quantify the effects of putative fibrogenic agents on collagen deposition in wounded embryonic tissue.

The role of transforming growth factor-beta in the conversion from "scarless" healing to healing with scar formation.

The objective of this study was to elucidate mediators responsible for conversion of "scarless" wound healing seen in wounded, day 14 fetal mouse limbs to healing with scar formation seen in wounded, day 18 fetal mouse limbs. Wounded, day 14 limbs were grown in a serum-free organ culture system in which either phosphate-buffered saline solution or human recombinant transforming growth factor beta-1 (1 microg/ml) was added daily. Wounded, day 18 limbs were also maintained in the same organ culture system with either phosphate-buffered saline solution or neutralizing antibody to transforming growth factor-beta (1 microg/ml) treatment. Limb cross sections were examined qualitatively with Masson's Trichrome stain and quantitatively by spectrophotometric analysis of Sirius Red and Fast Green dyes which bind to collagen and noncollagenous protein, respectively. Both qualitative and quantitative analyses showed the following: there was greater collagen deposition in day 18 versus day 14 limbs by 7 days after wounding, scar formation in day 18 limbs was attenuated by the addition of anti-transforming growth factor-beta, and there was the addition of transforming growth factor-beta-augmented collagenous scar formation in wounded regions of day 14 limbs. These results strongly suggest that transforming growth factor-beta present in the local wound environment is, at least in part, responsible for the conversion of "scarless" healing occurring in wounded, day 14 limbs to scar formation present in wounded, day 18 limbs.

Regional cerebral and neural lobe blood flow during insulin-induced hypoglycemia in unanesthetized rats.

The effects of hypoglycemia on regional cerebral blood flow (rCBF) were studied in awake restrained rats. The rats were divided into three groups consisting of a normoglycemic control group that received only saline, a hypoglycemic group A, which was given insulin 30 min before flow was measured, and a hypoglycemic group B, which was given insulin 90 and 30 min before flow was measured. Regional CBF was measured using 14C-iodoantipyrine. Mean plasma glucose was 8.76 mumol/ml in the control group, 2.63 mumol/ml in hypoglycemic group A, and 1.51 mumol/ml in hypoglycemic group B. Plasma epinephrine and norepinephrine concentrations increased to approximately 375% and 160%, respectively, of control values in hypoglycemic groups A and B. In the hypoglycemic group A, rCBF significantly increased in three brain regions. In the hypoglycemic group B, rCBF increased significantly in all brain regions measured, with the exception of the neural lobe, in which it decreased. The increase in rCBF ranged from 38% in the hypothalamus to 138% in the thalamus. Neural lobe blood flow significantly decreased by 31%. The neural lobe was the only brain region studied that is not protected by a blood-brain barrier. It may be sensitive to changes in the concentration of vasoactive agents in blood, such as epinephrine and norepinephrine.

Regional cerebral glucose utilization during insulin-induced hypoglycemia in unanesthetized rats.

Regional cerebral glucose utilization (rCMRgl) was studied during insulin-induced hypoglycemia in unanesthetized rats. Rats were surgically prepared using halothane and nitrous oxide anesthesia and allowed 5 h to recover from the anesthesia before rCMRgl was measured. The rCMRgl was measured using [6-14C]glucose in a normoglycemic control group and two hypoglycemic groups, A (30 min after insulin injection) and B (2 h after insulin injection). The mean plasma glucose level was 7.03 mumol/ml in the normoglycemic group, 1.96 mumol/ml in hypoglycemic group A, and 1.40 mumol/ml in hypoglycemic group B. The rCMRgl in hypoglycemic group A decreased 8-18% in 17 brain regions measured; five changes were statistically significant. The rCMRgl in hypoglycemic group B decreased significantly in all but one of the brain regions measured; the decrease ranged from 15% in the pyramidal tract to 36% in the motor and auditory cortices. The rCMRgl in every brain region decreased when the plasma glucose level fell below 1.5-2.5 mumol/ml. No brain region could maintain rCMRgl at plasma glucose concentrations lower than predicted by regional glucose influx described in previous studies. Glucose utilization in all brain regions appears to be limited by the influx of glucose.