Reversibility of abdominal wall atrophy and fibrosis after primary or mesh herniorrhaphy.

OBJECTIVE: To determine whether primary or mesh herniorrhaphy reverses abdominal wall atrophy and fibrosis associated with hernia formation. BACKGROUND: We previously demonstrated that hernia formation is associated with abdominal wall atrophy and fibrosis after 5 weeks in an animal model. METHODS: A rat model of chronic incisional hernia was used. Groups consisted of uninjured control (UC, n = 8), sham repair (SR, n = 8), unrepaired hernia (UR, n = 8), and 2 repair groups: primary repair (PR, n = 8) or tension-free polypropylene mesh repair (MR, n = 8) hernia repair on postoperative day (POD) 35. All rats were killed on POD 70. Intact abdominal wall strips were cut perpendicular to the wound for tensiometric analysis. Internal oblique muscles were harvested for fiber type and size determination. RESULTS: No hernia recurrences occurred after PR or MR. Unrepaired abdominal walls significantly demonstrated greater stiffness, increased breaking and tensile strengths, yield load and yield energy, a shift to increased type IIa muscle fibers than SR (15.9% vs 9.13%; P < 0.001), and smaller fiber cross-sectional area (CSA, 1792 vs 2669 µm(2); P < 0.001). PR failed to reverse any mechanical changes but partially restored type IIa fiber (12.9% vs 9.13% SR; P < 0.001 vs 15.9% UR; P < 0.01) and CSA (2354 vs 2669 µm(2) SR; P < 0.001 vs 1792 µm(2) UR; P < 0.001). Mesh-repaired abdominal walls demonstrated a trend toward an intermediate mechanical phenotype but fully restored type IIa muscle fiber (9.19% vs 9.13% SR; P > 0.05 vs 15.9% UR; P < 0.001) and nearly restored CSA (2530 vs 2669 µm(2) SR; P < 0.05 vs 1792 µm(2) UR; P < 0.001). CONCLUSIONS: Mesh herniorrhaphy more completely reverses atrophic abdominal wall changes than primary herniorrhaphy, despite failing to restore normal anatomic muscle position. Techniques for hernia repair and mesh design should take into account abdominal wall muscle length and tension relationships and total abdominal wall compliance.

Early laparotomy wound failure as the mechanism for incisional hernia formation.

BACKGROUND: Incisional hernia is the most common complication of abdominal surgery leading to reoperation. In the United States, 200,000 incisional hernia repairs are performed annually, often with significant morbidity. Obesity is increasing the risk of laparotomy wound failure. METHODS: We used a validated animal model of incisional hernia formation. We intentionally induced laparotomy wound failure in otherwise normal adult, male Sprague-Dawley rats. Radio-opaque, metal surgical clips served as markers for the use of x-ray images to follow the progress of laparotomy wound failure. We confirmed radiographic findings of the time course for mechanical laparotomy wound failure by necropsy. RESULTS: Noninvasive radiographic imaging predicts early laparotomy wound failure and incisional hernia formation. We confirmed both transverse and craniocaudad migration of radio-opaque markers at necropsy after 28 d that was uniformly associated with the clinical development of incisional hernias. CONCLUSIONS: Early laparotomy wound failure is a primary mechanism for incisional hernia formation. A noninvasive radiographic method for studying laparotomy wound healing may help design clinical trials to prevent and treat this common general surgical complication.

Loss of mechanical strain impairs abdominal wall fibroblast proliferation, orientation, and collagen contraction function.

BACKGROUND: Laparotomy wound load forces are reduced when dehiscence and incisional hernia formation occur. The purpose of this study was to determine the effects of strain loss on abdominal fascial fibroblast proliferation, orientation, and collagen compaction function. METHODS: Cultured rat linea alba fibroblasts were subjected to continuous cyclic strain (CS), CS interrupted at 24 or 48 hours followed by culture at rest (IS-24 and IS-48) or were cultured without mechanical strain (NS). Cell number was measured and images analyzed for cell orientation. Fibroblasts from these groups were seeded onto the surface of (FPCL-S) or mixed into (FPCL-M) a collagen gel matrix and gel area was measured over time. RESULTS: Continuous strain stimulated proliferation when compared with the nonstrained cells. The loss of strain (IS) delayed proliferation compared with CS throughout (P < .05). CS fibroblasts aligned perpendicular to the direction of strain within 12 hours. Within 12 hours of strain loss, IS-48 fibroblasts became significantly less aligned (P < .0001), and seemed similar to the randomly organized NS fibroblasts 48 hours after strain removal. The CS and IS-24 groups demonstrated faster and greater overall FPCL-M compaction than both the IS-48 and NS groups (P < .0002). The CS group contracted the gel faster than the NS group in FPCL-S (P = .029). CONCLUSION: Mechanical strain rapidly induces a proliferative, morphologic, and functional response in abdominal wall fibroblasts that is dependent on the continued presence of the strain signal and quickly lost when the load force is removed. The loss of wound edge tension that occurs during laparotomy wound separation and hernia formation may contribute to impaired wound healing through loss of a key stimulatory mechanical signal with important implications for abdominal wall reconstruction.

Impaired laparotomy wound healing in obese rats.

BACKGROUND: Obesity increases the risk of laparotomy dehiscence and incisional hernia. The aim of this study was to measure the biological effect of obesity on laparotomy wound healing and the formation of incisional hernias. METHODS: Normal-weight Sprague-Dawley (SD) and obese Zucker rats were used in an established laparotomy wound healing and incisional ventral hernia model. Mechanical testing was performed on abdominal wall strips collected from laparotomy wounds. Hernia size was measured by digital imaging. Picrosirius staining for collagen isoforms was observed with polarized microscopy. Abdominal wall fibroblasts were cultured to measure collagen matrix remodeling and proliferation. RESULTS: Laparotomy wound healing was significantly impaired in obese rats. Mechanical strength was lower than in normal-weight rats. Yield load was reduced in the obese group at all time points. Picrosirius red staining showed increased immature type III collagen content and disorganized type I collagen fibers within laparotomy wounds of obese rats. Wound size was significantly larger in the obese group. Collagen matrix remodeling was impaired with fibroblasts from obese rats, but there was no difference in fibroblast proliferation between the obese and normal-weight groups. CONCLUSIONS: We observed for the first time that laparotomy wound healing is impaired in obese rats. The recovery of laparotomy wound strength is delayed due to abnormal collagen maturation and remodeling, possibly due to a defect in fibroblast function. Strategies to improve outcomes for laparotomy wound healing in obese patients should include correcting the wound healing defect, possibly with growth factor or cell therapy.

The use of amnion-derived cellular cytokine solution to improve healing in acute and chronic wound models.

OBJECTIVE: Growth factors demonstrate mixed results improving wound healing. Amnion-derived multipotent cells release physiologic levels of growth factors and tissue inhibitors of metalloproteinases. This solution was tested in models of acute and chronic wound healing. METHODS: Acute model: Sprague-Dawley rats underwent laparotomy incisions. The midline fascia was primed with phosphate-buffered saline, unconditioned media, or amnion-derived cellular cytokine suspension prior to incision. Breaking strength of laparotomy wounds was tested with an Instron tensiometer. Incisional hernia formation was measured after 28 days. Chronic model: Chronic, infected granulating wounds were produced in rats by excising full thickness burn eschars inoculated with Escherica coli. Granulating wounds were treated with unconditioned media or amnion-derived cellular cytokine suspension. Treatments were applied either on day 0 and day 7 or day 0 and then every other day. Wounds were traced every 72 hours and biopsied for quantitative bacteriology. RESULTS: Acute model: Priming with amnion-derived cellular cytokine suspension increased the breaking strength of laparotomy incisions in comparison with phosphate-buffered saline or unconditioned media (P < .05). Acute wound failure and incisional hernia formation was 100% in the phosphate-buffered saline and unconditioned media groups and 18% in the amnion-derived cellular cytokine suspension-treated group (P <.05). Chronic model: The rate of wound closure was accelerated in amnion-derived cellular cytokine suspension-treated chronic wounds (P < .05). Multidosing improved the effect. CONCLUSIONS: A physiologic solution of cytokines and tissue inhibitors of metalloproteinases improves healing in models of acute and chronic wounds. Such a cocktail can be produced from amnion-derived multipotent progenitor cells.

Amnion-derived multipotent progenitor cells increase gain of incisional breaking strength and decrease incidence and severity of acute wound failure.

OBJECTIVE: Acute wound failure is a common complication following surgical procedures and trauma. Laparotomy wound failure leads to abdominal dehiscence and incisional hernia formation. Delayed recovery of wound-breaking strength is one mechanism for laparotomy wound failure. Early fascial wounds are relatively acellular, and there is a delay in the appearance of acute wound growth factors and cytokines. The objective of this study was to accelerate and improve laparotomy wound healing using amnion-derived multipotent cells (AMPs). AMPs' nonimmunogenic phenotype and relative abundance support its role as a cell therapy. METHODS: AMPs were injected into the load-bearing layer of rat abdominal walls prior to laparotomy, and cell viability was confirmed. Wound mechanical properties were measured over 28 days. The incidence and severity of laparotomy wound failure was measured in an incisional hernia model. RESULTS: AMP cells were viable in laparotomy wounds for at least 28 days and did not migrate to other tissues. Laparotomy wound-breaking strength was increased by postoperative day 7 following AMP therapy. AMP therapy reduced the incidence of hernia formation and the size of hernia defects. Histology suggested stimulated wound fibroplasia and angiogenesis. CONCLUSIONS: AMP cell therapy reduces the incidence of laparotomy wound failure by accelerating the recovery of wound-breaking strength. This results in fewer incisional hernias and smaller hernia defects.

Promoter elements responsible for antioxidant regulation of MCP-1 gene expression.

Monocyte chemoattractant protein-1 (MCP-1) is produced by different cells in response to inflammatory stimulation. In the present study, a series of human MCP-1 promoter reporter genes were constructed to illustrate elements involved in antioxidant dimethyl sulfoxide (DMSO) inhibition of MCP-1 gene expression. MCP-1 secretion and mRNA expression and transcription activity stimulated by TNF-alpha or IL-1beta were significantly inhibited by 1% DMSO in alveolar type II epithelial cells (A549). Deletion of -7537 to -2741 caused a 77% decrease in reporter activity, but DMSO inhibition was still present. Deletion of -7537 to -2616 containing the A1 NF-kappaB binding site resulted in a complete loss of MCP-1 stimulation. Deletion of -2585 to -74 decreased reporter activity by approximately 50%, and DMSO inhibited this induction. Deletion of -2614 to -74 containing the A2 NF-kappaB binding site completely abolished responses to stimulation. Mutations of either of the NF-kappaB binding sites decreased promoter activity, which could still be inhibited by DMSO, whereas deletion of both NF-kappaB binding sites abolished induced transcriptional activity. Mutation or deletion of the NF-kappaB binding sites significantly decreased or abolished reporter activity in response to reactive oxygen intermediates (ROI), generated by xanthine plus xanthine oxidase. In conclusion, DMSO inhibits MCP-1 gene expression through both NF-kappaB binding sites located far upstream of the 5'-flanking region of the MCP-1 promoter.

Mechanisms of oxidant regulation of monocyte chemotactic protein 1 production in human whole blood and isolated mononuclear cells.

Previous work has demonstrated that reactive oxygen intermediates (ROIs) play an important regulatory role in the induction of monocyte chemotactic protein 1 (MCP-1) in certain cells. This study investigated the mechanisms of ROI regulation of MCP-1 gene expression in whole blood and isolated peripheral blood mononuclear cells (PBMCs). The antioxidants dimethyl sulfoxide (DMSO), N-acetyl cysteine, and dimethyl thiourea significantly inhibited lipopolysaccharide (LPS)-induced MCP-1 production in either whole blood or isolated blood cells. In contrast, interleukin 6 and tumor necrosis factor production were not affected and interleukin-1beta levels were actually increased with DMSO treatment. Exogenous ROI (either hydrogen peroxide or O2 generated by xanthine/xanthine oxidase) stimulated MCP-1 production, which was also inhibited by DMSO. To confirm the biological relevance of these findings in vivo, mice treated with DMSO before LPS challenge had significantly lower plasma levels of MCP-1. The level of inhibition was addressed in experiments which demonstrated that DMSO significantly decreased MCP-1 mRNA induced by LPS in whole blood and PBMCs. Cycloheximide treatment did not abolish the DMSO inhibition of MCP-1 mRNA, demonstrating that de novo protein synthesis is not required. Treatment with actinomycin D showed that DMSO did not increase the decay rate of MCP-1 mRNA, indicating that ROI did not change the stability of MCP-1 mRNA. These results provide evidence that in whole blood and PBMCs, DMSO regulates MCP-1 gene expression by decreasing the induction of MCP-1 mRNA.

Mechanisms of dimethyl sulfoxide augmentation of IL-1 beta production.

Expression of the inflammatory cytokine IL-1beta occurs in various inflammatory diseases, and IL-1beta production is regulated at multiple levels. There are conflicting reports about the effects of antioxidants on IL-1beta production. In this study, we investigated the regulatory role of the antioxidant DMSO on LPS-stimulated IL-1beta gene expression in human PBMC and in vivo. This study demonstrated that 1% DMSO increased LPS-stimulated (50 ng/ml) IL-1beta secretion in a dose- and time-dependent manner without altering TNF or IL-6. DMSO also elevated IL-1beta secretion by PBMC in response to exogenous superoxide anions. Despite the increase in IL-1beta, there was no augmentation of NF-kappaB with the addition of DMSO. The steady state mRNA coding for IL-1beta following LPS stimulation was also increased. Cycloheximide studies demonstrated that the DMSO augmentation of IL-1beta mRNA did not require de novo protein synthesis, and studies with actinomycin D showed that DMSO did not alter the half-life of IL-1beta mRNA, suggesting that DMSO did not change the stability of IL-1beta mRNA. Experiments using a reporter vector containing the 5'-flanking region of the human IL-1beta gene revealed that DMSO augmented LPS-induced IL-1beta reporter activity. In vivo, treatment of mice with DMSO significantly increased plasma levels of IL-1beta after endotoxin challenge. These data indicate that DMSO directly increases LPS-stimulated IL-1beta protein production through the mechanisms of augmenting promoter activity and increasing mRNA levels.

Neutrophils as firemen, production of anti-inflammatory mediators by neutrophils in a mixed cell environment.

Neutrophils represent critical components of the innate immune system that bear primary responsibility for phagocytosis and killing of invading pathogens. Following stimulation of human whole blood, robust production of multiple cytokines and cytokine inhibitors occurs. We attempted to define the cell population responsible for the synthesis of different mediators by first stimulating whole blood and then isolating pure populations of granulocytes and monocytes. Monocytes produced mRNA coding for the classic pro-inflammatory cytokines tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and IL-6, while mRNA for these cytokines was not detectable in the isolated neutrophils. In contrast, neutrophils produce significant quantities of cytokine inhibitors such as the type 2 TNF soluble receptor and the IL-1 receptor antagonist. Both neutrophils and monocytes produced mRNA coding for IL-8. These data indicate that following stimulation of a mixed cell population the monocytes primarily produce pro-inflammatory mediators while the neutrophils synthesize a significant portion of the anti-inflammatory mediators. The neutrophils may be compared to firemen who bring the resources necessary to put out the flame of acute inflammation.

Relative cytokine and cytokine inhibitor production by mononuclear cells and neutrophils.

Mononuclear cells represent the major source of cytokines in blood; however, it has been postulated that neutrophils may produce and/or release modest amounts of cytokines. In this study, we compared the production of cytokines and cytokine inhibitors in lipopolysaccharide (LPS)-stimulated whole blood, peripheral blood mononuclear cells (PBMCs), and neutrophils (PMNs) separated by density gradient centrifugation. Contamination of PBMCs in the isolated PMNs was less than 0.1% as determined by morphological analysis and flow cytometry. LPS (50 ng/mL) induced a strong increase of the proinflammatory cytokines tumor necrosis factor, interleukin (IL)-1beta, IL-8, and IL-6 in whole blood. In the isolated cell preparations, proinflammatory cytokine production was significantly greater in the PBMCs compared with the PMNs. On a per cell basis, PMNs produced less than 1.5% of these cytokines compared with PBMCs. For cytokine inhibitors, TNF-soluble receptor type II and IL-1 receptor antagonist were increased by LPS stimulation in whole blood, PBMCs, and PMNs. On a per cell basis, LPS-induced TNF-soluble receptor type II and IL-1 receptor antagonist production by PMNs was 9.8% and 15.4% of those of PBMCs, respectively. These data show that a highly purified population of PMNs makes a relatively minor to nonexistent contribution to the production of proinflammatory cytokines.

Production and secretion of calcitonin gene-related peptide from human lymphocytes.

Calcitonin gene-related peptide (CGRP) is a 37-amino acid neuropeptide, which is mainly present in primary sensory nerves. Although our previous study has shown that rat lymphocytes can synthesize beta-CGRP, there is no evidence demonstrating whether CGRP can be synthesized by human lymphocytes. In this study, the production of CGRP from human lymphocytes from spleen and blood were investigated by using CGRP-specific radioimmunoassay (RIA), and RNase protection assay (RPA). The results showed that human T lymphocyte mitogen, such as phytohemagglutinin (PHA), could time- and dose-dependently induce hCGRP secretion; rhIL-2 alone did not effect hCGRP secretion, but it could potentiate PHA-evoked hCGRP secretion from human spleen lymphocytes. RPA showed that alpha- and beta-CGRP mRNA were both constitutively expressed in unstimulated human peripheral blood mononuclear cells (PBMC). PHA could cause beta-hCGRP but not alpha-hCGRP mRNA increase in a time-dependent manner. In addition, hCGRP(8-37), a CGRP(1) receptor antagonist, enhanced PHA or human interleukin-2 (rhIL-2), induced the proliferation of splenocytes and PBMC. These results suggest that hCGRP is produced and secreted by human lymphocyte. Lymphocyte mitogen can induce the elevation of beta-CGRP synthesis and secretion. The lymphocyte-derived beta-CGRP may inhibit, at least in part, lymphocytes proliferation, which are then involved in the modulation of human T lymphocyte function in response to immune stimulation.

Comparison of calcitonin gene-related peptide release from rat lymphocytes and dorsal root ganglia neurons.

Calcitonin gene-related peptide (CGRP), a neuropeptide contained in primary sensory neurons, has been demonstrated to be synthesized and released by rat lymphocytes in our previous studies. In this study, the release properties and molecular characteristics of CGRP such as immunoreactivity (CGRP-LI) from lymphocytes were compared with those from dorsal root ganglia (DRG) neurons by using CGRP-specific RIA, reverse-phase HPLC, and RT-PCR. Con A and IL-2 could trigger CGRP-LI release from lymphocytes in a time-dependent manner. After 3 days stimulation with 4 microg/ml Con A, the level of CGRP-LI released by lymphocytes was increased from 77.4 +/- 9.6 pg/10(8) cells to 191.1 +/- 13.6 pg/10(8) cells and increased further to 374.5 +/- 38.3 pg/10(8) cells after 5 days. Stimulation with 750 U/ml human IL-2 recombinant (rhIL-2) caused a significantly elevated CGRP-LI release from 75.4 +/- 6.5 pg/10(8) cells to 266.2 +/- 16.2 pg/10(8) cells after 3 days and to 469.1 +/- 43.2 pg/10(8) cells after 5 days. Con A and IL-2 also augmented CGRP mRNA expression in lymphocytes. In the tested period (1-5 days), Con A and rhIL-2 had no stimulating effect on CGRP release from DRG neurons. In contrast, a high concentration of potassium and LPS could induce an acute release of CGRP from DRG neurons, but not from lymphocytes. Lymphocyte-released CGRP-LI was shown to coelute with synthetic rat CGRP (rCGRP) and DRG neuron-released CGRP by reverse-phase HPLC. In addition, to displace (125)I-CGRP from CGRP antibody by lymphocyte-released CGRP-LI was similar to that by synthetic rCGRP. These data suggest that lymphocyte- and nerve-derived CGRP-LI are similar in terms of immunological characteristics, molecular size, and polarity. However, lymphocytes secrete CGRP-LI in response to different stimuli compared to nerve-derived CGRP.