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The mitotic activity of blood monocytes cultured in autologous wound fluid.

Wound fluid was obtained from the subcutaneous tissue of sheep by implanting a perforated Teflon sampling chamber. A catheter from this chamber was brought to the surface. The types and numbers of cells in this fluid were recorded. Cell free fluid was used at either 100% concentration, or 25% concentration in plasma, as a medium for the culture of autologous blood monocytes. Its ability to support mitosis was compared with that of 100% autologous plasma. Wound fluids taken upon consecutive post-implantation days were compared. Cultured monocytes survived in small numbers in wound fluid collected on the days immediately following operation. When mitosis occurred the mitotic indices remained low. The wound fluid did not exhibit that mitogenicity which is apparent in autologous plasma taken on the third or fourth days following implantation. The results suggest that, following the implantation of Teflon, the wound fluid contains a component which is toxic for blood monocytes cultured upon glass.

Animals

Antimicrobial systems of the surgical wound. II. Detection of antimicrobial protein in cell-free wound fluid.

Human wound fluid contains heat-stable proteins with moderate antibacterial activity against Staphylococcus aureus and Escherichia coli, and different heat-labile proteins with antibacterial activity against E coli. Blood serum also contains heat-labile antibacterial substances, but little heat-stable activity against Staphylococcus aureus. Both blood serum and wound fluid have bacteriostatic activity against S epidermidis, and early growth of Streptococcus fecalis occurs in serum and wound fluids. The concentration or activity of antimicrobial proteins increases during the first week in the fresh wound and then decreases as the wound matures.

Blood Bactericidal Activity

Antibiotic concentration in human wound fluid after intravenous administration.

Since the wound is the most common focus of infection in the surgical patient, adequate levels of antibiotic within the wound ar essential. This study examines the concentrations of antibiotic achieved in human wounds. Fluid was collected at timed intervals on the first postoperative day from the wounds of 56 patients receiving antibiotics after regional lymph node dissection. Antibiotic concentration was determined by bioassay. Six antibiotics were studied: cephalothin, cefazolin, cephapirin, oxacillin, ampicillin and clindamycin. The cephalosporins and penicillins showed similar patterns of appearance in the wound fluid. The peak level occurred early (1--1 1/2 hours) with subsequent slow decrease. Clindamycin produced nearly constant levels in wound fluid. The concentration of each antibiotic in wound fluid surpassed the serum levels after 2.5 hours. At the dosages studied each antibiotic produced wound fluid concentrations greater than the MIC for most susceptible organisms. Higher doses provided higher wound fluid levels. The rate of appearance and the levels achieved should be considered in the choice of antibiotics in the surgical subject.

Anti-Bacterial Agents

[Antibiotic concentration in postoperative wound fluid during short-term prevention with cephalosporins].

Antibiotic concentrations were measured in wound fluid from Redon-drains and in serum during short-time prophylaxis in orthopaedic surgery. 1. Chephaloridine and cephazolin concentrations in wound fluid rose to therapeutic levels. 3 h after i.v. application of 1 g concentrations in wound fluid and in serum were nearly identical. 6 and 12 h after application the concentrations in wound fluid were significantly higher than those in the serum. 2. At the end of the 12-h interval of administration the mean concentrations of both antibiotics as well in wound fluid as in serum remained at therapeutic levels. 3. For reasons of their good diffusion into the wounds both cephaloridine and cefazolin should be appropriate for short-time prophylaxis in orthopaedic surgery.

Aged

Prevention of wound infections. A case for closed suction drainage to remove wound fluids deficient in opsonic proteins.

Fluids collecting in surgical wounds in both dogs and man have been shown to lose progressively the ability to opsonize bacteria for phagocytosis and killing of bacteria by normal neutrophils. Since the collection of fluids in potentially contaminated wounds also interferes with access of phagocytic cells to contaminating bacteria and provides a pablum for growth, their removal seems to be indicated to minimize the risk of infection. This can be accomplished easily and safely with the use of closed suction drainage as demonstrated in 100 patients undergoing bilateral nephrectomy, splenectomy, and renal transplantation.

Animals

Effect of a deproteinized blood extract on experimental granulation tissue.

The present work was undertaken to study the effects of a deproteinized extract of calves' blood (Solcoseryl) on developing granulation tissue in rats. Cylindrical hollow viscose cellulose sponge implants were used as an inductive matrix for the growth of granulation tissue. In the first, sham group the implants were treated daily by withdrawing 1 ml of wound fluid from the central dead space of the implant and then re-injecting the fluid. In the second, experimental group the aspirated wound fluid was replaced by a corresponding volume or Solcoseryl. Analyses of wound fluid and granulation tissue were carried out 4, 10 and 21 days after implantation. A statistically significant increase of granulation tissue hemoglobin (+21%) was observed at 10 days in the Solcoseryl group as compared with the sham-treated rats, indicating an enhanced capillary ingrowth. Concurrently, the mean amount of DNA in the Solcoseryl-treated tissues was elevated by 48% over the level of the sham-treated group, demonstrating an augmented cellularity of granulation tissue. At 21 days the mean amount of collagen hydroxyproline of the Solcoseryl group was 31% above the level measured in the sham-treated animals. PO2, PCO2 and pH in the wound fluid and the amounts of RNA and uronic acids showed no essential differences between the two groups. These data demonstrate a stimulatory effect of Solcoseryl on several aspects of granulation tissue formation: augmented vascularization, elevated cellularity and subsequent enhancement in the accumulation of collagen.

Animals

Quantitation of local acidosis and hypoxia produced by infection.

Increased strength of healing incisions infected with E coli was demonstrated in this experiment. Efforts to measure respiratory gas tensions and pH in these incisions were unsuccessful. Therefore, these moieties were measured in normal and infected wound fluid contained in implanted wire cylinders. The wound fluid from infected cylinders was consistently more acidotic and had a lower pO2 and a higher pCO2 than fluid from unifected wound cylinders.

Animals

Prostaglandins and the sources of their production in a mild inflammatory lesion in sheep.

1. Wound fluid was collected from a subcutaneous lesion in the shoulder region of sheep by implanting a PTFE sampling chamber from which a catheter led to the skin surface. 2. The neutrophil granulocyte numbers and prostaglandin content of the wound fluid were studied, prostaglandins being identified by thin layer chromatography and measured by bio-assay. The major prostaglandin component was E2. 3. There was no clear relation between the amounts of prostaglandin present and the numbers of neutrophils in the fluid. 4. A subcutaneously implanted double chamber permitted perfusion of the lesion and thereby the study of the rate of prostaglandin production and the rate of disappearance of isotopically labelled prostaglandin from the lesion. The rate of leucocyte entry into the wound fluid could also be measured. 5. When the cutaneous muscle through which the chamber had been implanted was biopsied and incubated in vitro it was found to produce prostaglandin E2 in quantities which could be correlated with the weight of the incubated muscle. 6. These results suggest that a major contributor to the prostaglandin content of the wound fluid is the cutaneous striped muscle.

Animals

Local hyperalimentation of experimental granulation tissue.

The effect of local hyperalimentation on developing granulation tissue was studied in rats. Cylindrical hollow viscose cellulose sponge implants were used subcutaneously as an inductive matrix fro the growth of granulation tissue. In the first, control group the implants were kept untouched while the second, "sham" group was treated daily by withdrawing 1 ml of wound fluid from the central dead space of the implant and then injecting the fluid back. In the third, hyperalimentation group the aspirated wound fluid was substituted with a corresponding volume of sterile, nonpyrogenic solution containing a mixture of amino acids (Le-7402 A) and glucose, electrolytes and vitamins (Le-7402 B). Within the first week of tissue growth daily application of these nutritional substances caused a changeover of local tissue from predominantly anaerobic towards more oxidative metabolism. Measurement of nucleic acid and hydroxyproline contents indicated enhanced accumulation of cells and collagen in tissues receiving local hyperalimentation. The results combined with earlier data from our laboratory strongly suggest that several types of wounds, especially those containing a marked dead space or large regenerative area, exist in chronic lack of oxygen and other nutrients. Therefore, the healing process in these wounds can be stimulated, to a certain extent, by exposure to increased oxygen tension and/or by local hyperalimentation.

Amino Acids

High-Throughput and High-Sensitivity Biomarker Monitoring in Body Fluid by Fast LC SureQuant IS-Targeted Quantitation.

Targeted proteomics methods have been greatly improved and refined over the last decade and are becoming increasingly the method of choice in protein and peptide quantitative assays. Despite the tremendous progress, targeted proteomics assays still suffer from inadequate sensitivity for lower abundant proteins and throughput, especially in complex biological samples. These attributes are essential for establishing targeted proteomics methods at the forefront of clinical use. Here, we report an assay utilizing the SureQuant internal standard-triggered targeted method on a latest generation mass spectrometer coupled with an EvoSep One liquid chromatography platform, which displays high sensitivity and a high throughput of 100 samples per day. We demonstrate the robustness of this method by quantifying proteins spanning six orders of magnitude in human wound fluid exudates, a biological fluid that exhibits sample complexity and composition similar to plasma. Among the targets quantified were low-abundance proteins such at tumor necrosis factor A and interleukin 1-β, highlighting the value of this method in the quantification of trace amounts of invaluable biomarkers that were until recently hardly accessible by targeted proteomics methods. Taken together, this method extends the toolkit of targeted proteomics assays and will help to drive forward mass spectrometry-based proteomics biomarker quantification.

Humans

The effect of differing ambient oxygen tensions on wound infection.

Wound infections were studied in rabbits using two standard inocula (approximately equal to 10-4 and approximately equal to 10-6) of Pseudomonas aeruginosa injected into subcutaneous wound dead space made by implantation of standard wire mesh cylinders. The inoculation was done on the fourth day after implantation of the cylinders in animals kept from the day of implantation in atmospheres of 12%, 21%, or 45% oxygen content. Samples of wound fluid (0.2 ml) were removed for quantitative culture just before inoculation and 3, 7, 14, and 21 days later. No positive cultures resulted from samples taken before inoculation. One uninoculated wound served as a control in each animal. None of these control wounds became infected. Culture counts were significantly highest in the anoxic group and lowest in the hyperoxic group. Established infections were significantly lowest in the hyperoxics and highest in the hypoxics. The percent of wounds showing a significant culture count showed a similar trend. The mechanisms of this effect is not known, but a possible mechanism lies in the relative inability of leucocytes to kill this bacterium under hypoxic conditions.

Animals

Anaerobic metabolism and wound healing: an hypothesis for the initiation and cessation of collagen synthesis in wounds.

The implanted cylinder model was used to measure LDH activity and lactate and pyruvate concentrations in the extracellular fluid of wounds and wound tissue. Total LDH activity corresponded directly to lactic acid concentration and inversely to oxygen availability. LDH isoenzymes in wound fluid were in an anaerobic pattern soon after injury and evolved toward the aerotic pattern as oxygen supply more nearly matched metabolic capacity. Lactate levels in the wound space are elevated soon after wounding and remain elevated far above those in blood. These data again indicate that wound metabolism is characterized by a relatively poor oxygen supply. Current data from several sources indicate that lactate found in the hypoxic area of the wound may stimulate collagen synthesis in fibroblasts lying in the high lactate environment. We postulate that elevated concentration of lactate in wounds is a major signal for collagen synthesis and repair.

Anaerobiosis

Mechanism of obstruction of closed-wound suction tubing.

The mechanism of obstruction of closed-wound drainage tubing was investigated by means of coagulation tests performed on wound drainage fluid and by examination of the contents of the tubes after their removal. Although clotting is commonly thought to be responsible for the obstruction, the wound drainage fluid was found to be essentially incoagulable and little fibrin was evident within the tubes. By contrast, bits of tissue were frequently found within the tubes, and these frequently virtually occluded the lumen. This observation, that tissue fragments are responsible for tube obstruction, permits a rational approach to the solution of this problem. For example, meticulous wound flushing and irrigation, or perhaps tubing of different design, might lead to a reduced incidence of tube failure.

Blood Vessels

The importance of C5 and the role of the alternative complement pathway in leukocyte chemotaxis induced in vivo and in vitro by Bacteroides fragilis lipopolysaccharide.

Chambers implanted subcutaneously in C5 normal (C5 N) and C5 deficient (C5 D) mice were used to examine the migration of polymorphonuclear leukocytes (PMNs) into the wound chamber fluid in response to injected Bacteroides fragilis lipopolysaccharide (LPS). The difference in PMN migration was highly significant between the two mouse strains, the C5 D mice showing no initial, but a low, delayed migration. The results from the study indicated that chemotaxis plays a major role in the accumulation of PMNs in the acute inflammatory response. Intraperitoneal endotoxin stimulation also showed a significantly lower total number of leukocytes in the exudate from C5 D mice as well as a delayed migration of cells compared to C5 N mice. No leukotactic mediators were elaborated in C5 D serum or exudate upon incubation with LPS when tested in a modified Boyden chamber. However, endotoxin-induced wound chamber fluid in C5 D mice showed an increasing leukotactic activity at the same time as the acute inflammatory response subsided in C5 N mice. Incubation of B. fragilis LPS in C4 deficient (C4 D) guinea pig serum indicated that the LPS was able to activate complement components to generated split products chemotacic for rabbit PMNs via the alternative complement pathway.

Animals

Comparison between moist swab and tissue biopsy methods for quantitation of bacteria in experimental incisional wounds.

The relationship between tissue-associated and wound fluid-associated levels of bacteria in closed, healing experimental incisional wounds seeded with Escherichia coli was examined in 200 rats. Tissue specimens and moist swab specimens were taken simultaneously and cultured by a single plate serial dilution method. Colony counts of approximately 10(5) bacteria/gm of tissue were found to be equivalent to colony counts of 10(3) bacteria/ml of specimen obtained on a moist swab. Moist swab sampling of an incisional wound therefore offers a direct and simple method of ascertaining infection (defined by others as greater than 10(5) bacteria/gm of tissue). Moist swab sampling obviates the necessity of surgical manipulation of the patient and subsequent weighing and grinding of tissue in the laboratory.

Animals