Manabu Fukasawa

The mitogenic activity of peritoneal tissue repair cells: control by growth factors.

The purpose of this study was to determine the proliferative activity of tissue repair fibroblasts recovered directly from injured peritoneum at various times after surgery and to test the mitogenic response of tissue repair cells (TRC) to growth factors. Rabbits underwent bilateral peritoneal abrasion (5 X 5 cm) with sterile gauze until punctate bleeding developed. Postsurgical (Days 2, 5, 7, and 10) tissue repair cells were recovered from the injured peritoneum by scraping with a scalpel blade. Although tissue repair cells consisted of a mixed cell type after 4 days in culture, recovered cells were essentially fibroblasts. These TRC were then pulsed with [3H]thymidine after 4 days in culture. The incorporation of thymidine into Postsurgical Day 5 TRC increased significantly compared to that of Day 2 TRC (P less than 0.05). Incorporation then decreased with time following surgery. Fibroblast growth factor (FGF) and epidermal growth factor (EGF) stimulated the incorporation of thymidine into TRC. However, the response of Postsurgical Day 7 and 10 TRCs to 1 microgram/ml EGF was significantly greater than those of Postsurgical Day 2 and 5 TRCs (Day 2 TRC, 166 +/- 7.4; Day 10 TRC, 420 +/- 96% of control cells without EGF, P less than 0.05). Platelet-derived growth factor (PDGF, 10 ng/ml) also stimulated the incorporation of thymidine into Day 10 TRCs, but this stimulatory activity (129.9 +/- 8.5% of control) was less than EGF or FGF. IL-1 alpha and IL-2 did not stimulate the incorporation of thymidine into TRC at a concentration of 100 pg/ml, but these cytokines did stimulate protein synthesis by TRC.(ABSTRACT TRUNCATED AT 250 WORDS)

The hemostatic effect of deacetylated chitin membrane on peritoneal injury in rabbit model

In this study, we determined the effect of 80% deacetylated chitin (DAC-80) membrane on postsurgical bleeding after visceral and parietal peritoneal abrasion. Japanese white rabbits underwent a midline laparotomy followed either by a bilateral peritoneal sidewall abrasion (4×4 cm) or an abrasion of liver surface (3×2 cm). The injured surface was then covered with a 0.2 mm thick DAC-80 membrane. On postsurgical day 2, the rabbits were sacrificed and the amounts of postsurgical bleeding was determined by quantitating the number of red blood cells recovered in 50 ml peritoneal lavage fluid. The DAC-80 membrane was found to reduce postsurgical bleeding after the abrasion of liver surface (treated with DAC-80 membrane: 2.9±0.8; control: 24.6±5.9×108 cells/peritoneal cavity, P<0.005). This same hemostatic activity was not observed after application in the peritoneal sidewall abrasion model. We also measured plasminogen activator activity (PA) and urokinase inhibitory (PAI) activity in the spent culture media of macrophages recovered from the postsurgical peritoneal exudate. The DAC-80 membrane reduced the PA secretion from postsurgical macrophages after liver surface abrasion (treated with DAC-80: 2.8±0.7; control: 3.9±0.9 mPU/ml). The DAC-80 membrane also showed similar effects on PA secretion after peritoneal sidewall abrasion. No significant effects were found in the secretion of PAI by postsurgical macrophages in both surgical models.These findings suggest that the DAC-80 membrane may have hemostatic activity through the modulation of fibrinolytic activity of peritoneal exudative macrophages.

Superoxide anion production by postsurgical macrophages

In order to characterize further the metabolic activity of postsurgical macrophages, we examined their potential to produce superoxide anion (O2-) in response to phorbol myristate acetate. Rabbits underwent resection and reanastomosis of their ileum after which peritoneal exudative cells (PEC) were collected at various times after surgery. Macrophages were isolated from the PEC with a discontinuous Percoll gradient. Thereafter, O2- release by these cells in response to phorbol myristate acetate (PMA) was determined by cytochrome c reduction. Macrophages recovered on postsurgical Days 3-7 expressed three- to fourfold greater O2- release than resident (nonsurgical) macrophages (P less than 0.001). In a time-course study of O2- release, maximum release occurred at 6 hr postsurgery, dropped to half of peak levels by Day 1, increased to peak levels again on Day 4, and remained increased until Day 7. The ability of macrophages to release O2- than gradually decreased, reaching control levels by Day 13. These data suggest that resident peritoneal macrophages are rapidly primed to produce enhanced amounts of superoxide anion in response to appropriate stimuli after surgery, and that macrophages subsequently recruited into the peritoneal cavity as a result of surgical trauma may also be primed for enhanced O2- release as they differentiate into modulator macrophages for tissue repair.

A cardiac myocyte culture system as an in vitro experimental model for the evaluation of hypothermic preservation

In cardiac transplantation, the donor heart is exposed to severe hypothermic and ischemic conditions. The purpose of the present study was to evaluate the functional and biochemical effects on cardiac myocytes cultured under hypothermic conditions. Cardiac myocytes were isolated from neonatal rat ventricles and cultured for 4 days, then incubated (1.5×106 myocytes/culture flask) for 24 h in media at 4, 10, 15, 20, and 37°C. In addition, myocytes were incubated at 4°C for 6, 12, 18, 24, 36, and 48 h. After each incubation, creatine phosphokinase (CPK) and lactate dehydrogenase (LDH) were measured and the myocytes then cultured for an additional 24 h at 37°C to evaluate the recovery of the myocyte beating rate. The recovery ratio of the myocyte beating rate following 24 h of varying temperature incubations was complete for the 10, 15, 20, and 37°C groups, although it was markedly decreased in the 4°C group, at 25.1% of the control; taken as the beating rate prior to hypothermic incubation. The release of CPK and LDH in the 4°C group showed a three-fold increase compared to the other four groups, with a CPK of 147.2 mIU/flask and a LDH of 487.5 mIU/flask. The recovery of the beating rate for varying time incubations at 4°C was complete for the 6- and 12-h groups, but decreased significantly in the other four groups, being 59.0% at 18 h, 28.2% at 24 h, 16.3% at 36 h, and 0% at 48 h. The CPK and LDH levels increased gradually over 24 h, then markedly at 36 and 48 h, to 301.3 and 940.5 at 36 h, and 1143.6 and 1942.9 at 48 h, respectively. Thus, 4°C hypothermia induced myocyte injury both functionally and biochemically which increased with the incubation time.

Modulation of proline and glucosamine incorporation into tissue repair cells by peritoneal macrophages

The purpose of this study was to determine the patterns of [14C]proline and [14C]glucosamine incorporation by tissue repair cells (TRC) as modulated by postsurgical macrophages. Rabbits underwent a midline laparotomy followed by resection (2.0 cm) and reanastomosis of their ileum. Another group of rabbits underwent peritoneal wall abrasion with sterile gauze until punctate bleeding developed. Postoperative (1-28 days) exudate cells (PEC) were recovered from the peritoneal cavity after reanastomosis, and (TRC) were obtained directly from the injured peritoneal surface after abrasion. Since the postsurgical exudate was composed mainly of macrophages, we examined the effect of postsurgical macrophage-spent media on the incorporation of [14C]proline, [14C]glucosamine, and [3H]thymidine by TRC. After 7 days of culture, Postsurgical Day 7 TRC were incubated with spent media from postsurgical PEC (greater than 90% macrophages). When TRC were cultured with macrophage-spent media, the number of TRC increased significantly compared to that of fresh medium-treated controls. The incorporation of [3H]thymidine by TRC was also enhanced by macrophage-spent media. The incorporation of [14C]proline and [14C]glucosamine by TRC was also enhanced when incubated with macrophage-spent medium. However, when data were expressed on a per cell basis, incorporation of [14C]proline and [14C]glucosamine by TRC cultured with macrophage-spent media was the same or less than that by cells incubated with fresh medium. These data suggest that the increase in incorporation of glucosamine and proline into connective tissue protein by postsurgical repair cells may be directly modulated by macrophages recruited in response to surgical injury and that this increase is due to the fibroproliferative effect of postsurgical macrophages.

Production of protease inhibitors by postsurgical macrophages

The deposition and lysis of fibrin are important processes in normal peritoneal healing. Since macrophages secrete a neutral plasminogen activator, we studied the production of plasminogen-dependent, fibrinolytic activity by postsurgical macrophages. Peritoneal exudate macrophages were collected from rabbits after resection and reanastomosis of their ileum. Intracellular plasminogen activator (PA) activity of resident (nonsurgical) macrophages was 8.4 +/- 1.8 milli-Plough units (mPU)/10(6) cells. Postsurgical Day 1 macrophages had significantly less activity (0.33 +/- 0.056 mPU/10(6) cells) compared to resident cells. Thereafter, the PA activity gradually increased and reached control levels by Postsurgical Day 7. The PA activity secreted by postsurgical macrophages into serum-free medium after 48 hr of culture was also determined. Conditioned medium from macrophages collected on Postoperative Days 1-5 exhibited less PA activity than buffer controls. PA activity was detected after acid treatment of the conditioned medium to remove acid-labile inhibitors. The activities of PA in acid-treated conditioned medium increased gradually and reached nonsurgical levels by Postsurgical Day 7. In spent medium from macrophages collected on Postsurgical Days 1-3, high levels of urokinase inhibitory activities were secreted; production gradually decreased during the later postoperative period. This inhibitory activity of macrophage-conditioned medium on urokinase-like PA activity was partially diminished by acidification of the media. These results support the hypothesis that macrophages in the postsurgical exudate may play an important role in the fibrinolytic process during peritoneal wound healing, perhaps through production and secretion of plasminogen activator as well as acid-labile and resistant protease inhibitors.

Mitogenic and protein synthetic activity of tissue repair cells: control by the postsurgical macrophage.

It is well known that fibroblasts are a main source of extracellular matrix synthesis necessary for tissue repair. In addition, macrophages secrete products that are known to modulate synthesis of extracellular matrix. Accordingly, we studied the incorporation of [3H]thymidine, [3H]proline, and [35S]sulfate into macromolecules produced by fibroblasts recovered from the site of peritoneal tissue repair cultured with and without spent media from postsurgical peritoneal macrophages. Rabbits underwent resection and reanastomosis of their small intestines. Peritoneal exudative cells (PEC) were then collected on postsurgical day 5 and day 10 as well as from nonsurgical controls, separated by discontinuous Percoll gradient centrifugation, and cultured for 48 h. A second group of rabbits underwent peritoneal wall abrasion from which fibroblast tissue repair cells (TRC) were collected from the site of injury at postsurgical day 7 and maintained in culture for varying times. Incorporation of radiolabeled precursors into DNA, collagen, and sulfated proteoglycans was determined. Incorporation of [3H]thymidine and [3H]proline into untreated TRC gradually decreased with culture duration. Conversely, [35S]sulfate incorporation gradually increased during prolonged culture. Macrophage spent media increased the levels of [3H]thymidine incorporation by the TRC. [3H]Proline and [35S]sulfate incorporation into TRC were also stimulated by macrophage spent media. However, this stimulation may be due to the enhanced proliferation of TRC by macrophage spent media. In conclusion, tissue repair fibroblasts are activated for postsurgical repair at the site of injury by many factors including secretory products from postsurgical macrophages.

Incorporation of thymidine by fibroblasts: evidence for complex regulation by postsurgical macrophages.

Macrophages and fibroblasts are major components of the postsurgical repair process. In order to understand more fully the interaction between these two cell types, we studied the modulation by macrophages of the incorporation of [3H]thymidine into postsurgical fibroblasts recovered from the site of peritoneal injury. Peritoneal exudate cells (PEC):(greater than 95% macrophages) were collected from rabbits 4 and 7 days after resection and reanastomosis of the small intestine. PEC were suspended in Medium 199 (M-199) with 3% fetal calf serum (FCS) and incubated for 48 hr. Fibroblasts were obtained from rabbits that underwent abrasion of the parietal peritoneum 7 days previously, and were cultured for 7 days in M-199 with 3% FCS. Fibroblasts were then replated and incubated with macrophage-spent medium. Incorporation of [3H]thymidine into fibroblasts was significantly suppressed after 24 hr of incubation with macrophage-spent media compared to the incorporation by fibroblasts incubated with fresh medium (control). This suppression was most profound when fibroblasts were incubated with resident (nonsurgical) macrophage-spent medium. The incorporation of thymidine by macrophage-spent media groups then increased rapidly and reached control levels at 48 hr of incubation. After 54 hr of incubation, the incorporation of thymidine by fibroblasts incubated with media from postsurgical macrophages was significantly higher than that of control. Morphological changes in fibroblasts also appeared as the culture with macrophage-spent media progressed. Initially, fibroblasts were shaped like pine needles, but after 7 days of culture, fibroblasts assumed a spherical shape. Round-shaped fibroblasts returned to the original morphology (pine needle shape) after incubation for 48 hr with macrophage-spent medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of proliferation of peritoneal tissue repair cells by peritoneal macrophages

Macrophages produce soluble mediators which modulate fibroblast growth during tissue repair. Interaction between tissue repair fibroblasts (TRC) and regulatory proteins from surgically elicited macrophages is important for peritoneal reepithelialization. In this study, we compared the effects of an extract from postsurgical macrophage spent medium with those of known growth factors on TRC collected from injured peritoneum to evaluate certain characteristics of macrophage secretory products on peritoneal healing. Rabbits underwent a midline laparotomy followed by resection and reanastomosis of the ileum or abrasion of the abdominal wall. TRC were then collected at various times after surgery. Peritoneal macrophages recovered from nonsurgical or postsurgical rabbits were cultured for 2 days in vitro. The peak of thymidine incorporation by TRC occurred on day 5 after surgery; this gradually decreased with extended postsurgical times. Fibroblast growth factor and epidermal growth factor stimulated, whereas TGF-beta inhibited, [3H]thymidine incorporation into TRC. Maximal thymidine incorporation occurred when TRC from Postsurgical Day 5 were cultured with an extract from postsurgical macrophage spent medium. However, when TRC recovered from Postsurgical Days 2 and 10 were cultured with an extract of postsurgical macrophage spent medium, they showed greater stimulation than Day 5 TRC. These data suggest that postsurgical macrophages may produce an array of factors that stimulate fibroblast growth and differentiation and may in turn affect tissue repair throughout the wound healing process.

A clinical study of postoperative infections following open-heart surgery: occurrence and microbiological findings in 782 cases.

A total 782 consecutive patients underwent open-heart surgery with CPB between January, 1979 and December, 1988, at the Yamagata University Hospital. We assessed the incidence of postoperative infections in relation to age, the duration of surgery and antibiotic prophylaxis, and examined the causative organisms, after which the types of infecting flora were compared between the 1st period, from 1979 to 1983 and the 2nd period, from 1984 to 1988.Postoperative infection occurred in 104 of the 782 patients (13.3 per cent); in the form of a wound infection in 41 (5.2 per cent), pneumonia in 33 (4.2 per cent), urinary tract infection in 9 (1.2 per cent), prosthetic valve endocarditis in 6 (0.8 per cent), and other infections in 15 (1.9 per cent). Patients aged under 12 months or over 60 years showed a higher incidence of infection, being 17.4 per cent and 19.2 per cent, respectively. Patients who underwent an operation of over 8 hours duration also had a significantly higher incidence compared to those whose operation time was less than 4 hours, being 32.9 per cent and 6.3 per cent, respectively (p<0.0001). There was no significant difference in the incidence of postoperative infection between patients given or not given preoperative prophylaxis. A total 123 species of organisms were isolated from the 104 patients, 52.8 per cent being gram-negative bacteria (GNB), and 43.9 per cent grampositive bacteria (GPB), and a remarkable increase in the incidence of GPB was seen in the 2nd period compared to the 1st period from 31.7 per cent to 50.0 per cent.There has been a recent increase in the number of high risk patients compromised by the severity of an underlying disease. Thus, to control infection, the surgical environment and aseptic technique seem more important than antibiotic prophylaxis.