Improved survival with splenic autotransplantation and fibronectin therapy following endotoxin administration in rats

The risk of overwhelming infections is greatly increased after splenectomy. In this experimental study in rats, we investigated whether the administration of fibronectinrich cryoprecipitate can improve the survival rate of splenectomized autotransplanted rats subjected to an intravenous challenge with endotoxin. Inbred Lewis rats were divided into four groups: A, splenectomy; B, splenectomy + splenic autotransplantation; C, splenectomy, splenic autotransplantation + fibronectin treatment, and D, sham. Five months after surgery, rats were challenged intravenously with Escherichia coli endotoxin. Immunoglobulin (IgG, IgM, IgA), complement and fibronectin levels were measured before surgery and endotoxin challenge, and 48 h after endotoxin challenge. The survival rate of splenectomized rats was not significantly improved by autotransplantation of splenic tissue, but was significantly (p less than 0.05) improved by autotransplantation and fibronectin treatment. The levels of fibronectin, immunoglobulins and/or complements were significantly decreased after endotoxin challenge in control and in autotransplanted fibronectin-treated rats. The survival improvement of autotransplanted rats treated by fibronectin is probably due to increased endotoxin phagocytosis and clearance.     

Optimal site and amount of splenic tissue for autotransplantation.

Clinical and basic studies have documented a high susceptibility to pneumococcal infection in asplenic humans and animals. It has been suggested that autotransplantation of splenic tissue might be a method of providing host resistance when total splenectomy is necessary. However, the effect of splenic autograft has remained controversial. This study was performed to evaluate the most effective site and amount of splenic autograft using rats. Rats were divided into five groups for the purpose of determining the site of splenic autotransplantation: splenectomy, sham operation, implantation into the omental pouch, intraperitoneal implantation, and intramuscular implantation. For determining the amount for autotransplantation, the rats were divided into seven groups: splenectomy, sham operation, and implantations of 25, 50, 100, 200, or 300 mg of splenic tissue. All animals were challenged with Streptococcus pneumoniae type 6, 16 weeks after surgery. Howell-Jolly bodies appeared postsplenectomy, but disappeared in the implanted rats 16 weeks after the operation. Histologically, the implanted tissue was indistinguishable from that of a normal spleen. Pneumococcal clearance from the bloodstream and survival rate were significantly higher in rats implanted in the omental pouch as compared with splenectomized rats. Intraperitoneal and intramuscular implanted rats did not show a significant difference from the splenectomized rats. More than 50% of splenic tissue for autograft showed a significant increase in pneumococcal clearance and survival rate as compared with that of splenectomized rats. It was suggested that the most effective site of autotransplantation is the omental pouch and approximately 50% of the whole spleen would be necessary for prevention from sepsis.     

Overwhelming pneumococcal infection in rats immediately after splenectomy

A rat model was used to evaluate the possibility that a nonspecific factor of splenic origin, promoting opsonization and/or antibody production, could affect the susceptibility to pneumococci after splenectomy. Streptococcus pneumoniae type 1 4 × 10³ CFU was injected intravenously in Sprague-Dawley rats. In Experiment I, two groups of previously splenectomized rats (15 at 7 weeks and 15 at 14 weeks of age) were challenged with pneumococci at the age of 15 weeks. All these rats succumbed with no difference in survival time between the two groups. In contrast, the entire control group of 10 nonsplenectomized (sham-operated) rats challenged peroperatively with pneumococci survived. In experiment II, 62 animals were divided into two equal groups. One group was splenectomized when 9 weeks old, and the other was subjected to omental resection (sham operation) at the same time. Two weeks later splenectomy was performed on previously oment-resected animals and the remaining animals were sham-operated. At the second operation all animals were challenged with pneumococci. In each group 74% died and survival times did not show any difference between the two groups. In experiment III splenectomy was performed on 37 9-week-old rats. Two weeks later 20 of these were subjected to omental resection, and in the remaining 17, intraabdominal deposition of homologous dispersed splenic tissue was carried out. Peroperatively, pneumococci were injected intravenously. No difference between the two groups as regards mortality rate or survival times was registered. These experiments revealed no factor remaining briefly after splenectomy that could affect the susceptibility to intravenous injection of Streptococcus pneumoniae type 1.     

Immune consequences of nonoperative treatment of splenic trauma in the rat model

To investigate the immunologic consequences of non-operative management of splenic injury, three parameters were studied: survival following pneumococcal sepsis, clearance of blood-borne bacteria, following Hemophilus influenzae challenge, and antibody response to type III pneumococcal capsular polysaccharide. Two hundred twenty-five Sprague-Dawley rats were divided into three groups and subjected either to a splenectomy, a sham operation, or standard blunt trauma. A significant increase in mortality was noted in the splenectomized group as compared with both the traumatized and control groups when challenged with Streptococcus pneumoniae. In both the control and trauma groups, H influenzae cleared significantly within 18 hours. Blood-borne bacteria persisted at the same level for 72 hours in the splenectomized animals. Four and 11 days later, the antibody level in both traumatized and control groups was higher than in the splenectomized subjects (P less than .001). There was no difference in the serum antibody level between the control and trauma groups at four days. However, at 11 days the trauma group showed a significant decrease in the antibody level (P less than .05). It can be concluded that following spontaneously-healing splenic trauma in the rat model, survival, bacterial clearance, and antibody response were all superior to that observed in the splenectomized subjects. In addition, the healed splenic disruption did not impair clearance of blood-borne encapsulated bacteria.     

Influence of splenectomy, partial splenectomy and splenic artery ligation on E. coli sepsis in rats

Male Sprague-Dawley rats were allocated to four groups--sham operation, partial splenectomy, splenic artery ligation or total splenectomy, and 4 weeks after the operation 3 x 10(8) colony-forming units of Escherichia coli were injected intraperitoneally. Among the splenectomized rats the mortality was significantly (p less than 0.02) increased compared with the controls, while both partial splenectomy and splenic artery ligation did not influence survival. Blood clearance and organ (liver, spleen and lungs) uptake of intravenously injected, radiolabelled, heat-killed E. coli were determined 1 hour after the intraperitoneal challenge. Splenectomy caused a significant decrease in blood clearance. Splenic uptake of radiolabelled E. coli was significantly reduced following partial splenectomy and splenic artery ligation. The splenic operations increased hepatic uptake expressed per gram tissue. Splenectomy thus resulted in reduced blood clearance and increased mortality in Gram-negative sepsis, while the reduced splenic uptake following partial splenectomy or splenic artery ligation did not influence blood clearance of E. coli or mortality.     

Splenic autotransplantation provides protection against fatal sepsis in young but not in old rats.

Splenectomy increases the risk of contracting infections with high mortality. Thus, splenic tissue should be repaired orthotopically whenever possible. If all attempts fail, splenic autotransplantation might be a suitable method for splenic salvage. The protective function of such transplants in adults has been questioned, leading to a decreased frequency of splenic autotransplantations. However, the regeneration of splenic tissue is better in the young organism than in the old, suggesting that the protection provided by regenerated splenic tissue might be more reliable in children than in adults. In addition, children are at a higher risk in the case of overwhelming postsplenectomy sepsis. The protection warranted by regenerated splenic tissue after autotransplantation at different ages was examined using a highly standardized animal model. Sham operation, splenectomy, and splenic autotransplantation were performed on adult, weanling, and newborn rats, and Streptococcus pneumoniae was applied intranasally 9 months after the operation. After pneumococcal challenge about 80% of the splenectomized animals in the different age groups died of infection, whereas only 20% of the sham operated rats died. Regenerated splenic tissue resulting from splenic autotransplantation performed on adult or weanling rats demonstrated no protective function. However, in newborn rats with transplanted splenic tissue, both survival rate and survival time were increased significantly. Determination of lymphocyte subsets in the blood did not allow the protective role of splenic transplants to be predicted. This study indicates that disappointing results of splenic autotransplantation in adult patients should not lead to false pessimism about the role of this operation in children.     

Reimmunization and splenic autotransplantation: A long-term study of immunologic response and survival following pneumococcal challenge

Splenic autografts have phagocytic function and increase survival after experimental sepsis. The long-term effect of transplant viability, phagocytic capacity, and immunologic responsiveness were evaluated. Rats were divided into experimental groups: control, splenectomized, and splenic autotransplant rats. Approximately one-half of the rats were immunized against pneumococcus. Twelve months later, the rats were reimmunized, and the pneumococcal antibody titers were measured. The effect of operation and immunization was determined by challenging rats with intravenously administered pneumococci. Bacterial clearance from the bloodstream was measured and mortality recorded. Spleens were weighed and examined histologically. In unimmunized rats, pneumococcus was cleared from the bloodstream of control rats, whereas splenectomized and splenic autotransplant rats demonstrated a progressive increase of pneumococci in the bloodstream. However, splenic autotransplant rats grew fewer bacteria after challenge (P < 0.05). All control rats survived. Thirty-three percent of splenic autotransplant rats were alive, but significantly fewer splenectomized rats (6%) survived (P < 0.05). After reimmunization, highest antibody titers were noted in control rats (P < 0.05). Splenic autotransplant rats had higher antibody titers than did splenectomized rats (P < 0.05). Reimmunized splenic autotransplant rats had greater survivorship (71%) when compared with reimmunized splenectomized rats (26%) (P < 0.003). At 1 year, transplants were smaller than control spleens (P < 0.001), although histologic integrity was maintained. Splenic autotransplantation results in better phagocytic function, improved response to reimmunization, and increased survival after pneumococcal challenge and may be an important measure in preventing postsplenectomy sepsis.     

Intraportal splenic autotransplantation in rats: Feasibility and effectiveness

This experiment was designed to see whether or not normal host resistance to infection could be reestablished in splenectomized animals by intraportal autotransplantation of homogenized splenic tissue. Part I studied the feasibility of the technique. Within 1 hr of splenectomy, 16 adult Lewis rats received an intraportal injection of autogenous splenic tissue which had been passed through a 500-μm screen. Five rats died acutely from hemorrhage at the site of injection. The others tolerated the infusion well, both acutely and chronically. The animals developed only transient elevations in liver enzymes; chronic portal hypertension did not occur. Histologically, splenic tissue could be demonstrated within terminal portal venules. Part II assessed the effectiveness of intraportal splenic autotransplantation. Eight to twelve months after splenectomy, autotransplantation, or sham operation, 103 Sprague-Dawley rats were challenged with intravenous boluses of 10⁵ to 10⁸ pneumococci. Mortality was 91% for splenectomized animals, 88% for animals bearing autotransplants, and 59% for controls. Thus intraportal splenic autotransplantation is technically feasible in rats. The grafts are well tolerated by the liver, and splenic tissue is preserved in intimate contact with the blood stream. Even after 8 to 12 months, however, such autografts are not capable of providing normal protection against massive pneumococcemia.     

The effect of site and technique of splenic tissue reimplantation on pneumococcal clearance from the blood

The technique and site of reimplantation of splenic tissue influences survival of laboratory animals following intravenous injection of pneumococci. Splenic tissue was prepared by slicing, mincing, or grating the spleen. The tissue was placed subcutaneously, intraperitoneally, retroperitoneally, or in an omental pouch. This study was designed to determine the rate of pneumococcal clearance from the blood stream 16 weeks following splenic reimplantation by four different methods. All animals were challenged with an intravenous 1 mL bolus containing 10(7) bacteria. The New Zealand white rabbits were divided into six groups: intact spleen; splenectomized; spleen slices in an omental pouch; minced spleen in an omental pouch; splenic tissue implanted subcutaneously; and bits of spleen dropped into the peritoneal cavity. Animals with an intact spleen and those with spleen slices implanted into an omental pouch cleared bacteria during the first hour and all bacteria had disappeared at three hours. Bacteremia persisted longer than three hours in the other groups. Splenic tissue had regenerated in all animals with omental pouch implants, in four of six with minced spleen dropped into the peritoneal cavity but in only one with a subcutaneous implant. Reimplanted splenic tissue clears pneumococci from the blood stream best when thin slices of spleen are placed in an omental pouch. This technique also assures successful regeneration of splenic tissue.     

Splenic resection or heterotopic transplantation of splenic tissue as alternatives to splenectomy. Regeneration and protective effect against pneumococcal septicemia

In 82 male Sprague-Dawley rats, divided into eight groups according to surgical procedure performed (total splenectomy, sham operation and six different modes of splenic conservation), resistance to intravenous injection of 4 X 10(3) CFU of Streptococcus pneumoniae type I was evaluated 16 weeks after the surgical procedures. Significant regeneration of the spleen and almost normal resistance to pneumococci was seen 16 weeks after a two-thirds resection. Pieces of the spleen, implanted subcutaneously or into the greater omentum, also showed marked regeneration; though survival time was prolonged, the mortality among these animals following injection with pneumococci did not, however, differ from that of totally splenectomized animals. Dispersed splenic tissue, injected subcutaneously, intramuscularly, or retroperitoneally, showed less sign of regeneration and had no effect on mortality or survival time in partially vis-à-vis totally splenectomized rats.     

脾切除对肺内细菌清除和移位的影响

目的:探讨脾切除对肺内细菌清除及移位的影响,同时观察自体脾组织移植的应用效果.方法:将Wistar大鼠90只随机分为假手术组、脾切除组和半脾移植组,采用肺炎球菌悬液雾化吸入方法攻击动物,观察肺组织学病变,肺内细菌清除和移位状况.结果:脾切除组动物肺组织严重充血肿胀,炎性细胞浸润少,肺内细菌清除功能降低,细菌向肺门淋巴结移位和侵入血流加快,与假手术组比较有显著性差异(P<0.05),但半脾移植组动物能基本恢复对肺炎球菌的抵抗能力.结论:脾切除后动物肺抗菌功能降低,而自体脾组织移植是保留脾功能的有益术式.     

Comparison of omental splenic autotransplant to partial splenectomy. Protective effect against septic death

The possible benefit of either partial splenectomy or splenic autotransplantation as protection against post-splenectomy sepsis was investigated. Sprague-Dawley rats were challenged with intravenous Streptococcus pneumoniae and the incidence of bacteremia and mortality were recorded. Animals were divided into four groups based upon the amount of splenic tissue conserved: total splenectomy (0%), partial splenectomy (62%), splenic autotransplantation (27%), or sham celiotomy (100%). A statistically significant (P 0.05) decrease in the incidence of septic death was seen in comparing the total splenectomized animals (63%) to the autotransplant group (27%), the partial splenectomy (4%) and the control group (4%). This diminishing mortality is inversely proportional to the amount of splenic remnant in the respective groups. There was a similar, parallel relationship in the incidence of Streptococcus pneumoniae bacteremia. Thus, the greater the amount of remaining splenic tissue, the lower the incidence of bacteremia and subsequent mortality, implying the preservation of immunologic function with splenic conservation.     

Effect of splenectomy and hemisplenectomy on pneumococcal infection and bacteria clearance in the rat

Median survival times 1 month after intraperitoneal inoculation of Streptococcus pneumoniae Felton UC 41 show that the difference between hemisplenectomized and control rats is not at all significant (chi 2 = 0.04). On the other hand, comparing splenectomized rats with hemisplenectomized and control rats taken together, there is a significant difference: 10% level (chi 2 = 2.84; 1 degree of freedom; p less than 0.10). The blood concentrations of pneumococci at different time intervals after inoculation do not differ between control and hemisplenectomized rats (F = 0.02; 1 and 135 degrees of freedom), but they differ very significantly when the splenectomized rats are compared with the hemisplenectomized rats and controls taken together (F = 10.00; 1 and 135 degrees of freedom; p less than 0.01).     

Serum antibody responses to pneumococcal vaccine after splenic autotransplantation

Immunization with pneumococcal capsular polysaccharide vaccines is advocated after splenectomy; however, experimental and clinical data suggest an impaired antibody response in splenectomized individuals. This study examined the value of splenic autotransplantation at various sites in augmenting the antibody response to Type III pneumococcal capsular polysaccharide in mice immunized 3 months after operation. Splenectomy resulted in impaired antibody responses compared to sham-operated mice (p less than 0.001) using an enzyme-linked immunosorbent assay. Mice with intraperitoneal splenic autotransplants, but not mice with subcutaneous or intramuscular transplants, had greater antibody responses compared to splenectomized mice (p less than 0.05). Antibody responses were elevated only in mice autotransplanted with 50% or more of the original splenic mass. Since autotransplantation of splenic tissue augments the antibody response to pneumococcal capsular polysaccharides, the combination of splenic autotransplantation and pneumococcal vaccination may confer more protection than either modality alone in individuals who must undergo splenectomy.     

Evaluation of phagocytic function of macrophages in rats after partial splenectomy

Background: Understanding the immunologic properties of the spleen has enabled surgeons to practice splenic conservation surgery. If the upper pole of the spleen can be preserved solely on the upper short gastric vessels, will phagocytic function of macrophages in remnant splenic tissue be affected? The aim of this experimental study was to evaluate the phagocytic function of macrophages in partially resected spleens, with hilar excision preserving the short gastric vessels.

Study Design: Forty-eight female Wistar albino rats were divided into four groups. Groups 1 and 2 underwent sham operations and groups 3 and 4 underwent partial splenectomy. One milliliter of sodium chloride 0.9% was injected into the abdomen of the rats in groups 1 and 3 and 1 mL of Streptococcus pneumoniae type III as an antigenic stimulus was injected into the abdomen of the rats in groups 2 and 4, 6 weeks after the first operation. Forty-eight hours later, relaparotomy was performed in all animals. India ink was used to determine the capacity of uptake in the splenic phagocytes. To evaluate the phagocytic function of the splenic tissues, histologic examinations were performed according to a macrophage grading system.

Results: All spleens in all four groups were stained black after injection of India ink. Phagocytic activity of macrophages was reduced in the partially splenectomized groups, compared with intact spleen groups (group 3 versus group 1; p < 0.0001, group 4 versus group 2; p < 0.0001). There was a significant difference between groups 1 and 2 according to phagocytic function of macrophages (p = 0.0121). Also, after Streptococcus pneumoniae type III injection as an antigenic stimulus in group 4, we found that the phagocytic functions of macrophages increased compared with those of the sodium chloride 0.9%-injected group 3 after partial splenectomy (p < 0.0001).

Conclusions: Phagocytic function of macrophages in rats decreased after partial splenectomy. Nevertheless, the remnant spleens in rats could be stimulated when challenged with an antigenic stimulus.     

The importance of the spleen for the immunosuppressive action of cyclosporine in transplantation

The spleen plays an important role in the response of the recipient's immune system to a primarily vascularized graft and cyclosporine treatment is known to alter this response. To investigate the interaction between the splenic immune response and CsA's immunosuppressive actions more thoroughly, Lewis recipients of Brown-Norway heterotopic heart grafts were treated i.p. daily with normal saline or with CsA doses of 0.75, 1.5, or 3.0 mg/kg/day from day 1 through day 50 or until rejection. Rats treated with 3 mg/kg were splenectomized intraoperatively (i.o.) or not splenectomized. Rats in subgroups of the other treatment groups were splenectomized i.o., on day 5, not splenectomized, or the recipient's spleen cells were reinfused after i.o. splenectomy. In non-CsA-treated rats, i.o. splenectomy (median survival time, [MST] = 11 days) and day 5 splenectomy (MST = 11 days) prolonged graft survival minimally in comparison with nonsplenectomized animals (MST = 7 days). Reinfusion of the spleen cells reversed this effect (MST = 7 days). Most interestingly, the immunosuppressive efficacy of 1.5 mg/kg of CsA (MST = 91 days) was reduced by day 5 splenectomy (MST = 24 days) and completely abolished by i.o. splenectomy (MST = 11 days). Spleen cell reinfusion partially restored the effect of CsA treatment (MST = 88 days). Since splenectomy resulted in a complete abrogation of the immunosuppressive efficacy of 1.5 mg/kg CsA, our results support the hypothesis that certain spleen cells augment immunosuppression by CsA. These findings provide additional evidence that the immune system's own regulation of its antigraft response can be an important component of the overall suppression of rejection that is associated with the use of certain immunosuppressive drugs.     

Conservative management in blunt splenic trauma

We reviewed for analysis the charts of two groups of adults patients with blunt splenic injuries issued from two University Hospital Centers; the group 1 (G1) of 22 patients and the group 2 (G2) of 20 patients. The results of actually therapeutic procedures concerning blunt splenic injuries and subsequently the effectiveness of non operative treatment were evaluated. Splenectomy was performed in G1 for 11 patients, instead of 19 patients in G2 (p = 0.0003), whereas, the non surgical treatment was done in 9 patients and 1 patient, respectively (p = 0.02). The mean Splenic Injury Score (SIS) was 2,95 in G1 and 3.47 in G2 (p = 0.03). The spleen was preserved in G1 for 8 patients, instead 1 patient in G2 (p = 0.04). In G1, the non operative treatment was successfully accomplished in 66% of patients. It was obtained with lack of mortality, with a lower overall morbidity and a lower length of hospital stay than in splenectomized patients, but the latter group accounted higher values of Injury Severity Scores (p < 0.05). If proper selection criteria for non operative management are used, more than a third of patients with blunt splenic injury can be treated by splenic preservation at least as safely as splenectomized patients.     

Evaluation of splenectomy in total gastrectomy for gastric cancer

It has been well accepted that extensive prophylactic lymphadenectomy is certainly effective for elevating cure rate after gastric cancer surgery, however, regarding to the prophylactic splenectomy the arguments are controversial. We studied the value of splenectomy in total gastrectomy for gastric cancer by examining the late survival rates, the accuracy of intraoperative judgement of splenic hilar lymph node metastasis and postoperative changes of serum immunosuppressive factors. In curatively resected stage III cases without splenic hilar lymph node metastasis, the nonsplenectomized group showed a significant better late survival rate than the splenectomized group, 5-year survival rate being 59.9% in the former and 30.8% in the latter. In cases with splenic hilar lymph node metastases, 2 of 9 splenectomized patients survived more than 10 years. In cases of noncuratively resection, splenectomy did not enhance the survival rate. Although further clinical randomized study is needed to draw a definitive conclusion, we had better take a splenic reserve operation for the patients without splenic hilar lymph node metastasis. On the other hand, splenectomy should be performed in cases with splenic hilar lymph node metastases.     

Penicillin and natural immunity protect against postsplenectomy sepsis

Prophylactic penicillin, splenic autotransplantation, and immunization using pneumococcal vaccine have all been shown to reduce the incidence and mortality of postsplenectomy sepsis. However, little is known regarding the effect of penicillin in established infection or the effect of prior infection in either asplenic controls or animals with autotransplanted splenic tissue. An animal model with bacterial introduction via the lungs was used to investigate the effect of penicillin, splenic autotransplantation, and previous exposure to the infecting organism on the mortality of postsplenectomy sepsis. One hundred fifty-nine rats underwent either sham celiotomy, intraperitoneal splenic autotransplantation, or splenectomy. Twelve weeks postoperatively all animals were challenged using Streptococcus pneumoniae delivered transtracheally. Half of each group received procaine penicillin by intramuscular injection for 5 days beginning 24 hr post bacterial inoculation and mortality was observed. Eight weeks later surviving rats that had received penicillin were reinoculated with the same organism and mortality was again observed. Splenic autotransplantation reduced the early mortality in postsplenectomy sepsis. Prior bacterial exposure reduced the mortality in postsplenectomy sepsis, even in splenectomized animals. Treatment with penicillin produced a marked reduction in mortality even when administration was postponed for 24 hr after bacterial inoculation.     

Heterotopic splenic autotransplantation in prevention of overwhelming postsplenectomy infection.

An experimental study was undertaken to evaluate the protective effect of heterotopic splenic autotransplantation in weanling rats. Rats were divided into three experimental groups: splenectomy, control, and splenic autotransplantation. Rats were challenged with i.v. type I pneumococcus. Bacterial bloodstream clearance and survival were determined. Splenic bacterial uptake was measured by determining the isotopic activity of technetium-99m-labeled pneumococci. Autoradiographs and material stained with hematoxylin and eosin and Gram strains were examined for histologic features. All autografts survived and were histologically compatible with normal splenic tissue. Bloodstream clearance of pneumococci was significantly greater in rats with splenic autotransplantation. Splenic autografts had 10 to 30 times greater uptake of pneumocci than did the liver. Rats with autotransplantation had a prolonged survival time. Heterotopic splenic autotransplantation may prove to be an important adjunctive surgical measure in the treatment of children undergoing splenectomy.