Influence of donor and host age on the regeneration and blood flow of splenic transplants

After splenectomy there is an increased risk of fatal overwhelming postsplenectomy sepsis, especially in children. If all alternatives to splenectomy fail, autotransplantation of splenic fragments is indicated. These fragments regenerate after a necrotic phase to small splenic nodules. Regulatory factors governing the regeneration process are largely unknown. Inbred rats were used as a model to define the influence of recipient and donor age on the regenerated mass and the blood flow of transplanted splenic fragments. These are both important factors for the protective function of the spleen. Fetal, newborn, weanling, or adult spleens were implanted into the greater omentum of newborn, weanling, or adult rats. The younger the recipient and donor, the better the regeneration and perfusion of transplants. However, these did not reach more than 40% of the normal splenic mass. In addition, no experimental group achieved more than one third of the normal splenic blood flow. There is an obvious age dependency in splenic regeneration and blood flow, but the transplants are far from attaining a normal splenic mass and perfusion.     

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.     

Immune cell subpopulations in regenerated splenic tissue in rats.

BACKGROUND: Asplenic patients have an increased risk of infections. Operations such as autotransplantation or splenic artery ligation have been suggested to ensure retention of functional splenic tissue after splenectomy, but their protective value is unclear. Immune responses, such as production of antibody, remain impaired in humans and animals even when such tissue is present, and phagocytosis is less efficient than by normal spleen tissue. In the present study the cellular composition of regenerated tissue is determined. METHODS: Splenic tissue was obtained from rats 6-9 months after splenic autotransplantation, splenic artery ligation or sham operation. The lymphocyte and macrophage subpopulations were labelled using a panel of monoclonal antibodies and analysed by flow cytometry. RESULTS: Both the total number of cells and the number of cells per gram of tissue were significantly reduced. There was a substantial reduction in the percentage of some of the cells examined (CD4+ and CD8+ T lymphocytes subsets), but not all (B lymphocytes, ED1+ and ED2+ macrophages, OX2+ and OX6+ cells). CONCLUSIONS: The reduction in the T lymphocyte subsets in regenerated splenic tissue compared with the normal spleen might explain the immunological dysfunction which persists after splenic autotransplantation. The reduction in the number of macrophages may be responsible for the alteration in phagocytic efficiency of regenerated splenic tissue.     

IMMUNE CELL SUBPOPULATIONS IN REGENERATED SPLENIC TISSUE IN RATS

Background : Asplenic patients have an increased risk of infections. Operations such as autotransplantation or splenic artery ligation have been suggested to ensure retention of functional splenic tissue after splenectomy, but their protective value is unclear. Immune responses, such as production of antibody, remain impaired in humans and animals even when such tissue is present, and phagocytosis is less efficient than by normal spleen tissue. In the present study the cellular composition of regenerated tissue is determined. Methods : Splenic tissue was obtained from rats 6–9 months after splenic autotransplantation, splenic artery ligation or sham operation. The lymphocyte and macrophage subpopulations were labelled using a panel of monoclonal antibodies and analysed by flow cytometry. Results : Both the total number of cells and the number of cells per gram of tissue were significantly reduced. There was a substantial reduction in the percentage of some of the cells examined (CD4⁺ and CD8⁺ T lymphocytes subsets), but not all (B lymphocytes, ED1⁺ and ED2⁺ macrophages, OX2⁺ and OX6⁺ cells). Conclusions : The reduction in the T lymphocyte subsets in regenerated splenic tissue compared with the normal spleen might explain the immunological dysfunction which persists after splenic autotransplantation. The reduction in the number of macrophages may be responsible for the alteration in phagocytic efficiency of regenerated splenic tissue.     

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.     

Decreased phagocytic capacity of autotransplanted splenic tissue

Background: Asplenic patients have an increased risk of infections. Operations such as autotransplantation have been proposed to restore functional splenic tissue after splenectomy, but the protective value of this tissue is unclear. Immune responses such as production of antibody remain impaired in humans and animals even when such tissue is present, and clearance of particles from the blood is reported to be less efficient than by normal spleen tissue. The present study investigated the phagocytic capacity of cells in the regenerated tissue in vitro, free of the confounding effects of hepatic clearance. Methods: Single cell suspensions were prepared from splenic tissue from rats 6 months after splenic autotransplantation or sham operation. Phagocytosis of killed, fluorescein‐labelled bacteria was measured by flow cytometry. Results: Autotransplanted tissue contained fewer phagocytic cells than normal tissue, and these cells phagocytosed less per cell. Phagocytosis by spleen cells was dependent on heat‐labile opsonic factors. Conclusions: Autotransplanted splenic tissue does not restore the phagocytic capacity lost following splenectomy.     

Splenic tissue autotransplantation in rabbits: no restoration of host defense

BACKGROUND: The loss of spleen may increase the incidence of overwhelming sepsis. To prevent this, splenic autotransplantation has been performed in humans and experimental animals. However, there is still controversy about the effectiveness of regenerated splenic tissue in preventing infection. This study explored the effectiveness of splenic tissue autotransplantation in restoring host defense. MATERIALS AND METHODS: Rabbits were divided into three groups: splenic autotransplantation, sham operation, and total splenectomy. Histomorphology, T-lymphocyte count, serum lysozyme levels, hemolysin titers, and pneumococcal clearance were observed as read-out parameters over 24 weeks. RESULTS: Histological study showed that the white pulp was poorly developed and central arterioles were missing in the regenerated splenic tissue of the autotransplanted rabbits. The weight of regenerated spleens recovered 6 months later in the splenic autotransplantation group was 11% of that in the sham operation group and was significantly less than the weight at implantation. There was no significant difference in the number of T lymphocytes or level of serum lysozyme between the three groups. A poor antibody response by the rabbits in the splenic autotransplantation and total splenectomy groups was noted after the primary intravenous administration of sheep red blood cells compared to those of sham operation group. After the challenge with type 3 pneumococci intravenously, pneumococcal clearance from the bloodstream in the splenic autotransplantation group did not differ significantly from that in the total splenectomy group, but was markedly delayed compared with that in the sham operation group. CONCLUSIONS: The low quantity and poor quality of the regenerated splenic tissue contribute to the inferior immunoprotective ability of animals autotransplanted with one-third of the original spleen. This suggests that the regenerated spleen cannot compensate for the immunological function of the original one, especially host resistance to infection.     

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.     

Experimental study on function of regenerated splenic tissue after splenic autotransplantation

To prevent postsplenectomy overwhelming sepsis, splenic autotransplantation has been clinically attempted. However, function of regenerated splenic tissue after splenic autotransplantation has not been completely understood. Changes in weigh of regenerated splenic tissue, splenic blood flow, splenic immune responses and phagocytic function were studied for one year after splenic autotransplantation using Sprague-Dawley rats. At one year after autotransplantation, the weight of regenerated splenic tissue was increased to 80% of the originally implanted spleen and the blood flow was increased to 80% of the control spleen. The counts of lymphocytes and macrophages in the regenerated splenic tissue were significantly low at eight weeks after transplantation, however lymphocytes was increased to 58.8% and macrophages was increased to 29.5% of the control spleen at 16 weeks after transplantation. The blast formation of splenic lymphocytes was lower at the early stage after transplantation, thereafter, it was increased at the later time after transplantation. Microangiography of the regenerated spleen showed new capillaries around the implanted tissue 2 weeks after transplantation. These results suggested that the transplanted splenic tissue was regenerated to the similar structure to normal spleen and immunological function was recovered close to the normal splenic tissue.     

Does survival depend on the amount of autotransplanted splenic tissue?

Susceptibility to Streptococcus pneumoniae infection was studied in 11 groups of rats allocated to sham operation, splenectomy, or splenic autotransplantation of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the removed spleen. Three months later, all rats were exposed intravenously to type 1 Streptococcus pneumoniae (median lethal dose, LD50, for control group). Survivors were killed 13 days after the bacterial challenge. Autopsy showed that more splenic tissue was recovered in rats that received less than 50% splenic tissue compared with those that received 50% or more. More survivors were found among sham-operated rats (47.5%; 95% confidence intervals, 32 to 68) and rats that had 40% splenic tissue implanted (35%; confidence interval, 20 to 54) or those that were found to have regenerated 40% splenic tissue. We conclude that 40% of the spleen should be autotransplanted to protect the rat optimally against infection after splenectomy.     

Response to immunization after partial and total splenectomy

Survival after infection from Streptococcus pneumoniae in both animals and man is influenced by the amount of splenic tissue. We investigated the effect of differences in splenic weight upon the antibody response to immunization and the effect of immunization upon survival after pneumococcal challenge. Young Sprague-Dawley rats had either sham operation, hemisplenectomy, splenectomy with splenic autotransplantation, or total splenectomy. Nine weeks later, rats were immunized with a heat- and formalin-killed type-specific pneumococcal vaccine. Antibody response measured by radioimmunoassay was similar in all operative groups and was significantly higher than in nonimmune rats (P less than 0.01). Splenic weight was less after hemisplenectomy or autotransplantation than in sham-operated animals (P less than 0.01). Immunization improved survival after live pneumococcal challenge in rats that had autotransplantation and total splenectomy (P less than 0.001). Our results demonstrate that splenic weight does not affect the antibody response to pneumococcal immunization in rats. Immunization improves survival after bacterial challenge in susceptible animals and minimizes the detrimental effect of reduction in splenic mass.     

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.     

The influence of splenic weight and function on survival after experimental pneumococcal infection.

Splenectomy impairs survival after pneumococcal challenge in rats, while preservation of sufficient splenic tissue can be protective. This study investigated the effects of methylcellulose on stimulation of splenic weight, splenic histology, reticuloendothelial (RE) activity, and survival after pneumococcal infection. Methylcellulose increased spleen weight four- to five-fold but did not improve RE function or survival after infection. These parameters correlated best with the weight of the remnant in animals that did not receive methylcellulose. The functional limitations of splenic autotransplants were not corrected by methylcellulose stimulation of splenic weight. Preservation of a splenic remnant with intact blood supply is preferable to autotransplantation of the spleen to conserve RE capability.     

Relative merits of partial splenectomy, splenic reimplantation, and immunization in preventing postsplenectomy infection.

Partial splenectomy, splenic autotransplantation, and immunization with pneumococcal vaccine have been reported to protect patients against overwhelming postsplenectomy infection, and this study was undertaken to evaluate these therapeutic alternatives. For this purpose 136 rats were divided into experimental groups: 34 controls, 34 splenectomy, 34 partial splenectomy, and 34 splenic autotransplantation animals. Five weeks after operation, two-thirds of the animals were immunized with killed pneumococci. The effects of operation and immunization were studied by challenging the animals intravenously with pneumococci. Pneumococcal antibody titers were determined, and phagocytic uptake of pneumococci by the spleen and liver was measured. Immunization impressively increased the survival rate in all groups. At low-challenge doses autotransplantation prolonged survival. At higher-challenge doses only partial splenectomy increased survival. Partial splenectomy and control animals had higher antibody titers than did splenectomy and autotransplantation rats. Animals with the highest antibody titers had the greatest splenic and hepatic phagocytic uptake of pneumococci. Partial splenectomy was more efficient in removing pneumococci than was autotransplantation. Thus immunization is one of the most important factors contributing to survival after splenectomy. Partial splenectomy is preferable to splenic autotransplantation because it is associated with higher antibody titers after immunization, better pneumococcal splenic uptake, and improved survival rates.     

腹膜后自体脾移植术在肝硬变门静脉高压症治疗中的临床研究

目的　观察腹膜后自体脾移植联合食管下段横断术治疗肝硬变门静脉高压症的临床效果。方法　将20例肝功能Child A、B级的肝硬变门静脉高压症患者随机均分为自体脾移植组和切脾组。自体脾移植组采用自体带蒂脾组织腹膜后移植联合改良的食管下段横断术,切脾组则采用脾切除联合改良的食管下段横断术。以患者术前的情况为对照,在术后2~6 个月观察患者的一般情况、脾扫描、肝功能、血清促吞噬素(tuftsin)及IgM水平。结果　术后第6天切脾组死亡1例,术后第10天脾移植组出现再出血1 例。自体脾移植组术后血清tuftsin、IgM水平高于切脾组,差异有显著性意义(P<0.01),而对肝功能无明显影响。结论　腹膜后自体脾移植能维持脾脏的基本免疫功能,且能长期存活,在临床上推广应用是可行的。     

Enhanced regeneration of transplanted splenic tissue by increased work load to the splenic compartments

Autologous splenic tissue regenerates after subperitoneal transplantation in laboratory animals and in man. Qualitatively it resembles normal splenic tissue but the quantity usually only attains a small proportion of the normal spleen. In the prevention of overwhelming post-splenectomy sepsis a critical mass and a considerable blood flow to the regenerated spleen seem to be essential. By increasing the work load to splenic transplants in rats, significantly more splenic tissue was regenerated. This was achieved by: stimulating the white pulp by repetitive injections of xenogeneic red cells; stimulating the red pulp by damaging red cells with phenylhydrazine; a combination of 1 and 2; and stimulating the reticuloendothelial system by IP injections of methylcellulose. Stimulating the red pulp and the reticuloendothelial system were more effective than the injection of antigens. As the splenic mass is obviously regulated by the work load, we conclude that this effect should be used to attain the critical splenic mass and to increase the blood flow for effective clearance of bacteria from the blood.     

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.     

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.     

自体脾组织移植后病理学变化的实验研究

目的　研究自体脾组织移植后不同时相点病理形态学变化规律。　方法　健康Wistar大鼠 5 6只 ,雌雄不限 ,体重 10 0～ 12 0 g ,随机分为 7组 ,每组 8只中又设脾切除自体组织大网膜内脾移植组 5只 ,假手术组 3只 ,分别于术后 7、14、3 0、60、90、12 0、180d取脾组织 ,光镜、透射电镜观察。　结果　移植脾组织之重量在术后 7d最轻 ,为 0 .0 72 g ,其后逐渐增加 ,180d时为 0 .5 11g ,各时相点之间有明显差异 (P <0 .0 5 )。病理检查提示移植脾组织经历急性期、缓解期、修复期三个病理时期 ,逐步恢复并接近正常的组织结构。　结论　自体脾组织大网膜内移植术是简便有效的脾移植方法。自体移植脾组织再生过程可分为 :急性期、缓解期与修复期     

Complications of splenic tissue reimplantation.

Splenic tissue reimplantation employing the omental implantation technique was applied in 23 patients undergoing splenectomy for traumatic or iatrogenic splenic injury. Four complications were encountered after autotransplantation (17.4%). Two of these consisted of small bowel obstruction due to postoperative adhesions and were successfully managed by lysis of the adhesions. The other two complications were aseptic necrosis of the splenic transplants and were treated with ablation of the autolysed transplants. A case of abnormal splenic tissue reimplantation in a male patient with unsuspected myelofibrosis is also discussed. He underwent an emergency laparotomy for rupture of a subcapsular splenic haematoma. It is concluded that splenic tissue implantation in the greater omentum is associated with important early morbidity and this should be taken into account whenever application of the method is considered.