Splenic Autografts Research Articles

Spleen regeneration after subcutaneous heterotopic autotransplantation in a mouse model

BackgroundSplenectomy may lead to severe postoperative complications, including sepsis and cancers. A possible solution to this problem is heterotopic autotransplantation of the spleen. Splenic autografts rapidly restore the regular splenic microanatomy in model animals. However, the functional competence of such regenerated autografts in terms of lympho- and hematopoietic capacity remains uncertain. Therefore, this study aimed to monitor the dynamics of B and T lymphocyte populations, the monocyte-macrophage system, and megakaryocytopoiesis in murine splenic autografts.MethodsThe model of subcutaneous splenic engraftment was implemented in C57Bl male mice. Cell sources of functional recovery were studied using heterotopic transplantations from B10-GFP donors to C57Bl recipients. The cellular composition dynamics were studied by immunohistochemistry and flow cytometry. Expression of regulatory genes at mRNA and protein levels was assessed by real-time PCR and Western blot, respectively.ResultsCharacteristic splenic architecture is restored within 30 days post-transplantation, consistent with other studies. The monocyte-macrophage system, megakaryocytes, and B lymphocytes show the highest rates, whereas the functional recovery of T cells takes longer. Cross-strain splenic engraftments using B10-GFP donors indicate the recipient-derived cell sources of the recovery. Transplantations of scaffolds populated with splenic stromal cells or without them afforded no restoration of the characteristic splenic architecture.ConclusionsAllogeneic subcutaneous transplantation of splenic fragments in a mouse model leads to their structural recovery within 30 days, with full reconstitution of the monocyte-macrophage, megakaryocyte and B lymphocyte populations. The circulating hematopoietic cells provide the likely source for the cell composition recovery.

Experimental splenosis in the liver and lung spread through the vasculature.

To demonstrate that intra-organ splenosis can engraft and develop after being distributed through the vasculature, tiny fragments of splenic tissues were injected into the inferior vena cava or the portal vein to induce intrapulmonary and intrahepatic splenosis in rats. After 1 month, splenic autograft structures in the lung and liver were assessed for structure by histology, for immunologic compartments by immunohistochemistry, for phagocytic function by carbon uptake and for vascular formation by Microfil (a silicon rubber compound) injection. Intrapulmonary and intrahepatic splenoses were indeed able to spread through the vasculature. The intrapulmonary splenic autografts were trapped and spread out in the interstitium, without forming a capsule. White pulp was markedly developed, showing lymphocyte aggregations that consisted in B cells surrounding the dilated vessel. Splenic sinuses were not definitively observed. Although macrophages were detected by immunohistochemistry, they showed no indication of having phagocytized carbon particles from the vessels, implying a closed circulation. In contrast, intrahepatic splenic autografts formed well-developed capsules, trabeculae and red pulp with splenic sinuses. Macrophages detected by immunohistochemistry were observed capturing carbon particles, which clearly revealed an open system circulation, as seen in normal rat spleen. The development of white pulp was poor and lymphocytes consisting in B cells aggregated in the peripheral margins. These results demonstrate that intra-organ splenosis can spread through the vasculature and that the morphologic and immunologic structures formed in these regenerated autografts are influenced by the organ vasculature and extracellular matrix wherein the tissue fragments settled.

Regeneration of Splenic Autotransplants Attached on Liver by a Tissue Adhesive

Background The regeneration of splenic autotransplants remains unsatisfactory. The liver has a rich blood supply and is prone to form collateral vessels to nearby ischemic tissues. It should be valuable to study whether it is a new site to facilitate splenic regeneration. Materials and Methods One hundred twenty Sprague-Dawley rats were randomly divided into 12 groups. After splenectomy, each weighed spleen was attached to the diaphragmatic side of the left hepatic lobe using an adhesive. The rats were sacrificed on postoperative day(POD)s 1, 3, 7, 14, 21, 28, 35, 42, 49, 56, 70, and 84. Six spleens in each group were dissected for weighing and another 4 for histologic analysis (H&E). Splenic autografts of another 12 rats attached to the mesenterium were sacrificed on POD 28, 56, and 84 for histologic analysis (H&E). Results The spleens attached to the liver underwent almost complete necrosis and then regenerated centripetally into structures comparable to a normal spleen, with typical red and white pulps as well as a marginal zone on POD 35. The mass of the implants recovered to more than two fifths of the original (44.2% ± 3.6%) on POD 84. For splenic autografts attached to mesenterium, typical red and white pulps were observed on POD 56. The marginal zone was not clear on POD 84. Conclusion The liver proved to be an appropriate site for splenic autotransplantation. Regeneration was histologically superior to that of splenic tissue attached to the mesenterium. The application of a tissue adhesive may be a new technique for splenic autotransplantation.

Aspectos histológicos de fragmentos esplênicos autotransplantados em ratos

Estudou-se a viabilidade do autotransplante de tecido esplênico, um terço do baço, associado à esplenectomia parcial, e verificou-se a cinética evolutiva de sua regeneração, sob o aspecto macroscópico e histológico. Foram utilizados 28 ratos Wistar, adultos, machos, com média de peso de 300g, distribuídos em quatro grupos experimentais. Cada animal foi submetido à esplenectomia parcial, e um fragmento de baço foi transplantado para o omento maior, por períodos de 30, 60, 90 e 120 dias. Após cada período pré-estipulado, os tecidos esplênicos autotransplantados foram coletados e analisados histologicamente. Os resultados mostraram regeneração da cápsula esplênica, dos vasos sanguíneos e das polpas branca e vermelha. Aos 90 dias, a arquitetura microscópica apresentava-se semelhante à do baço normal.

Ultrastructural analysis of TiO 2 nanotubes with photodecomposition of water into O 2 and H 2 implanted in the nude mouse

To analyze the biocompatibility and O2 generation of TiO2 nanotubes via photodecomposition of water into O2 and H2 in vivo, samples were implanted under the inguinal skin of the nude mouse. Venous oxygen saturation (SvO2) of the inguinal skin over the implanted region was measured with a tissue oximeter and the ultrastructures were examined with an electron microscope. Four weeks after the implantation, SvO2 of the inguinal skin of the groups with TiO2 nanotubes was 30-40% higher than that of the opposite control region (54%). SvO2 of the other groups, comprising splenic autografts, fetal cardiac tissue transplantation and surgical procedure without TiO2 nanotubes, was roughly the same as that of controls. Ultrastructurally, TiO2 nanotubes were phagocytized by the macrophage and promoted filament formation in its cytoplasm. Neither death of the cell nor destruction of the tissue was recognized. These findings indicate excellent biocompatibility and O2 generation of TiO2 nanotubes in vivo.

The risks of total pancreatectomy and splenic islet autotransplantation.

The intraportal site is the most common site for islet transplantation. Many other sites have been tried experimentally, including the spleen, which has successfully lead to insulin independence in a number of animal models. Nevertheless, there are no detailed reports of total pancreatectomy and splenic islet autotransplantation in humans. Five patients underwent total pancreatectomy and splenic islet autotransplantation for chronic pancreatitis. Four patients had a pylorus-preserving total pancreatectomy and one patient a duodenal-preserving pancreatectomy. In three cases islets were embolized into both the portal vein and spleen. Two patients received splenic islet transplants alone. Islets were transplanted by retrograde venous infusion via the short gastric veins (n = 3), splenic vein stump (n = 1), and the left gastroepiploic vein (n = 1). The total volumes of transplanted pancreatic digest in those receiving combined intraportal and splenic autografts (n = 3) were 15.8, 13.0, and 13.5 ml. The volumes in those receiving a splenic-alone autograft (n = 2) were 12.0 and 5 ml. The mean rise in portal pressure was 18 cm of water. Complications related to the splenic autograft included a wedge splenic infarct, an emergency splenectomy, and a portal vein thrombosis in one patient having a combined intraportal and splenic autograft. Two patients developed insulin independence. two patients were still insulin independent at 1-year follow-up, and all had normal HbA1c levels (mean 5.6, range 5.2-6.3). Splenic islet autotransplantation, after total pancreatectomy, does lead to insulin independence. However, in our experience the combined procedure has a high morbidity because of splenic infarction and venous thrombosis.

Kinetics of neovascularisation of splenic autotransplants in mice.

The aim of this study was to examine the kinetics of neovascularisation of splenic autoimplants into the abdominal cavity after splenectomy in mice. Sixty-eight female Swiss mice were submitted to splenectomy. The spleen from each animal was sliced and the slices were implanted into the abdominal cavity. Groups of animals were killed after 1, 2, 3, 5, 7, 15, 21, 28, 42, 56, 70 and 84 d. Fluorescent polystyrene microspheres were injected via the orbital venous plexus before killing and the splenules were removed 5 min later for light and electron microscopy. Mesenteric blood vessels were injected with coloured latex to study the origin of the nutrient vessels. Three days after the implant the microspheres were observed at the periphery and then migrating to the internal parts of the implant in the subsequent days. The blood supply to the implants originated from branches of the splenic, short gastric, mesenteric and gastroepiploic arteries. It is concluded that revascularisation of splenic autografts proceeds centripetally, starting as early as 3 d after implantation.

Late Results of Heterotopic Autotransplantation of Splenic Tissue into the Greater Omentum

There is still controversy about the beneficial effects of splenic autotransplantation. As a result, replantation of the spleen has almost been abandoned for surgical treatment of splenic injuries. To learn more about the long-term effects we have analyzed eight patients 9.2 +/- 1.2 years after autotransplantation of the spleen into the greater omentum. Splenic replants could be detected by scintigraphy in seven patients (87.5%). Splenic size, measured by single-photon emission computed tomography, varied between 19.344 and 471.705 units of volume (Uov). Two patients with the smallest replants had pathologic changes of leukocytes and lymphocyte subsets in 12 of 16 measurements (75%). These changes closely resembled a spleenless state and were similar to those in the patient with no detectable splenic autograft. In contrast, five patients with large splenic replants (49.192-471.705 UoV) revealed pathologic findings in only 2 of 40 measurements (5%). Based on an extensive review of the literature and our own results, it can be concluded that splenic autotransplantation into the greater omentum is superior to splenectomy but undoubtedly less effective than in situ preservation of the spleen. Therefore splenic replantation should still be considered, particularly in pediatric surgery, when in situ preservation of the spleen is not possible by other techniques.

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.

Morphological analysis of neovascularization at early stages of rat splenic autografts in comparison with tumor angiogenesis

This study was undertaken to reveal the neovascularization at early stages of splenic autografts three-dimensionally, to illustrate the differences between it and tumor angiogenesis, and to establish its origin. Early vascular formation after transplantation of the rat spleen or Walker tumor into the major omentum was examined by using a video macroscope, vascular casting methods and the organ culture technique. A complex vascular network layer (vascular cortex) was first formed beneath the capsule of an autograft; later, vascular buds grew from this network toward the necrotic center. They anastomosed and changed into a form resembling withered twigs (vascular medulla). Tumor angiogenesis did not present such morphological features and was characterized by capillary loop formation with a columnar vertex resembling an "inverted V". This fundamental structure did not change throughout angiogenesis except for dilation and irregularity of vascular diameter. The organ culture technique demonstrated that the preliminary vasculature was formed in splenic autografts by regeneration of preexisting vessels in the graft and not by invading capillaries. Transmission electron microscopy showed that the cells present had characteristics of sinus endothelial cells. These results suggest that preexisting sinus endothelial cells rearrange themselves after devascularization and reconstruct a new vasculature that anastomoses with the penetrating capillaries. This mechanism establishes vascular circulation at an early stage, and accelerates regeneration of the splenic autograft before complete necrosis.

Splenic autotransplantation in nude mice

Splenic fragments were autotransplanted into the greater omentum of nude mice (nu/nu) and normal mice to investigate the formation of white pulp in splenic regeneration. The formation of white pulp began from day 5 after transplantation in both groups. Two patterns were found. In one pattern, follicles containing only B cells were first formed and developed into white pulp with poorly developed periarterial lymphoid sheaths (PALS). Most of the white pulp in nude mice and about one third of white pulp formed at day 28 in normal mice followed this pattern. In the second pattern, PALS were first formed and afterwards follicles appeared in the periphery of PALS. This was mainly seen in normal mice. Thus splenic autografts in normal mice contain white pulp with poorly developed PALS, and the homing of T cells begins later than that of B cells. This suggests the possibility of incomplete cellular immunity.

Dynamic Change of the Vascular Wall from Transmural to Intercellular Passage of Red Blood Cells Observed in Splenic Regeneration

Rat splenic tissues were autotransplanted into the major omentum, and the operated animals were treated with phenylhydrazine to investigate the passage of erythrocytes through the vascular wall during splenic regeneration. Both ways of the passage were differentiated during regeneration. From day 1 to day 5 after transplantation, the pores were formed in the endothelial cells, through which erythrocytes (chiefly reticulocytes) migrated, and were closed by the basal lamina when erythrocytes did not penetrate. From day 7 to day 10, endothelial cells proliferated, and some of them were transformed into sinus endothelial cells containing condensed microfilaments and formed the interendothelial slit, but few erythrocytes passed there at this stage yet. On and after day 11, when the sinus endothelial cells exhibited well-developed microfilaments, reticular cells contained moderately developed microfilaments and the basal lamina developed well, the slits were opened, where a large number of erythrocytes passed. These results showed, concerning the passage of blood cells, that the vascular wall in the splenic autografts changed from the transmural pattern to the intercellular one after a marked proliferation of endothelial cells and that the effective passage of erythrocytes was closely associated with the development of microfilaments in the cytoplasm of endothelial cells and basal lamina as well as the interaction of reticular cells.

Splenic implants: influence of particle size and fibrin fixation on vascularization and angioarchitecture

Splenic autotransplantation in tissue is a clinical method for partial preservation of splenic function following total splenectomy. Microangioarchitecture and vascular density of the implants as prerequisites for splenic function and the influence of particle size and fixation on neo-and revascularization have not been analyzed in detail. Homologous spleen particles of different sizes were implanted into transparent dorsal skinfold chambers in 30 golden hamsters. By means of intravital microscopy and computerized video analysis techniques, the process of capillary sprouting could be quantitatively analyzed in the awake animal over a 14-day period following implantation. Measurements included vessel diameter, vascular density, and tissue surface pO2. Based on the results obtained, we suggest limiting the size of splenic autografts to a volume that can be nourished by diffusion until the implants become vascularized. The use of fibrin glue for implant fixation does not interfere with vascularization of the graft.

Compensatory spleen growth and protective function in rats.

To quantify compensatory spleen growth after reduction in splenic mass and to determine the effect of age and vascular supply on remnant growth, we examined adult and weanling rats for remnant regeneration and adult rats for host protection against bloodstream pneumococcal challenge at intervals up to 180 days after partial resection or autograft implantation. In adults, subtotally resected spleens with remnant blood flow and segmental anatomy otherwise intact (eutopic remnants) displayed prompt but relatively limited gains in remnant weight and nucleated cell number, and growth after 60 days was minimal; survival after pneumococcal challenge was directly related to initial remnant weight and unrelated to length of postoperative interval. Splenic autografts (ectopic remnants), by comparison, underwent necrosis early after devascularization, recovered initial remnant weight slowly, fell far short of restoring original cellularity, and even 6 months after implantation barely attained initial size; moreover, host protection against pneumococcaemia was no different in rats with autografts than in those with no residual spleen. Remnants in situ (both partial resection and whole spleens) grew more vigorously in weanling than adult rats. The results suggest that splenic regeneration after either partial resection or implantation is much more limited than previously suggested, at least in disease-free rats, and is regulated by size and integrity of the initial remnant spleen, adequacy of vascular inflow, and age of the host. Whereas eutopic remnants provide protection against bloodborne pneumococcal challenge in proportion to initial residual size, ectopic remnants even with prolonged maturation fail to display immunoprotective function.

Neovascularization in the splenic autograft transplanted into rat omentum as studied by scanning electron microscopy of vascular casts

Neovascularization during regenerating process in splenic tissue autotransplanted into rat omentum was studied by means of scanning electron microscopy of vascular casts. The results obtained indicated that neovascularization is classified into the following four steps; capillarization in the connective tissue surrounding the degenerating autograft, connection of blood vessels between the still surviving splenic cords and capillaries in the surrounding tissue, rebuilding of the splenic sinuses and remodelling of regenerated blood vessels. This neovascularization process is specific in that the preexisting splenic cords play an important role in angiogenesis during regenerative process in the autograft.

Scintigraphic imaging of autotransplanted splenic grafts by 99mTc-labeled heat-damaged erythrocytes

Splenectomy is known to increase the risk of bacterial infection. Recently splenic autotransplantation has been suggested as a method of preserving splenic function. In order to demonstrate the viability of transplanted tissue, spleen scintigraphy using 99mTc labeled heat damaged erythrocytes were carried out. So far 21 studies have done in 12 patients. Spleen scans were positive 1 month after surgery, though images showed poor contrast against considerable background of bone marrow and blood pool. The quality of the images much improved five to twelve months after surgery. Functioning splenic autografts could be also shown by scintigraphy using 99mTc sulfur colloid, but the image quality was poorer, particularly within the early stage after operation. Labeling yields were 79.8% on the average, ranging from 45.6-92.3%, that affected little the quality of images. Important techniques in the splenic autotransplantation imaging include a thorough elimination of free 99mTcO4- before injection and to use comparatively small volume of damaged erythrocytes.

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.

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.