Long-term following-up of viability of spleen autotransplants in the Beagle canine model.

PURPOSE: To examine the possible late complications of splenectomy or spleen autotransplantation in large laboratory animal model, in which we need non-invasive or minimal-invasive methods for long-term monitoring of the experimental animals. METHODS: Experimental groups of beagle dogs were: non-operated control, sham-operated control, splenectomy, spleen autotransplantation with 5 or 10 spleen-chips taken into the greater omentum (Furka's technique). Prior to operations, on the 1(st) postoperative week, monthly till the 6(th) as well as in the 9(th) and 12(th) month, hemorheological examinations were performed. In postoperative 12(th) month colloid scintigraphy and diagnostic laparoscopy were carried out. At the end of the investigation comparative morphological examinations were performed, too. RESULTS: From the 4(th)-5(th) postoperative month filtration function of spleen-autotransplants showed particular restoration compared to splenectomy group. However, the functional results did not reach the values of the control or sham-operated groups. Sham-operated control's scintigraphy nicely showed activity in the spleen. In spleen autotransplantation-groups scintigraphy indicated well the activity of spleen-chips. During diagnostic laparoscopy spleen-chips with their blood supply were found. Histologically, the structure of spleen-autotransplants was similar to normal splenic tissue. CONCLUSIONS: The autotransplants are regenerated, their functions have been partly restored, and thus spleen autotransplantation may prevent the possible complications of splenectomy. These parameters and the presented investigative protocol are suitable for long-term following-up of viability of the spleen-autotransplants.

Long-term following-up of viability of spleen autotransplants in the Beagle canine model / Avaliação em longo prazo da viabilidade do autotransplante de baço em cães Beagle

PURPOSE: To examine the possible late complications of splenectomy or spleen autotransplantation in large laboratory animal model, in which we need non-invasive or minimal-invasive methods for long-term monitoring of the experimental animals. METHODS: Experimental groups of beagle dogs were: non-operated control, sham-operated control, splenectomy, spleen autotransplantation with 5 or 10 spleen-chips taken into the greater omentum (Furka's technique). Prior to operations, on the 1st postoperative week, monthly till the 6th as well as in the 9th and 12th month, hemorheological examinations were performed. In postoperative 12th month colloid scintigraphy and diagnostic laparoscopy were carried out. At the end of the investigation comparative morphological examinations were performed, too. RESULTS: From the 4th-5th postoperative month filtration function of spleen-autotransplants showed particular restoration compared to splenectomy group. However, the functional results did not reach the values of the control or sham-operated groups. Sham-operated control's scintigraphy nicely showed activity in the spleen. In spleen autotransplantation-groups scintigraphy indicated well the activity of spleen-chips. During diagnostic laparoscopy spleen-chips with their blood supply were found. Histologically, the structure of spleen­autotransplants was similar to normal splenic tissue. CONCLUSIONS: The autotransplants are regenerated, their functions have been partly restored, and thus spleen autotransplantation may prevent the possible complications of splenectomy. These parameters and the presented investigative protocol are suitable for long-term following-up of viability of the spleen-autotransplants.

OBJETIVO: Examinar as possíveis complicações tardias da esplenectomia ou do autotransplante de baço em modelo animal de grande porte, no qual faz-se necessário o uso de métodos não invasivos ou minimamente invasivos para monitorizar os animais de experimentação. MÉTODOS: Grupos experimentais de cães beagle foram: não-operados controle, sham-operados controle, esplenectomia, autotransplante de baço com 5 ou 10 fatias de baço colocados no grande omento (técnica de Furka). Antes das operações, na 1ª semana de pós­operatório, mensalmente até 6o.assim como no 9º. e 12º. mês, foram realizados exames hemorreológicos. No 12º. mês de pós-operatório, cintilografia colóide e laparoscopia diagnóstica foram realizadas. Ao final do experimento, exames morfológicos comparativos foram realizados também. RESULTADOS: A partir do 4º-5º mês pós-operatório, a função de filtração dos baços autotransplantados mostraram particular restauração comparados ao grupo esplenectomia. Entretanto, os resultados funcionais não alcançaram os valores dos grupos controle ou sham-operados. A cintilografia dos controles sham-operados mostraram atividade no baço. Nos grupos de autotransplante, a cintilografia indicou bem a atividade das fatias de baço. Durante a laparoscopia diagnóstica, as fatias de baço com seu suprimento sanguíneo foram encontrados. Histologicamente, a estrutura dos autotransplantes de baço foi similar ao tecido normal de baço. CONCLUSÕES: Os autotransplantes são regenerados, suas funções foram parcialmente restauradas, e então ao autotransplantate esplênico pode prevenir as possíveis complicações da esplenectomia. Estes parâmetros e o protocolo experimental são adequados para o seguimento em longo prazo da viabilidade de autotransplantes esplênicos.

Hemorheological consequences of hind limb ischemia-reperfusion differs in normal and gonadectomized male and female rats.

It is known that hemorheological parameters show gender differences that might be altered by gonadectomy (GoE). Since micro-rheological parameters (erythrocyte deformability and aggregation) sensitively change during and after ischemia-reperfusion (I/R), the question arises whether the hemorheological effects of I/R may show gender differences and further changes might be expected when GoE and I/R are additive. Sprague-Dawley rats were divided into six groups: Control males and females, I/R males and females with 1-hour hind limb ischemia, GoE + I//R males and females when 3 months after bilateral gonadectomy the I/R was induced. Before and just after ischemia, and on the 1st-3rd-5th-7th postoperative days blood samples were taken (lateral tail vein, 0.3-0.5 mL) for analyzing hematological parameters, erythrocyte's deformability (slit-flow ektacytometer) and aggregation (light-transmission aggregometer). Leukocyte and platelet counts raised markedly in gonadectomized animals during the investigated days. Hemorheological changes of I/R showed gender differences: significant impairment of erythrocyte deformability was found on the 1st-3rd postoperative days, expressed mostly in females. In gonadectomized females the postischemic deformability values were impaired. Erythrocyte aggregation index significantly raised by the 1st postoperative day, dominantly in males. It is suggested that gonadectomy may act as an additional rheological 'risk factor' related to blood micro-rheological parameters in ischemia-reperfusion.

Morphological, hemodynamical and hemorheological changes of mature artificial saphenous arterio-venous shunts in the rat model.

Artificial femoral arterio-venous (AV) shunts are widely used in rodent models for studying shunt maturation and to optimize various surgical techniques. However, little is known about complex circulatory, microcirculatory, and hemorheological effects of end-to-side saphenous AV shunts. We aimed to study these parameters in mature AV shunts. Studying these questions in CD rats, end-to-side anastomoses were made between the left saphenous artery and vein. On the right-side the nonoperated saphenous vessels served as own control. Furthermore healthy control animals were also investigated. On the 8th to 12th postoperative week microcirculatory and blood flow measurements were performed and blood samples were taken both from the shunt's arterial and venous limbs and from the nonoperated side vessels. Hematological parameters, erythrocyte aggregation, and deformability were determined. The entire shunt and the control vessels were removed for histological examinations. The skin microcirculation on shunt side slightly increased on thigh and decreased on paws versus the nonoperated side. Blood flow measurements made directly on the vessels showed that arterial to venous blood flow rate ratio was 1.59 ± 0.29 on nonoperated side and 1.2 ± 0.13 on the shunt side, and 1.49 ± 0.05 in control animals. Erythrocyte aggregation and deformability worsened on the shunt side. Histologically increased number of smooth muscle elements and connective tissue were found in venous limb of the shunts. The artificial AV shunt between the saphenous artery and vein seems to be a suitable model for further functional-morphological and hemorheological examinations of hemodialysis in various states and diseases.

Examination of aggregation of various red blood cell populations can be informative in comparison of splenectomy and spleen autotransplantation in animal experiments.

Using "Furka's spleen-chip" autotransplantation technique, in a follow-up study we investigated erythrocyte aggregation and its influencing factors regarding to asplenic-hyposplenic conditions. Healthy beagle dogs were equally divided into 4 groups: "Sham"- sham operated control, "SE"-splenectomy, "AU-5" and "AU-10"-autotransplantation with 5 or 10 spleen chips by Furka-method after splenectomy. Blood sampling for current investigation occurred on the 12th, 18th, 20th and 24th postoperative months, besides comprehensive structural and functional examinations. In this study we focused on the aggregation properties of erythrocytes in native samples and of density separated "young" versus "old" erythrocytes (Myrenne MA-1 aggregometer). Fibrinogen concentration (Sysmex CA-500 coagulometer) and erythrocyte sedimentation rate (ESR) were also determined. SE group showed significantly elevated ratio of "old" and "young" erythrocyte aggregation index values on the 20th and 24th postoperative months (1.5 and 2.16, respectively) comparing to the AU (1.14 and 1.05) and Sham groups (1.2 and 1.27). In SE group fibrinogen concentration was mildly higher and ESR almost doubled versus control. The greater differences experienced between "young" and "old" erythrocytes may reflect decreased splenic filtration function. Consequently, the comparison of aggregation and deformability properties of "young" and "old" erythrocytes may give important information about the function of the spleen.

Splenic function and red blood cell deformability: The beneficial effects of spleen autotransplantation in animal experiments.

Erythrocyte deformability could be a suitable parameter reflecting hyposplenic-asplenic conditions after splenectomy or spleen autotransplantation. The aim of this study was to analyze the efficacy of spleen autotransplantation using filtrometry and ektacytometry for testing erythrocyte deformability, besides complex structural, functional, hematological, hemostaseological and immunological examinations. Healthy beagle dogs were subjected to the study in the following experimental groups: "Sham"-sham operated control, "SE"-splenectomy, "AU-5" and "AU-10"-autotransplantation with 5 or 10 spleen chips using "Furka's spleen chip" technique after splenectomy. Blood samples were taken before the operations and monthly afterwards for 1 year and bi-monthly till the end of the 2nd postoperative year. Erythrocyte deformability was measured by bulk filtrometry (relative cell transit time, RCTT) and on the 20th-24th postoperative months by ektacytometry (elongation index, EI), too. RCTT was elevated in operated groups till the 12th month. On the 20th/24th months "SE"-group expressed the highest level, while the "AU"-groups were close to "Sham"-group. Using ektacytometer we found that on the 20th/24th month both EImax and SS1/2 showed the lowest values (because of unusual curve characteristic) in "SE"-group and "AU"-groups almost reached the "SH"-group. Spleen autotransplantation partially restored the normal rheological conditions. Both filtrometry and ektacytometry was suitable to investigate hyposplenic-asplenic conditions.

Application of leukocyte antisedimentation rate calculation in investigation of spleen salvaging experimental surgical techniques.

Clinical studied showed leukocyte antisedimentation rate (LAR) changes in various conditions that modulate the immune system. There is a lack of LAR data in animal experiments concerning splenic function after splenectomy or spleen preserving operations, thus we aimed to include LAR in our ongoing inbred canine study on following-up spleen salvaging surgical techniques. Blood samples of healthy beagle dogs served as normal control. The experimental groups were: "SH"-sham operated, "SE"-splenectomy, "R1/3" and "R2/3"-one-third/two-third of the spleen resected, "AU-5" and "AU-10"-autotransplantation with 5/10 spleen chips using Furka's techniques. On the 12th postoperative month Rabigen-Mono+Vanguard-Plus-5 vaccines were given to the animals, which provoked immune response. Blood samples were taken before and one week after the vaccination. LAR was calculated by Bogar's method. In normal control group LAR was 3.11, leukocyte count was 12.04 G/l. In the experimental groups LAR varied between 2.4-7.3 before vaccination. After vaccination LAR increased in all groups. Although "SE" group expressed the largest changes (LAR = 16.56; 135%), the leukocyte count increased only by 12%. In "AU" groups the increase was smaller, while "R" groups showed slight changes only. LAR can be applicable in animal experiments and may serve as a supplementary parameter in hemorheological investigations of hyposplenic-asplenic states.

Changes of local and systemic hemorheological properties in intestinal ischemia-reperfusion injury in the rat model.

The aim of this study was to investigate intestinal ischemia-reperfusion and its local and systemic hemorheological relations in the rat. Ten anaesthetized female CD outbred rats were equally divided into 2 experimental groups. (1) Ischemia-reperfusion (I/R): the superior mesenterial artery was clipped for 30 minutes. After removing the clip, 60 minutes of the reperfusion was observed before extermination. Blood samples were taken from the caudal caval vein and from the portal vein before ischemia, 1 minute before and after clip removal, and at the 15th, 30th, and 60th minutes of the reperfusion. (2) Sham operation: median laparotomy and blood sampling were done according to the timing as in I/R group. Hematological parameters, red blood cell aggregation, and deformability were determined. Leukocyte count and mean volume of erythrocytes increased slightly but continuously in portal venous samples during the reperfusion period. Red blood cell aggregation values were higher in portal blood by the end of ischemia, and then became elevated further comparing to the caval venous blood. Both in caval and portal venous samples of I/R group red blood cell deformability significantly worsened during the experimental period compared to its base and Sham group. In portal blood red blood cell deformability was impaired more than in caval vein samples. Histology showed denuded villi, dilated capillaries, and the inflammatory cells were increased after a 30 minutes ischemia. In conclusion, intestinal ischemia-reperfusion causes changes in erythrocyte deformability and aggregation, showing local versus systemic differences in venous blood during the first hour of reperfusion.