Transplantation In Dogs Research Articles

Recombinant human granulocyte colony-stimulating factor accelerates hematopoietic recovery after DLA-identical littermate marrow transplants in dogs

We studied whether treatment of dogs with recombinant human granulocyte colony-stimulating factor (rhG-CSF), after 920 cGy total body irradiation (TBI) and transplantation of 3.3 +/- 1.0 x 10(8) bone marrow cells per kilogram from a DLA-identical littermate, accelerated hematopoietic recovery and influenced the incidence of subsequent marrow graft failure or graft-versus-host disease (GVHD). Ten animals were treated with 100 micrograms rhG-CSF/kg/d from days 1 through 10 after TBI. Results were compared with those of historical control of 14 dogs not administered rhG-CSF. Neither group of dogs received GVHD prophylaxis. The median time to recovery of 1,000 neutrophils/mm3 was 8 days for dogs administered rhG-CSF compared with 14 days in controls (logrank test: P less than .03). The median time to reach 100 monocytes/mm3 was 17 days in G-CSF-treated dogs compared with 49 days in controls (P less than .002). The median time to attain 500 lymphocytes/mm3 was 15 days versus 31 days, respectively (P less than .01). The median time to reach 20,000 platelets/mm3 was 26 versus 20 days (P = .68). Graft failure occurred in 1 of 10 G-CSF-treated dogs versus 2 of 14 controls (two-tailed Fisher's exact test: P = 1.00). GVHD was seen in 4 of 9 rhG-CSF-treated dogs compared with 1 of 12 controls (P = .12). Two G-CSF-treated dogs died of GVHD versus none of the controls (P = .17). No unusual toxicities were seen in dogs receiving rhG-CSF. In summary, rhG-CSF significantly accelerated recovery of neutrophils, monocytes, and lymphocytes after DLA-identical littermate marrow transplantation without altering platelet recovery. Graft failure was not seen more often than in controls, but there was a trend toward an increased incidence of GVHD.

Oxygen free radical scavengers decrease reperfusion injury in lung transplantation

An in vivo canine model was used to assess the ability of an oxygen free radical scavenger to decrease reperfusion injury in lung transplantation. In 12 dogs, the left lungs were transplanted after they had been preserved for 24 hours at 4 °C after pulmonary artery flushing with modified Eurocollins solution. In 6 dogs, dimethylthiourea, a potent oxygen free radical scavenger, was added to the flush solution and was also given to the recipients just before reperfusion. In all animals, the contralateral pulmonary artery and bronchus were ligated and lung function was assessed for 12 hours or until death. Three dogs died prematurely in the control group, whereas only 1 dog died prematurely in the dimethylthiourea group. This resulted in a statistically significant difference in the average length of survival ( p < 0.05). Pulmonary artery and right atrial pressures were significantly lower in the dimethylthiourea group during the first 6 hours ( p < 0.05). Treatment with dimethylthiourea resulted in a significantly higher arterial oxygen tension at 4 hours, and intrapulmonary shunt tended to be lower. Thus, it would appear that dimethylthiourea has a protective effect on lungs preserved for 24 hours before transplantation in dogs.

Orthotopic Liver Transplantation in Dogs by Veno - venous bypass using Centrifugal Pump

Orthotopic Liver Transplantation in Dogs by Veno - venous bypass using Centrifugal Pump

Contribution of Partial Pancreatectomy, Systemic Hormone Delivery, and Duct Obliteration to Glucose Regulation in Canine Pancreas: Importance in Pancreas Transplantation

Our aim was to isolate and determine the contribution of partial pancreatectomy, systemic delivery of pancreatic hormones, and duct obliteration to glucose regulation after segmental pancreas transplantation in dogs. Fasting, postprandial, and intravenous glucose-stimulated glucose, insulin, glucagon, pancreatic polypeptide (PP), and cholecystokinin (CCK) and intravenous bombesin-stimulated PP levels were studied in beagles at three successive intervals in a crossover design. The first was 6 wk after partial (approximately 70%) pancreatectomy with intact regular enteric exocrine drainage from the duodenal pancreatic remnant, the next was 2 wk after venous transposition with systemic delivery of pancreatic hormones, and the third was 6 wk after in situ duct obliteration of the remnant. With partial pancreatectomy, K values were modestly diminished (30%), and a concomitant reduction of second-phase intravenous glucose-stimulated insulin release was observed. Other parameters were not significantly affected. Venous transposition doubled peripheral plasma levels of insulin under all conditions. Fasting glucose, PP, and CCK levels decreased slightly. Other parameters were not affected. Duct obliteration of the systemic draining pancreatic remnants seriously impaired glucose sensitivity, resulting in a 50% reduction of K values and fasting and sustained postprandial hyperglycemia (approximately 8 mM) and a 70-50% reduction (acute and overall responses, respectively) of intravenous glucose-stimulated insulin. Fasting hormone and postprandial insulin, glucagon, and CCK levels were not affected. The postprandial PP response was severely reduced, and bombesin-stimulated PP release was abolished by duct obliteration. We conclude that histological changes associated with duct obliteration are the major determinants of glucose regulation in segmental pancreas transplantation.(ABSTRACT TRUNCATED AT 250 WORDS)

Cold storage preservation of pancreatic tissue prior to and after islet preparation in a dog autotransplantation model

Utilizing an intrasplenic canine islet autotransplant model, the effects of cold storage preservation on pancreatic tissue prior to and after collagenase dispersion were examined. A control series, in which freshly retrieved and prepared tissue was transplanted, yielded a 75% success rate (6/8). In contrast, when the pancreas was stored in modified silica gel filtered plasma (SGF I) for 24 hr, no autotransplant was successful (0/6). However, when the islet tissue was prepared following pancreatectomy and then stored in a mannitol-containing modification of SGF (SGF III), autotransplantation was successful in 83% (5/6) after 24 hr of preservation and in 60% (3/5) after 48 hr of preservation. Similarly, the islet tissue was stored in a hyperkalemic hydroxyethyl starch solution (HES) and this was successful in 20% (1/5) after 24 hr of preservation and in 50% (1/2) after 48 hr of preservation. Cold storage preservation techniques for the pancreas prior to islet isolation need to be refined, but dispersed islet-enriched pancreatic tissue can be successfully maintained at 4°C for up to 48 hr prior to transplantation in dogs using established pancreas preservation solutions.

Pulmonary flow-resistance relationships in allografts after single lung transplantation in dogs

To evaluate the applicability of single lung transplantation as a treatment of pulmonary hypertension, we investigated the relationships between the pulmonary blood flow and resistance in allografts 10 days after single lung transplantation in dogs. In eight surviving recipients and five healthy dogs (control group), pulmonary flow-resistance relationships were investigated at flows in the range of 0.3 to 2.0 L/min at 0.1 L/min increments. Resistance decreased from 2585 to 746 dyne.sec.cm-5 in the successful group (five allografts receiving complete studies) and 2400 to 891 in the control group with an increase of flow. Lung water content of the successful group was 2.0 times that of the control group (successful group, 95.6 +/- 16.7 gm; control group, 47.8 +/- 7.5 gm), and water content of the other allografts (unsuccessful group), including allografts of dogs that died before investigations, was 2.2 times that of the successful group (unsuccessful group, 211.0 +/- 89.6 gm). The chest roentgenograms of the successful group showed mild or moderate consolidations, whereas those of the unsuccessful group showed severe consolidations, but no difference was found in pulmonary flow-resistance relationships between the successful and the control groups. These results show that the allografts, which have about twice the water content of normal lung, maintain adequate pulmonary vascular function. We concluded that single lung transplantation could be an effective treatment for pulmonary hypertension.

Orthotopic partial liver transplantation in dogs can be performed without cold perfusion of the donor liver. Evaluation of its feasibility in terms of energy metabolism.

To investigate the feasibility of obtaining grafts from living, genetically related adult donors without any complicated harvesting techniques, orthotopic partial liver transplantation (PLT) without cold perfusion of the donor graft was evaluated in beagles by assessing the graft viability using ketone body ratio (KBR). Ten PLT were performed under venovenous bypass. The left half of the donor liver was transected in situ followed by systemic heparinization, and immediately implanted with care taken to leave the recipient's inferior vena cava intact, and to maintain the normothermic state without cold perfusion. Four of ten dogs survived for 5 days or longer (longest survival was 9 days) and died of other causes than graft dysfunction. Four others died of thromboembolitic episode, accidental bleeding or technical failure. Two dogs died of graft dysfunction. The changes in KBR in 5 dogs without fatal complications after transplantation were maintained within normal range thereafter. These results suggest that the PLT without cold perfusion of donor graft is possible from the viewpoint of energy metabolism.

Single dose or fractionated total body irradiation and autologous marrow transplantation in dogs: Effects of exposure rate, fraction size, and fractionation interval on acute and delayed toxicity

Dogs were given single dose or fractionated total body irradiation (TBI) and autologous marrow grafts to prevent death from myelosuppression. Acute and delayed non-marrow toxicities were compared. Fifty-six dogs were given single dose TBI at 2.1 (n = 13), 5 (n = 12), 10 (n = 15), or 20 (n = 16) cGy/min. Acute radiation toxicity and mortality was related to the exposure rate; radiation doses resulting in 50% mortality at 7 days (LD 50/7) at 2.1, 5,10 and 20 cGy/min were 1692,1499,1261, and 1056 cGy respectively. Fifty-three dogs were given fractionated TBI, 200 cGy three times a day with 6-hour intervals at 2.1 (n = 13), 5 (n = 9), 10 (n = 13), or 20 (n = 18) cGy/min. The LD 50/7 at the four exposure rates were 1628, 1470, 1184, and 1320 respectively. Thus, for exposure rates of 2.1, 5, and 10 cGy/min, the tolerated doses were comparable for single dose and fractionated TBI. At 20 cGy/min dose fractionation appeared to offer some advantage, although this fractionation effect in part may have been due to random variation with small numbers of dog treated. Following recovery from the immediate TBI-related toxicity, eight dogs given single dose and four dogs given fractionated TBI died, generally from infections, 8–30 days following transplantation. There was a striking difference in regards to long-term survival dependent upon the TBI regimen. Among dogs given ≥1000 cGy of TBI and alive 30 days after transplant only 1 of 18 given single dose TBI became a long-term survivor compared to 19 of 22 given fractionated TBI. Causes of death included pancreatic fibrosis, malnutrition, hepatic failure, and a generalized wasting syndrome. All 5 dogs given a single dose of 800 cGy (at 20 cGy/min) became long-term survivors. Fourteen dogs were given increments of 150 cGy at 7 cGy/min every 3 hours for total doses of 1500–2400 cGy. The LD 50 7 was approximately 1900 cGy. All 6 dogs alive at 30 days became healthy long-term survivors. Four dogs were given increments of 600 cGy at 2.1 cGy/min every 48 hours for a total dose of 1800 cGy. All 4 dogs became long-term survivors. In conclusion, exposure rate and total dose are the most important parameters for acute toxicity associated with TBI. The effect of dose fractionation is minimal at low exposure rates and appears to be dependent also upon increment size and fractionation interval. Long-term survival after total doses of 1000 cGy or greater was significantly improved by dose fractionation.

Toxicity trial of prophylactic 9-[2-hydroxy-1-(hydroxymethyl)ethoxymethyl]guanine (ganciclovir) after marrow transplantation in dogs.

The effects of 9-[2-hydroxy-1-(hydroxymethyl)ethoxymethyl]guanine, or ganciclovir, administered during the immediate posttransplant period to dogs given total-body irradiation and autologous marrow transplants were studied. Doses of ganciclovir of 3.0 mg/kg (body weight) per day were well tolerated without detectable delay in hematopoietic recovery, whereas doses of 5.0 mg/kg per day were associated with delayed platelet recovery.

Cardiac transplantation in dogs: evaluation with gated MRI and Gd-DTPA contrast enhancement.

It is important to evaluate the severity and extent of cardiac rejection in heart transplantation. Six heterotopic heart transplantation models in the peritoneal cavity were prepared with 12 adult mongrel dogs and in vivo imaging of the donor heart was performed using gated magnetic resonance imaging, (MRI) and Gd-DTPA contrast enhancement. In cardiac rejection, high signal intensity was obtained in the rejected myocardium, especially from the right ventricular wall to the intraventricular septal wall. The rejected myocardium was clearly visualized after administration of Gd-DTPA (0.5 mmol/kg body weight) via the femoral vein in all donor hearts. The mean ratios of signal intensity calculated from the rejected and normal myocardium were also increased from 25% to 42% after administration of Gd-DTPA. On the other hand, in the cases with no rejection, whole myocardium was visualized homogeneously before and after Gd-DTPA and there was no high signal intensity. Thus, our method has the potential of assessing the extent and severity of cardiac rejection using gated MRI and Gd-DTPA contrast enhancement.

Heterotopic Heart–Unilateral Left Lung Transplantation in Dogs

A technique is presented for transplantation of the heart and left lung in the canine model. Preservation of the recipient's heart and right lung allowed heterotopic en bloc transplantation of the donor organs, thus preserving the normal respiratory pattern and obviating the need for cardiopulmonary bypass. Four experiments were performed to develop the surgical technique. The mean survival of the remaining 10 dogs studied was 23 ± 28 days (range 3 to 91 days). The transplanted heart and lung could be investigated as in orthotopic heart-lung transplantation; bronchoscopy with bronchoalveolar lavage and endobrachial biopsy as well as endomyocardial biopsy were performed by standard approaches. This model is suitable for physiological and immunological long-term observations of cardiopulmonary transplantation in dogs.

Engraftment of dogs with Ia-positive marrow cells isolated by avidin- biotin immunoadsorption

Previous work has shown failure of engraftment in lethally irradiated dogs when autologous marrow was depleted of Ia-positive cells with an anti-Ia antibody and complement before infusion. In the current study, we have utilized an avidin-biotin immunoadsorption procedure to obtain a population of highly enriched Ia-positive cells for autologous bone marrow transplantation in dogs given lethal irradiation. Dog marrow cells (2.4 to 7.0 X 10(9) cells) that contained 8.6% to 19.9% Ia- positive cells were treated successively with monoclonal antibody 7.2, which reacts with a framework determinant of Ia-antigen, and biotin- conjugated goat antimouse immunoglobulin. These treated cells were passed over a column of avidin-Biogel (polyacrylamide) and the adherent cells removed by mechanical agitation. Seven lethally irradiated dogs were transplanted with 5.9 to 33.4 X 10(6) recovered adherent cells per kilogram of which 69.0% to 88.0% were Ia-positive. All dogs had hematologic recovery; six are alive and well with durable engraftment and one died on day 15 posttransplant. They are immunologically normal as determined by lymph node and bone marrow biopsies, lymphocyte function, and immunophenotyping of peripheral blood and bone marrow cells. These data provide further evidence that canine hematopoietic stem cells express Ia-like antigens and that these cells are capable of complete hematopoietic and immunologic reconstitution in an autologous model.

Engraftment of Dogs With Ia-Positive Marrow Cells Isolated by Avidin-Biotin Immunoadsorption

Previous work has shown failure of engraftment in lethally irradiated dogs when autologous marrow was depleted of Ia-positive cells with an anti-Ia antibody and complement before infusion. In the current study, we have utilized an avidin-biotin immunoadsorption procedure to obtain a population of highly enriched Ia-positive cells for autologous bone marrow transplantation in dogs given lethal irradiation. Dog marrow cells (2.4 to 7.0 X 10(9) cells) that contained 8.6% to 19.9% Ia-positive cells were treated successively with monoclonal antibody 7.2, which reacts with a framework determinant of Ia-antigen, and biotin-conjugated goat antimouse immunoglobulin. These treated cells were passed over a column of avidin-Biogel (polyacrylamide) and the adherent cells removed by mechanical agitation. Seven lethally irradiated dogs were transplanted with 5.9 to 33.4 X 10(6) recovered adherent cells per kilogram of which 69.0% to 88.0% were Ia-positive. All dogs had hematologic recovery; six are alive and well with durable engraftment and one died on day 15 posttransplant. They are immunologically normal as determined by lymph node and bone marrow biopsies, lymphocyte function, and immunophenotyping of peripheral blood and bone marrow cells. These data provide further evidence that canine hematopoietic stem cells express Ia-like antigens and that these cells are capable of complete hematopoietic and immunologic reconstitution in an autologous model.

Reappearance of Hering-Breuer reflex after bilateral autotransplantation of the lungs.

In combined heart-lung transplantation the afferent nerve pathways inevitably are transsected. In previous studies with en bloc heart-lung transplantation in dogs, we found altered regulation of breathing-abolition of Hering-Breuer reflex and response to hypercapnia inhalation stimulus consisting of augmented tidal volume with no change in respiratory rate-shortly after the operation. The long-term effects of pulmonary denervation on breathing regulation were now studied in dogs after staged bilateral pulmonary autotransplantation. Mechanical and electrical activities of the respiratory muscles were recorded during spontaneous breathing and after deflation and inflation with varying volumes of air. Five months postoperatively the duration of the respiratory cycle increased 2.5 times on inflation with 600 ml of air and occlusion of the airways, compared with tenfold prolongation in intact control dogs, indicating a partial return of the Hering-Breuer reflex after the autotransplantation. The duration of the EMG bursts in respiratory muscles increased in intact dogs and in those with bilateral lung autotransplants. In impulse frequency the response to stretching was less evident after autotransplantation. The mechanism mediating reappearance of Hering-Breuer reflex warrants further study.

Glucose Metabolism during Liver Transplantation in Dogs

Arterial and hepatic venous blood levels of glucose were studied in 12 dogs during orthotopic liver transplantation performed under ketamine anesthesia without exogenous glucose administration. During the early part of surgery, arterial blood glucose levels were stable: 161 +/- 12 mg/dl (mean +/- SEM) after laparotomy and 183 +/- 16 mg/dl 5 min before the anhepatic stage. During the anhepatic stage, arterial blood glucose levels decreased progressively to 135 +/- 9 and 88 +/- 8 mg/dl, 5 min in the anhepatic stage and 5 min before reperfusion of the graft liver, respectively (P less than 0.05). Reperfusion of the graft liver resulted in an increase in arterial glucose levels to 206 +/- 17 and 240 +/- 24 mg/dl, 5 and 30 min after reperfusion, respectively (P less than 0.05). Hepatic venous blood glucose levels increased after reperfusion (405 +/- 37 and 346 +/- 41 mg/dl, 5 and 30 min after reperfusion, respectively) and were significantly higher than in arterial blood (P less than 0.05). Arterial plasma insulin, measured in five animals, did not change significantly during the procedure, whereas plasma glucagon levels, stable during the preanhepatic and anhepatic stages, increased steadily after reperfusion of the graft liver, from 66.1 +/- 14.2 to 108.4 +/- 38.1 pg/ml (P less than 0.05). This study shows that in dogs with ketamine anesthesia mild hypoglycemia occurs during the anhepatic stage of liver transplantation without exogenous glucose administration followed by hyperglycemia on reperfusion of the graft liver, possibly secondary to the release of glucose from the donor liver.

Segmental auxiliary liver transplantation in dogs: a search for an ideal graft--illusion or reality?

Segmental auxiliary liver transplantation (SALT) has been carried out in 13 mongrel dogs to assess the possibility of a certain size of liver segment to accept without sequelae the total splanchnic and arterial blood normally diverted to the liver of the host. Prednisone (1 mg/daily) and azathioprine (2 mg/kg daily) were used as immunosuppression. Five dogs died during the first hours after the operation. Three because of technical failure and two of acute portal hypertension secondary to total portal and arterial blood diversion in dogs with liver segments of 195 +/- 49 g as a result of overloading of the graft. The remaining 8 dogs were divided into: 4 dogs into which a liver segment (195 +/- 49 g) was transplanted (group A) and 4 dogs in which a liver segment (385 +/- 85 g) was used (group B). Partial portal and total arterial blood diversion in group A dogs was not associated with portal hypertension but resulted in poor function of the graft and in poor survival. In contrast, the graft in group B dogs was able to cope with both total or partial portal blood and with a normal arterial blood diversion. Infection and graft rejection prohibited long-term survival (8-28 days). Data from this study support the view that the present technique of SALT with a graft corresponding to 300-400 g in mongrel dogs of about 30 kg is a potential alternative as temporary liver support in the diseased animal.

Variation of treatment conditions alters the outcome of fetal liver transplantation in dogs.

Fetal liver transplants (FLT) were carried out in 25 beagles under various conditions. Graft recipients were prepared with fractionated total body x-irradiation (TBI) of 3 X 6 Gy or 2 X 6 Gy and rescued with cryopreserved fetal liver cells (FLC) from 51- to 52-day-old or 43- to 46-day-old, DLA-identical siblings or DLA-haploidentical, homozygous half-siblings. In all groups, FLC grafts contained comparable numbers of granulocyte-macrophage progenitor cells. Initial engraftment was achieved in all dogs. However, low TBI dose and DLA haplotype disparity between donor and recipient were associated with graft failure in 1/3 and 2/9 recipients, respectively, within 10-16 days of treatment. Lectin-responsive host type lymphocytes circulated for more than 5 weeks, whereas bone marrow metaphases were always of donor sex. Reduced TBI dose and young donor age were associated with delayed granulocyte recovery. Moreover, circulating platelets and total lymphocytes as well as T and B-cell numbers and rose more slowly in recipients of immature FLC grafts than in the other groups. Delayed cutaneous hypersensitivity reactions were normal one year after FLT, whereas the IgM component of the hemagglutinin response to sheep red blood cells was depressed. In mixed leukocyte culture, chimeric lymphocytes were tolerant to host antigens. Nonetheless, clinical and histological signs compatible with low-grade graft-versus-host disease were recorded in 10 or 25 animals. Thus FLT in dogs could be carried out, even across DLA barriers, without severe graft-versus-host disease. However, a low pretransplant TBI dose, incomplete DLA match and young age of the fetal donor were associated with graft rejection and protracted restoration of hemopoiesis and immune functions.

Comparison of two methods of islet preparation and transplantation in dogs

Comparison of two methods of islet preparation and transplantation in dogs

Comparison of Two Methods of Islet Preparation and Transplantation in Dogs

Seventy-nine mongrel dogs underwent total pancreatectomy. Fifteen dogs served as apancreatic controls and died 7.0 +/- 4.2 days later (mean +/- SD). The pancreases of 44 dogs (group 1) were intraductally distended by manual injection of Hanks' balanced salt solution (HBSS). Thereafter each organ was mechanically disrupted and subjected to collagenase digestion as described by Mirkovitch et al. The pancreases of 20 dogs (group 2) were intraductally distended and subsequently perfused with collagenase by a roller pump. The organs were then mechanically disrupted and filtered through screens as described by Horaguchi et al. The resulting tissue suspensions were injected into the spleens of the dogs as autotransplants in both groups, by direct punction of the splenic capsule in group 1 and by retrograde infusion via a splenic vein tributary in group 2. The functional outcome was better in group 2 than in group 1, as assessed by the number of animals that became normoglycemic after transplantation [15/20 (75%) vs. 13/44 (30%); P = .0025]. The degree of islet purification, as measured by an increase in the tissue insulin/amylase ratio, was higher in group 2, and in both groups it was higher in normoglycemic than in hyperglycemic animals. The percent engraftment [i.e., amount of insulin recovered from spleen as percent of tissue transplanted (mean, 15.4% in group 1 and 14.5% in group 2) or as percent of original pancreas (mean, 4.9% in group 1 and 4.4% in group 2)] was low in both groups but again was higher in normoglycemic than in hyperglycemic animals within each group. In conclusion, both the degree of engraftment and purification and the route of implantation influenced the functional outcome after dispersed pancreatic islet autotransplantation to the spleen of totally pancreatectomized dogs, with purified tissue injected retrogradely functioning better than unpurified tissue injected directly.

Liver transplantation in dogs after 30 minutes of warm ischemia.

In order to investigate the applicability of liver transplantation after warm ischemia, a partial auxiliary auto-transplantation of the liver after 30 min of warm ischemia was carried out in 9 mongrel dogs. Among them, 6 dogs survived longer than 7 days. Liver functions, blood coagulability and histological changes were investigated before and after the transplantation. Serum GOT, LDH, and GPT levels were elevated immediately after transplantation, and returned to normal within a few days. Hemostatic tests also showed significant abnormalities after transplantation. Although PT and thrombotest became normal within a week, PTT and antithrombin III remained slightly abnormal for longer periods. Microscopically, vacuolization and degeneration of hepatocytes were observed after transplantation but they recovered completely within 4 weeks. Based on these results, it could be said that the livers subjected to warm ischemia for 30 min ischemia were still acceptable for auxiliary transplantation.