Unloading the infarcted heart affect MMPs-TIMPs axis in a rat cardiac heterotopic transplantation model.

Ventricular assist devices may function as a bridge to recovery or heart transplantation, however, little is known about its mechanisms. This study examined the role of matrix metalloproteinases (MMP)-tissue inhibitors of metalloproteinases (TIMP) axis in the process of recovery after unloading in a rat ischemic-induce heart failure (HF) model. Myocardial infarction model was created with the coronary artery ligation. The infarcted rats hearts were unloaded by heterotopic cardiac transplantation (n=14). 2 weeks later, the function of normal and infarcted hearts with or without loading was evaluated by Langendorff perfusion model. The hearts were then harvested and prepared for the study of expression of MMPs and TIMPs. Developed pressure in the unloading group was higher than the loading group (P=0.0074). Unloading increased the ratio of TIMP-1-MMP-1(1.38±0.11 vs. 0.76±0.09, P<0.05), TIMP-2-MMP-2 (1.06±0.10 vs. 0.33±0.07, P<0.01), TIMP-3-MMP-9(1.07±0.08 vs. 0.59±0.06, P<0.05). Although MMP-1, 2, 9 were downregulated (P<0.01, 0.01, 0.05, respectively), TIMP-2 and TIMP-3 upregulated (P<0.01, 0.05, respectively), MMP-7 and TIMP-1 was not affected significantly. The infarcted cardiac function could be improved by unloading. It was attributed to downregulation of MMP-1, 2 and 9, and upregulation of TIMP-2 and -3, and furthermore, the ratio of TIMPs to MMPs was increased, which might be more sensitive than sole MMPs or TIMPs for the judgment of myocardial matrix homeostasis.

[Extracellular matrix accumulation and expression of gelatinases and their tissue inhibitors in a mechanically unloaded heart model].

AIM: To investigate the relationship between the expression of MMP-2, MMP-9, TIMP-1 and TIMP-2 and ECM accumulation in rat left ventricle in a mechanical unloaded heart model. METHODS: 12-week-old male Lewis rats were subjected to abdominal heterotopic heart transplantation to achieve pressure and volume unloading(mechanical unloading). Age and sex matched in situ heart of Lewis rats were used as control. Collagen volume fraction(CVF) was analyzed by picrosiris-red staining plus polarized microscopy. MMP-2 and -9 gelatinolytic activity were measured by gelatin-zymography. mRNA level of MMP-2, MMP-9, TIMP-1 and TIMP-2 were measured by real-time quantitative PCR. TIMP-1 and TIMP-2 protein level were measured by immunoblotting. RESULTS: Myocardial cross-sectional area of transplanted heart was significantly reduced, and accompanied by excessive ECM deposition (CVF 5.22% +/- 1.6% vs. 2.21% +/- 0.9%, P < 0.05) compared to in situ heart. MMP-2 and MMP-9 activity were significantly increased, as well as mRNA level of MMP-2, MMP-9, TIMP-1 and TIMP-2 compared to in situ heart. TIMP-1 and TIMP-2 protein level in mechanically unloaded heart were significantly upregulated compared to in situ heart, especially for TIMP-1. CONCLUSION: Mechanical unloading of left ventricle may lead to excessive ECM deposition, accompanied by imbalance between MMPs and TIMPs system, especially the upregulation of TIMPs.

Effects of blocking the chemokine receptors, CCR5 and CXCR3, With TAK-779 in a rat small intestinal transplantation model.

BACKGROUND: The effect of blocking lymphocyte chemokine receptors with TAK-779 was investigated using a rat intestinal transplantation model. METHODS: Dark Agouti rat small intestines were heterotopically transplanted into Lewis rats. The recipients were treated with TAK-779 (10 mg/kg per day) by subcutaneous injection. Graft survival, histologic changes, mixed lymphocyte reaction, and antibody production were examined. Furthermore, expression of the chemokine receptors on the graft-infiltrating lymphocytes in the mesenteric lymph node (MLN) and Peyer's patches (PP) were measured using fluorescence-activated cell sorter analysis. RESULTS: Average duration of survival was greater for group T (with TAK-779: 9.8+/-1.4) than group A (without TAK-779: 7.0+/-0.6). Histologic findings and immunohistochemistry of the graft were consistent with the prolonged survival in group T. In the fluorescence-activated cell sorter analysis, several CD4+ and CD8+ cells were significantly suppressed in the blood, spleen, and MLN of the recipient and in the PP of the graft on postoperative day (POD) 6, but recovered in recipient spleen and MLN by POD 9. However, double-staining of graft-infiltrating lymphocytes in MLN and PP showed a significant reduction in the numbers of T cells expressing CCR5 and CXCR3 by POD 9. On the other hand, mixed lymphocyte reaction and cytokine production, and the antidonor antibody titer were suppressed on POD 6 but not on POD 9. CONCLUSION: TAK779 diminished not only the number of the graft-infiltrating cells and their expression of CCR5 and CXCR3, but also the total number of recipient T cells that play a role in graft rejection. Further exploration of these effects will provide the novel treatment of the intestinal transplantation.

The subepicardial microcirculation in heterotopically transplanted mouse hearts: an intravital multifluorescence microscopy study.

OBJECTIVE: We developed a model for intravital microscopic analysis of the coronary microcirculation in transplanted murine hearts and assessed the influence of cold ischemia on postischemic microcirculatory dysfunctions. METHODS: Murine hearts were exposed to 60 (n = 12) and 240 minutes (n = 8) of cold ischemia before syngeneic heterotopic transplantation. Intravital fluorescence microscopy allowed detailed analysis of the right ventricular coronary microcirculation, including feeding arterioles, nutritive capillaries, and postcapillary venules. With this technique, we further studied leukocyte-endothelial cell interactions, microvascular permeability, tissue oxygenation, and microlymphatics. RESULTS: Cold ischemia of 240 minutes aggravated nutritive capillary perfusion failure, indicated by a significant reduction of functional capillary density and capillary flow velocity by 63% and 45% (P < .05 vs 60-minute cold ischemic isografts). The mitochondrial redox state, visualized by nicotinamide adenine dinucleotide hydrogen autofluorescence, was markedly deteriorated after 240-minute cold ischemia (P < .05), indicating a persistent mismatch between oxygen supply and demand resulting from pronounced capillary no-reflow. Prolonged ischemia further resulted in 6- and 11-fold higher numbers of rolling and firmly adherent leukocytes in postcapillary venules (P < .05), together with increased microvascular permeability. CONCLUSIONS: We introduce a novel approach to visualize in detail the murine coronary microcirculation in vivo by multifluorescence microscopy. Our data demonstrate that prolonged cold ischemia provokes posttransplant capillary no-reflow, leukocytic inflammation, and persistent tissue hypoxia.

Physiologic microcirculation of the heterotopically transplanted rat liver with portal vein arterialization depending on optimal stent diameter.

BACKGROUND: Clinical experience with portal vein arterialization (PVA) in liver transplantation is controversial. One reason for this is the lack of standardized flow regulation. The present experiments aimed to establish flow regulation in the arterialized portal vein for heterotopic auxiliary liver transplantation (HALT), to obtain physiological portal blood flow, and to compare this technique with orthotopic liver transplantation. MATERIAL/METHODS: Lewis rats were divided into 7 groups (n = 8 transplantations/group). Group: A I-IV: In HALT, the portal vein was anastomosed to the right renal artery using stents with different diameters (0.2, 0.3, 0.4, 0.5 mm). Afterwards, HALT with PVA using the stent diameter that had achieved the most physiological portal blood flow (group B II) was compared with orthotopic liver transplantation with porto-portal anastomosis (group B III) and to the sham group (B I). RESULTS: After reperfusion, only the 0.3 mm stent resulted in an average blood flow in the arterialized portal vein in HALT which was within the normal range (1.7+/-0.4 ml/min/g liver weight). The parameters of microcirculation and early graft function were significantly better in group B II than in group B III (functional sinusoidal density: 335+/-48 vs. 224+/-31/cm, diameter of sinusoids: 6.4+/-0.6 vs. 5.2+/-0.6 microm, diameter of postsinusoidal venules: 31.1+/-3.3 vs. 25.5+/-2.0 microm, bile-production: 27+/-8 vs. 19+/-5 microl/h/g liver weight). CONCLUSIONS: Using an optimal stent diameter in HALT with portal vein arterialization, an adequate flow-regulation can be achieved. Avoiding portal hyper- and hypoperfusion, good results for microcirculation and early graft function can be obtained.

Orthotopic retransplantation in heterotopic transplant recipients: 3 case reports.

We report 3 patients who initially underwent heterotopic transplantation due to a size mismatch but then later underwent orthotopic retransplantation because of heart failure. In each case, the heterotopic graft was left in place, the native heart was removed, and the new allograft was placed orthotopically. This technique resulted in reduced postoperative morbidity and excellent long-term outcomes.

Development of an ectopic site for islet transplantation, using biodegradable scaffolds.

Clinical islet transplantation in liver has achieved normoglycemia. However, this site may not be ideal for islet survival. To create a more optimal site for islet transplantation, we have developed a construct with biodegradable scaffolds. Islets were seeded in scaffolds and transplanted into the epididymal fat pad of diabetic BALB/c mice. Controls included islets transplanted underneath the kidney capsule or into the fat pad without scaffolds. All animals with islets in scaffolds or the kidney became normoglycemic and maintained this metabolic state. When islets were transplanted without scaffolds the time to achieve normoglycemia was significantly increased and less than 45% of mice survived. An oral glucose tolerance test was performed on the scaffold and kidney groups with similar blood glucose levels and area under the curve values between the groups. Grafts were removed at more than 100 days posttransplantation and all animals became hyperglycemic. There was no significant difference in insulin content between the grafts and all grafts were well vascularized with insulin-positive beta cells. Therefore, islets in scaffolds function and restore diabetic animals to normoglycemic levels, similar to islets transplanted underneath the kidney capsule, suggesting scaffolds can be used to create a site for islet transplantation.

Na+/H+ exchange inhibition and antioxidants lack additive protective effects after reperfusion injury in the working heterotopic rat heart isograft.

BACKGROUND: The utility of combining strategies of myocardial protection was studied in intact rat hearts subjected to 1 hour of ischemia and 40 minutes blood reperfusion. METHODS: Lewis rats (n = 48) were divided into 4 transplant groups. Twenty-four hearts were arrested by coronary perfusion with hypothermic Celsior solution at 60 mm Hg. The aortic valve was punctured to introduce volume into the left ventricle (LV), and the hearts were abdominally isografted. Animals were either given both the antioxidant probucol (300 mg/kg) and the sodium-hydrogen exchange inhibitor cariporide (5 mg/kg) (CP; n = 6), just cariporide (CAR; n = 6), just probucol (PROB; n = 6), or neither drug (CON; n = 6). After 40 minutes of blood reperfusion, transplanted hearts were rearrested. The control recipients' native hearts (native; n = 6) were also arrested. Postmortem LV compliance relations and myocardial water content (MWC) were measured. RESULTS: Grafts protected by probucol were significantly more compliant than controls and significantly less compliant than grafts protected by cariporide alone and with both cariporide and probucol (p = 0.0001, analysis of variance). Compliance relations for CP overlapped those for CAR. All grafts were less compliant than natives. MWC was significantly greater in controls and PROB than in natives. CONCLUSIONS: Pretreatment with cariporide in the setting of ischemia-reperfusion injury provides greater protection against the development of diastolic abnormalities than probucol when Celsior solution is used for both arrest and preservation. In this model, there is no advantage to combining the drugs, supporting the hypothesis that there is an overlapping mechanism of protection.

Evaluation of the hormonal function and histological features of heterotopic isogenic ovarian transplantation in rats.

The purpose of the study was to determine the feasibility of preserving ovarian function after heterotopic transplantation by means of microvascular anastomosis of the transplanted vascular pedicles to a set of preselected vessels. Six groups of 10 Sprague-Dawley inbred rats were used in this study. Group I underwent bilateral ovariectomy operation and served as the ovariectomy control. Group II underwent bilateral ovariectomy followed by heterotopic isogenic ovarian implantation. Group III underwent bilateral ovariectomy and isogenic heterotopic ovarian transplantation by means of microvascular anastomosis. Group IV served as the laparotomy sham-operated control. Group V served as the ovarian donor for group II. Group VI served as the donor of the ovarian-kidney vascular pedicle complex for group III. Postoperative ovarian estradiol levels were measured, and histological characteristics were elucidated in groups I, II, III, and IV. The results demonstrated that the estradiol level of the transplantation group was comparable to that of the sham operation group and was significantly higher than that of the implantation group. Histologically normal ovarian architecture was observed in the sham group (IV) and also in the transplantation group (III). Altered architecture was observed in the implantation group (II). These findings indicate that extraabdominal heterotopic ovarian transplantation with microvascular anastomosis led to normal ovarian hormonal function and was effective in preserving oocyte production capacity.

Inhibin and follicular development in heterotopical ovary transplants without vascular pedicle in syngeneic Lewis rats.

OBJECTIVE: To evaluate the role of inhibin in elevated base levels of FSH and follicular hyperplasia in ovarian autotransplantation in rats. DESIGN: Experimental animal study. SETTING: Unit of Experimental Research at the Barcelona University School of Medicine. ANIMAL(S): Female syngeneic Lewis rats aged 16 weeks. INTERVENTION(S): The animals were randomized into two groups: group A, control group undergoing only laparotomy (n = 5) and group B, oophorectomized with SC autologous heterotopic transplant (n = 5). The animals were killed and their ovaries removed for histologic, morphometric, and immunohistochemical analysis at 28 days after surgery in both groups. MAIN OUTCOME MEASURE(S): Serum levels of E2 and FSH were determined on day 0 (the day of surgery or baseline) and days 4, 7, 14, 21, and 28. Morphometric analysis of ovarian structure for evaluation of antral follicles and their granulosa cell area and immunohistochemistry for inhibin staining were also done. RESULT(S): The endocrinological function recovered at 28 days, and the FSH levels for the transplant group were significantly higher than for the group with normoinsert ovary. Morphometric analysis showed that the mean granulosa cell area was greater in group B when compared with the control group. Immunohistochemistry revealed almost null inhibin staining of the stroma in transplanted ovarian tissue. CONCLUSION(S): Tissue damage brought on by ischemia in the transplant of nonvascularized ovaries may bring about an inhibin deficit in the ovarian stroma, which might explain the increased levels of FSH. These increased levels, in turn, would be responsible for the follicular hyperplasia seen in this tissue when it recovers its function.

Immunobiology and long-term graft function in a transplant heterotopic renal rat model.

BACKGROUND: The Th1-Th2 paradigm proposes clonal expansion of Th2 lymphocytes as the basis of allograft tolerance. The Th2 cells have been found to be present in recipients with long-term allograft survival. However, the presence of Th2 cells and tolerance is not a uniform finding. Previously we have shown that pre-engraftment single dose rapamycin and a 7-d course of cyclosporin induce transplantation tolerance to 120 d. In the present study, we investigated the immunobiology of grafts in a long-term follow-up (>350 d). METHODS: Kidney allografts (n = 7), isografts (n = 5) and single nephrectomy (n = 3) groups were followed for 350 +/- 87 d. Heterotopic kidney transplant was performed by the same surgeon in the allograft group (ACI-Lewis) and the isograft group (Lewis-Lewis). The left kidney was removed in the single nephrectomy group. The allograft group was treated with pre-engraftment single dose rapamycin and a 7-d course of cyclosporin. A kidney biopsy was performed at midpoint time for histological study and tissue was frozen for measuring intragraft cytokine expression (IL-4, IL-10) in all animals. Prior to biopsy, serum blood urea nitrogen (BUN) and creatinine (Cr) levels were studied. Serum BUN, Cr levels, plus 24-h urinary protein (PRO) were measured prior to sacrifice. Randomly, four allograft rats received skin grafts (ACI, Lewis and Buffalo skin donors) after kidney biopsy. Skin grafts were studied for a mean of 6 weeks for signs of acceptance or rejection. Analysis of variance (ANOVA) with Tukey's test was used; p < 0.05 was considered statistically significant. RESULTS: The mean follow up was 352 +/- 87 d. BUN and Cr levels at biopsy time (mean 214 d) were not statistically different between the three groups (p = 0.19 and p = 0.66). At sacrifice (mean 352 d), BUN, Cr and PRO were statistically different between allograft and isograft groups (p = 0.013), and between allograft and single nephrectomy groups (p = 0.027). Functional and histological signs of graft loss occurred in three of seven (42.8%) of the allografts at 352 d. Using BANFF criteria, three allografts at biopsy time and seven allografts (100%) and four isografts (80%) at sacrifice time developed chronic histologic changes. Intragraft overexpression of IL-4 and IL-10 was seen at biopsy and sacrifice time in six of seven allografts and one of five isografts. All donor specific skin grafts (ACI-Lewis) on allografts were accepted and third party (Buffalo) donor skin grafts were rejected in all animals (>95% skin necrosis). CONCLUSIONS: This highly stringent, functional, renal transplant model yields 100% normal renal function as compared with isografts at 120 d follow-up. With the follow-up extended to 350 d, 43% of the allografts loose function and develop a chronic allograft histology despite a demonstrated intragraft Th2 cytokine dominance and donor specific skin graft acceptance.

Epithelial kinetics in mouse heterotopic tracheal allografts.

Obliterative bronchiolitis (OB) is the most important cause of graft dysfunction post-lung transplantation. It is likely that the small airway epithelium is a target of the alloimmune response, and that epithelial integrity is a crucial determinant of airway patency. Our goals are to elucidate epithelial cell kinetics in the heterotopic mouse trachea model and to determine potential mechanisms of cell death in allografts. Allografts and isografts were obtained by transplanting BALB/c tracheas into C57BL/6 and BALB/c immunosuppressed and non-immunosuppressed hosts, respectively and harvested from day 3-20. Morphometry, BrdU and TUNEL labeling, and EM studies were performed. Columnar epithelium in isografts and allografts sloughs during day 0-3, but regenerates in both sets of grafts by day 10. Subsequently, allografts become inflamed and denuded, while isografts retain an intact epithelium. Prior to airway denudation, allografts exhibited significantly increased epithelial cell density, BrdU labeling index (LI), and TUNEL positive cells. Epithelial apoptosis was confirmed by electron microscopy. Allograft percent ciliated columnar epithelium and lumenal circumference were significantly decreased. Cyclosporin delayed airway fibrosis but did not alter the progression of the allograft through the phases of early ischemic injury, airway epithelial cell regeneration, and eventual cell death. These studies quantitatively demonstrate that the allograft epithelium actively regenerates in the alloimmune environment, but succumbs to increased apoptotic cell death, underscoring the importance of the airway epithelium as a self-renewing source of alloantigen.

Long-term ovarian function evaluation after autografting by implantation with fresh and frozen-thawed human ovarian tissue.

The transplantation of ovarian tissue has recently been the focus of intense investigation with the aim of avoiding premature ovarian failure mainly in patients receiving chemotherapy or radiotherapy for malignant disease. Here, we present an evaluation of the long-term function of both fresh (patients 1, 2, and 3) and cryopreserved (patient 4) ovarian autografts in four premenopausal patients aged 46-49 yr who underwent heterotopic ovarian transplantation and were followed over a 1-yr period without receiving gonadotropins to stimulate follicular growth. In patients 1 and 2, approximately 1 cm(3) ovarian cortical autograft was placed sc in the inner aspect of the arm, whereas and in patients 3 and 4 minced ovarian tissue was placed into a muscle pocket in the abdominal wall. In patients 1, 2, and 4 the ovarian hormone secretion (as suggested by sequential estradiol and FSH serum measurements) was reestablished 3-4 months after autotransplantation, and graft function was not improved by immediate rather than delayed heterotopic ovarian autografting. Despite a reestablished ovarian function, a 2- to 7-fold increase in peripheral FSH concentration was evidenced. The cases reported here suggest that hormonal protection can be restored after fresh or cryopreserved heterotopic ovarian transplantation in women, albeit for only a short reproductive span.

Revascularisation of fresh compared with demineralised bone grafts in rats.

Revascularisation of bone grafts is influenced by both the anatomical origin and the pre-implantation processing of the graft. We investigated the revascularisation by entrapment of 141Ce (cerium)-labelled microspheres in large, fresh and demineralised syngeneic grafts of predominantly cancellous (iliac bone) or cortical (tibial diaphysis) bone three weeks after heterotopic implantation in rats. The mean (SD) 141Ce deposition index (counts per minute (cpm) of mg recovered implant/cpm of mg host iliac bone) was higher in fresh iliac bone grafts, 0.98 (0.46) compared to that of demineralised iliac bone, 0.32 (0.20), p < 0.001, and fresh tibial bone grafts, 0.51 (0.27), p = 0.007. We found no significant difference in the mean 141Ce deposition index between fresh tibial bone grafts and demineralised tibial bone grafts, 0.35 (0.42), p = 0.4, or between demineralised tibial grafts and demineralised iliac bone grafts, p = 0.8. The results suggest that whereas fresh cancellous grafts are revascularised more completely than fresh cortical grafts, there is no difference in the revascularisation of demineralised cancellous and cortical grafts. In addition, fresh cancellous bone is revascularised more completely than demineralised cancellous bone, whereas there is no difference between fresh and demineralised cortical bone.

Comparison of myocardial oxygen consumption using 11C acetate positron emission tomography scanning in a working and non-working heart transplant model.

OBJECTIVE: In acute cardiac rejection, changes in myocardial oxygen consumption occur; non-invasive detection of these metabolic changes would have obvious clinical utility. In the classic cervical, heterotopic, canine, transplant model, the heart is non-working. It has a low myocardial oxygen consumption. Creation of a working model with normal myocardial oxygen consumption would enhance validity of non-human studies. METHODS: Clearance of 11C acetate was determined by positron emission tomography (PET) scanning and compared with myocardial oxygen consumption in normal and transplanted canine hearts. Donor hearts from mongrel dogs (2.5-3 kg; n=4) were transplanted into the neck of adult beagles (12-15 kg; n=4), no immunosuppression was given. Two non-working hearts were modified to eject only coronary flow via the right ventricle. In two hearts, a novel working model was created with aortic regurgitation to load the left ventricle. Working and non-working hearts underwent PET scanning on post-operative days 2 and 4. Normal dog hearts (n=2) and native hearts of transplanted dogs (n=3) were used to validate the scanning technique. Coronary sinus and aortic oxygen saturation data along with myocardial blood flow (radiolabeled microspheres) confirmed that clearance of 11C acetate in normal and transplanted hearts followed a bi-exponential model. RESULTS: Myocardial oxygen consumption was correlated with the rate constant of 11C acetate rapid phase clearance (r=0.91) in normal and transplanted hearts. The working hearts had increased myocardial oxygen consumption compared to non-working hearts. CONCLUSIONS: This study (1) introduces a model of a working heterotopic cardiac transplantation with near-normal oxygen consumption; and (2) demonstrates that regional myocardial oxygen consumption in transplanted hearts can be detected by 11C acetate PET.

Heart transplantation changes the expression of distinct gene families.

We took advantage of the combination of a rat heart transplantation model with a modified differential display RT-PCR method to identify transcriptome changes in the right atria from transplanted compared with native hearts. Based on sequence homology search, the 37 cDNAs differentially displayed both 2 and 7 days posttransplantation were categorized into 7 unknown transcripts, 16 expressed sequence tags (ESTs), and 14 partially or completely characterized genes. The last group cDNAs, validated by relative RT-PCR, belonged to diverse gene families involved in specific metabolisms, protein synthesis, cell signaling, and transcription. Furthermore, we identified differential transcripts corresponding to denervation and fetal gene reexpression. We found coordinate downregulation of genes involved in energy metabolism and protein synthesis regulation, similar to that reported for senescent skeletal muscle. From these transcriptome changes, we propose that heart transplants and senescent muscles share common molecular mechanisms.