SEQUENTIAL ISOLOGOUS ORGAN TRANSPLANTATION IN INBRED RATS

Pancreaticoduodenal Transplants¹

Abstract

A total of 847 inbred Lewis rats of mixed sex were used in this pancreaticoduodenal (Pd) donor aging study. Pd grafts were taken from 9- to 12-month-old donors and transplanted into 3-month-old recipients (thus, the first generation Pd graft, or 1 Pd). After 9 to 12 months, the same Pd grafts were again harvested and transplanted into 3-month-old rats (thus the 2 Pd generation). This cycle was repeated to obtain the 3, 4, and 5 Pd series. Sequential transplantation was able to extend the Pd grafts' mean survival time to 32 months for fourteen 4 Pd grafts, and to 39.2 months for four 5 Pd grafts (the longest lived graft survived for 42 months).

The pancreas and duodenal sections of the grafts remained normal throughout the entire study. However, the aortic sections of the grafts (which were harvested to include the superior mesenteric and celiac arteries) all exhibited moderate to massive atherosclerotic changes by the 5 Pd mean survival age of 39.2 months. Such histological changes commenced even before 21 months of Pd graft age in some animals, gradually progressing to dilation of the aorta (and subsequent narrowing of aortic tributaries), as well as formation of an eggshell-like inner membrane shielding the aortic intima, by 42 months. Such atherosclerotic changes precluded transplantations beyond the 5 Pd series.

Previous investigators have demonstrated the viability of serially transplanting adrenal, ovary, and skin grafts from old to young rodents ^(1-3). Krohn, for example, was able to extend the age of skin grafts to 6.7 years using inbred strains of mice ⁽²⁾. These valuable investigations suggested that certain cells, tissues, and even organs, have the possibility of a life span far surpassing that of the host. The present study involves the sequential transplantation of pancreaticoduodenal (Pd^*) grafts into a series of syngeneic hosts to extend the life span of the pancreas far beyond the usual life expectancy of the host rat. Many factors have made this prolongation technically feasible, such as the availability of genetically histocompatible strains of rats, established and improving techniques for microvascular organ transplantation, and experience gained from 25 years of study in endocrine ^{(4, 5)} and exocrine ⁽⁶⁾ integrity after syngeneic and allogeneic Pd transplantation in the rat ⁽⁷⁾. For the first time, the Pd cluster has been transplanted by vascular anastomosis into a second, third, fourth, and even fifth sequential host. Each successive transplant added 9-12 months of intrinsic age to the transplanted pancreas.

The purpose of this report is to indicate the feasibility and availability of this model for scientific studies of the aging pancreas. The concept of sequential transplantation can help separate the effects of intrinsic age from those of the environment by keeping the continuously aging organ in a perpetually “young” environment. It can also help magnify the suspected effects of intrinsic aging, such as vascular degeneration.

At the clinical level, these experimental observations should help to determine which organs are suitable for harvesting from older donors. The organs of older donors are currently scorned because of perceived intrinsic degeneration due to aging. Further knowledge in this area may tap another source of organs in these days of donor organ shortage.

MATERIALS AND METHODS

Experimental Design

The experimental design elaborated in Table 1 incorporates the transplantation of aging organs into younger recipients. Briefly, primary procurement of grafts was performed on old LEW donors varying between 9 and 12 months in age. The recipients of these grafts and any succeeding transplants were 3 months of age. The first recipients of aging grafts were termed first generation transplants. After the original graft had resided for 9 to 12 months in the first generation recipient, it was re-procured and transplanted to a second recipient (thus, the second generation transplant). Likewise, this progression was followed until the graft was transplanted for the fifth and final time to a fifth young host (the fifth generation transplant). The use of the term “retransplantation” to describe this process was avoided as it represents more than one meaning to the transplant community.

Rats

Both male and female inbred Lewis (LEW) (RT1) rats weighing 200-250 g were commercially obtained from Charles Rivers Laboratories (Wilmington, MA), and also inbred in our laboratory. Rats were allowed unrestricted access to food and water and maintained in approved facilities. The animals were handled and cared for humanely according to the “Principles of Laboratory Animal Care” (NIH Publication No. 85-23, revised 1985). Both donor and recipient surgeries were performed under ether anesthesia using the nose cone technique. All surgical procedures were conducted using a clean technique. For the donor operations, a magnification of ×2.5 was adequate; the vascular anastomoses in the recipient were performed under a magnification of ×6.5 to ×10 using an operating microscope.

Surgical Model

The previously reported technique of Pd ⁽⁷⁾ transplantation developed at this laboratory was used with minor modifications, adapted for the purposes of the study (Fig. 1). Syngeneic transplants were performed between LEW rats. It was necessary to incorporate modifications into the original technique of grafting Pd because of the increasing difficulty encountered with each successive procurement and grafting procedure. The modifications that helped simplify each successive donor operation were (a) harvesting a longer segment of donor aortic segment containing the celiac and superior mesenteric arteries and an extended segment of donor jejunum with the primary graft at the time of primary procurement of the donor organ, and (b) procuring a longer venous segment of the donor inferior vena cava (IVC) about the portal venous (PV) anastomosis (to the recipient's IVC). These technical modifications allowed (a) adequate trimming of the grafted jejunum without compromising pancreatic integrity through dissection and subsequent anastomoses in the recipient, (b) an effective PV outflow orifice for subsequent transplantations, and (c) appropriate shortening of fibrotic or atherosclerotic arterial wall of the donor aorta.

Additionally, because 6-0 silk ligature was used to ligate minute vessels on and about the Pd graft during procurement, suture-related abscesses were frequently encountered at the time of reharvesting these Pd grafts the second time. These abscesses/granulomas were carefully resected when feasible. A portable electric cautery proved useful for the purpose of aiding dissection at each successive procurement. A meticulous microsurgical “no-touch” technique was exercised to procure the pancreas, in an effort to minimize bleeding during dissection from neighboring structures. The Pd was flushed through the aorta with 3-4 ml of saline, which achieved an effective washout of blood from the organ. Using this approach, we were able to standardize the procurement procedure.

After a minimal cold ischemic time, during which the recipient was surgically prepared, and its aorta and IVC were clamped together with a Lee Portacaval Shunt clamp, the graft was transplanted using a microsurgical technique with continuous 9-0 nylon microsuture.

For the first generation recipient operation, the graft's aorta and PV were anastomosed to the recipient's aorta and IVC below the level of the left renal vein. An end-to-side jejunoduodenostomy was then performed using 7-0 sutures. For the second and succeeding recipient procedures, the graft's aorta and PV confluent with the segment of IVC from the first recipient, were anastomosed end-to-side to the recipient's infrarenal aorta and IVC. A jejunoduodenostomy followed, as in the first operation. The recipient's own pancreas was not disturbed in this study. The recipient procedure was standardized so that revascularization of the graft was accomplished within 35 min. Transplanted grafts that did not seem to be satisfactorily vascularized immediately after reperfusion were excluded from the study.

Postoperative Assessment and End Points

Postoperatively, the animals were kept in a thermally regulated environment until they were able to regulate their body temperature, at which time they were transferred to holding cages and permitted food and water ad libitum. Body weight and clinical behavior were monitored on a daily basis for the first 2 weeks, followed by periodic biweekly assessments for 1 month. Transplanted animals were housed two to a cage. Grafts were re-procured from their second, third, or fourth host at the completion of 9-12 months of graft residence.

Tissue samples for biopsy were collected from Pd grafts during transplant procedures, or whenever the host seemed moribund. After collection, samples were fixed in 10% formalin for hematoxylin and eosin analysis.

RESULTS

From April 1990 to October 1995, a total of 847 animals were used in this study. There were 337 rats (3 months old) that received the first generation Pd grafts (1 Pd) from 9- to 12-month-old rats. Of these 337, 1 Pd grafts, a total of 117 grafts (2 Pd) were reharvested and transplanted into 3-month-old recipients, 9 to 12 months postoperatively. Every attempt was made to adhere to the original protocol (Table 1) as much as possible, but some donors fell ill before 9-12 postoperative months, and thus retransplantation was hurried in some cases (Table 2).

Nearly half of the above mentioned 1 Pd grafts were not able to be transplanted into a second host. In some cases, the graft was not of transplantable quality, and, in others, the host died before the requisite 9-12 months. Although the early deaths were due to poor surgical performance, deaths between 3 and 11 postoperative months resulted from out-breaks of infectious diseases in the colony.

Throughout the study, the hosts' vital organs (including pancreaticoduodenums) seemed normal. The most cumbersome donor complications were occurrences of single to multiple, small to large suture abscesses on and about the grafted pancreas. These abscesses were usually nonresectable, if the pedicles were broad and/or located on the main blood supplies to the Pd grafts.

Histological Examination

Tissue integrity. Periodic biopsies of transplanted Pd complexes were performed as indicated in Postoperative Assessment and End Points. At 6 postoperative months, 1 Pd grafts exhibited completely normal tissue. Subsequent biopsies, obtained after an additional 6 months, showed no apparent change in the structural morphology of the gland or cell viability. Pd grafts that were 21 months still exhibited a well preserved acinar and Langerhans islet structure (Fig. 2a). At 27 months, acini and islets of graft 3 Pd-34 (the 34th graft in the 3 Pd series) were normal, with the exception of patchy periductal infiltrates and minimal lymphocytic infiltrate throughout (Fig. 2b). At 36th months, graft 3 Pd-38 (the 38th graft in the 3 Pd series) displayed normal acini and islets (Fig. 3a). At 40.5 months, graft 5 Pd-2 showed a predominantly normal pancreatic histological integrity, with minor fibrotic changes accompanied by inflammatory cells on the second postoperative day, probably due to the recent surgical intervention (Fig. 3b). Before being transplanted into a young recipient to make 5 Pd-4, the 42-month-old Pd graft 4 Pd-8 exhibited normal gross appearance (Fig. 4, a and b).

The host's pancreas and other vital organs were examined at the time of surgery or death, but were not biopsied. Among these hosts, only one animal exhibited massive lymphomatous changes of thymus, spleen, and other lymphoid structures at 18 months of age, and neither the host's nor the transplanted pancreas was affected. No abnormalities were observed at any time in the duodenal section of the Pd grafts.

Atherosclerotic effects. Based on histological examination of 103 rats, the graft aortas underwent mild to moderate atherosclerotic changes beginning at about 21 months graft age, in some cases earlier (Table 3). Subsequent mild to marked atherosclerotic changes progressed by means of smooth muscle and endothelial cell proliferation (by 27 months in the case of 3 Pd-32). The aortic section taken from 4 Pd-14 at 35 months of graft age displayed a thickened intimal plaque, consisting predominantly of fibroblastic cells with few macrophages (Fig. 5, a-c). Older aortas, such as that procured from 5 Pd-4, displayed intimal plaque consisting of collagen, lipid deposits, macrophages, and calcium (Fig. 6, a and b).

DISCUSSION

Sequential grafting of the Pd cluster among syngeneic hosts demonstrates the feasibility of providing animals with healthy tissue that has an intrinsic age much greater than that of the current host. The applicability of sequential vascularized isotransplantation of other organs is currently under investigation in pilot experiments involving kidney, liver, and heart transplants. This “recycling” is technically demanding. It does carry a progressive attrition rate, because of technical problems with each added generation. The cumulative damage to each multiple-generation transplant can be extremely high, unless expert surgical microvascular expertise provides a reasonable yield from each transplanted generation. Needless to say, expertise and practice with the technique must previously be established if laboratories are to undertake the preparation of such “super-aged” organs. As described in Materials and Methods, special precautions must be taken in the harvesting of the original donor graft to provide extra long segments of duodenum and vascular stumps for these sequential grafts, because each subsequent graft progressively shortens these segments.

It is striking how well preserved the parenchymal tissue of the pancreas can remain throughout these sequential transplants. These findings testify strongly to the adequacy of old pancreatic tissue as a donor organ. Although the Pd histology is very reassuring, further careful studies must be performed to confirm the apparent health of these tissues.

The observed deterioration of the transplanted aortic vascular stump with atherosclerosis is notable. The observed damage seems to be primarily in the large bore vessel stumps. This indicates that residence in a young host does not completely protect a vessel from intrinsic aging. These findings do suggest the need for future experiments, involving sequential transplantation of a sleeve of aorta in rats, to pinpoint the effect of medications or diet on intrinsic vascular deterioration. Pilot experiments are already underway in this area.

It is noteworthy that although Lewis male and female rats lived to a median age of 24.2 months in the series of Feldman and Woda ⁽⁸⁾, we have thus far extended the graft age to 32.5 months among 14 survivors in the 4 Pd series, and 39.2 months among four survivors in the 5 Pd series, with the oldest Pd graft being 42 months old. If the arteries on and about the pancreatic grafts had been nonatherosclerotic at the time of transplantation, much longer survival of the Pd grafts would have been possible.

It is hoped that this first report will encourage investigators in gerontology to plan sequential transplantation experiments to isolate which effects are truly due to aging and which are due to other factors. Atherosclerosis, for example, is probably caused by a variety of factors other than age: mechanical injury due to surgical intervention, turbulence at the anastomotic site, immunosuppressants, diet, etc. Our study to this point may be considered a starting point, from which other researchers may pinpoint the exact factors contributing to atherosclerosis. The separation of the organ from the effects of an aging environment are clearly uniquely feasible with this model. Host factors, such as growth factors, cardiovascular changes, endocrine changes, and neurological conditions affecting grafts, are all maintained at the level of a young, unimpaired animal.

Various clinical organ transplantation centers around the world have different criteria for acceptable donor age, with specificity apparently dictated by organ supply and demand. Deaths due to long waiting lists cause busier transplant centers to adhere less strictly to age limits than other centers, and to expand these limits every year. Dunn et al. ⁽⁹⁾ studied living related kidney donors with an average age of 34.3 years (range 18-67 years). Williams et al. ⁽¹⁰⁾ used donors between 19-59 years old, whereas Kahn et al. ⁽¹¹⁾ used donors with a mean age of 26 years (the youngest was between 2 and 5 years, and the oldest was 65 years old). Evidently, transplant surgeons are willing to transplant aging organs if the need arises and the organ is morphologically within normal limits. To make a point, extrapolation of our experimental data into the clinical setting reveals that our longest surviving rat pancreas graft at 42 months of age is comparable to a human pancreas slightly more than 100 years old. Although such comparisons are clearly not precise, they do suggest that older organs may be beneficial in treating some end-stage illness.

Acknowledgments. We thank the Mercy Hospital Medical Media for reproduction of histological photographs.

Figure 1: A modified rat Pd transplant model (PdT). (A) The portal vein is harvested close to the liver to gain the longest segment possible. (B) A completed PdT, with the shaded area showing the intestinal anastomosis. Silk suture (7-0) is preferred because the sutures are absorbable and allow the site to heal better.

Figure 2: (a) Normal acini and islets are shown on a 21-month-old Pd graft. (b) A 26-month-old Pd graft showing minimal lymphocytic periductal infiltration (×400).

Figure 3: (a) A 36-month-old Pd graft exhibiting normal pancreas. (×200). (b) A 40.5-month-old Pd graft exhibiting minor fibrotic changes, with otherwise normal-appearing acini and islets. This slide was prepared 2 days after reharvesting and transplantation from 4 Pd to 5 Pd (×200).

Figure 4: (a) A 42-month-old Pd graft (displaying normal pancreatic integrity) is shown before transplantation into a 5th generation recipient (×200). (b) An enlarged view of panel a to show acini and islets (×400).

Figure 5: (a) A 35-month-old graft displaying thickened plaque adjacent to the intima (arrow) (×400). (b) Enlarged photo of panel a shows marked smooth muscle and endothelial proliferation (×800). (c) Further magnified view of panel b shows that the plaque contains predominantly fibroblastic cells with few macrophages.

Figure 6: (a) A 42-month-old Pd graft is shown (arrow) before transplantation into 5th generation recipient (5 Pd-4). (b) A section of aorta (4 Pd-8) at 42 months of graft age displays marked atherosclerosis consisting of smooth muscle, macrophages, collagen, lipid, and calcium (×400).