The bridge between transplantation and regenerative medicine: Beginning a new Banff classification of tissue engineering pathology.

The science of regenerative medicine is arguably older than transplantation-the first major textbook was published in 1901-and a major regenerative medicine meeting took place in 1988, three years before the first Banff transplant pathology meeting. However, the subject of regenerative medicine/tissue engineering pathology has never received focused attention. Defining and classifying tissue engineering pathology is long overdue. In the next decades, the field of transplantation will enlarge at least tenfold, through a hybrid of tissue engineering combined with existing approaches to lessening the organ shortage. Gradually, transplantation pathologists will become tissue-(re-) engineering pathologists with enhanced skill sets to address concerns involving the use of bioengineered organs. We outline ways of categorizing abnormalities in tissue-engineered organs through traditional light microscopy or other modalities including biomarkers. We propose creating a new Banff classification of tissue engineering pathology to standardize and assess de novo bioengineered solid organs transplantable success in vivo. We recommend constructing a framework for a classification of tissue engineering pathology now with interdisciplinary consensus discussions to further develop and finalize the classification at future Banff Transplant Pathology meetings, in collaboration with the human cell atlas project. A possible nosology of pathologic abnormalities in tissue-engineered organs is suggested.

Anergy for delayed-type hypersensitivity in preleukemic AKR mice.

A singular anergy for delayed-type hypersensitivity (DTH) in preleukemic AKR mice was discovered. This total anergy for DTH against allogeneic cells, which developed in the AKR mice by 4 to 5 months of age, was not due to an artifact of route of sensitization or of other assay parameters and was not found in other strains sharing H-2 or other genetic background. The mice had an intact capacity to be stimulated in mixed lymphocyte culture to produce cytotoxic effector cells. Although the relationship to lymphoma was not directly addressed in these experiments, the genetic and temporal characteristics of this anergy suggest a biologically important relationship to the preleukemic state.

Breath ethane: a specific indicator of free-radical-mediated lipid peroxidation following reperfusion of the ischemic liver.

A major component of the organ injury mediated by toxic oxidants, such as seen following reperfusion of the ischemic liver, is due to the peroxidation of polyunsaturated fatty acids, especially of cell membranes. We utilized the measurement of exhaled breath ethane, a metabolic product unique to oxidant-mediated lipid peroxidation, as a noninvasive indicator of this process in swine liver subjected to warm ischemia/reperfusion. Under rigorously controlled anesthesia conditions, pig livers were subjected to 2 h of warm total ischemia, followed by reperfusion in situ. Expired air was collected and its ethane content quantitated by a novel gas chromatographic technique. The time course of breath ethane generation correlated closely with the appearance of hepatocellular injury as measured by impairment of Factor VII generation and other measures of liver integrity. Moreover, the administration of the specific superoxide free radical scavenger, superoxide dismutase (SOD), significantly attenuated both the elaboration of ethane and the hepatocellular injury. These findings not only provide confirmation of the previously reported link between hepatocellular injury by free radicals generated at reperfusion, but also establish the use of expired breath ethane analysis as a sensitive, specific, and noninvasive indicator of the injury process in real time.

Primary acute renal failure ("acute tubular necrosis") in the transplanted kidney: morphology and pathogenesis.

"Acute tubular necrosis" (ATN) in the transplanted kidney, when properly differentiated from other causes of acute renal failure, appears to be a relatively benign condition. It has been widely assumed to be pathologically identical to ATN in the native kidney, but its histopathologic features have not been studied in detail. Because immunosuppressive therapy with cyclosporine adds an additional layer of complexity to the morphologic changes observed, in the present study we have confined our observations to patients immunosuppressed with steroids and azathioprine. Thirteen renal allograft biopsies from patients with ATN and 5 biopsies from patients with normal allograft function were compared with the previously obtained series of 57 native kidney ATN biopsies and 20 control biopsies. Both qualitative and quantitative differences between transplant and native kidney ATN were found. Compared with native kidney ATN, transplant ATN showed significantly less thinning and absence of proximal tubular brush border and less variation in size and shape of cells in individual tubular cross-sections. There were also significantly fewer casts and less dilatation of Bowman's space and a significantly greater number of polarizable crystals presumed to be oxalate in transplant ATN. In native kidney ATN the tubular injury sites were mostly characterized by desquamation of individual epithelial cells leaving areas of bare basement membrane (the "non-replacement" phenomenon). In transplant ATN, sites of tubular injury, although rare and affecting only short tubular segments, were characterized by the actual presence of identifiable necrotic tubular cells, a finding seldom seen in native kidney ATN. There also was a greater interstitial infiltrate of mononuclear inflammatory cells in transplant ATN compared to native kidney ATN. Electron microscopic studies of 9 transplant ATN biopsies showed a mild reduction in proximal tubular brush border compared with controls but this alteration was significantly less than that observed in native kidney ATN. There was no significant alteration in proximal or distal basolateral infoldings and this contrasted sharply with the marked reduction in basolateral infoldings of the plasma membrane observed in native kidney ATN. Disintegrated necrotic cells were found by electron microscopy in transplant ATN whereas these were not observed in native kidney ATN. There were significantly more cells with apoptosis (shrinkage necrosis) in transplant ATN than in native kidney ATN. There were significantly more cells with apoptosis (shrinkage necrosis) in transplant ATN than in native kidney ATN. On the other hand, there were significantly greater numbers of "non-replacement" sites in the distal tubules in native kidney ATN compared to transplant ATN.(ABSTRACT TRUNCATED AT 400 WORDS)

Rejection of murine cardiac allografts. I. Relative roles of major and minor antigens.

The relative contributions of incompatibilities for class I, class II, or minor antigens to primarily vascularized graft rejection have not been previously compared in large numbers of strains. In our experiments, murine primarily vascularized heterotopic cardiac graft rejection was studied in 16 donor-recipient strain combinations, representing different precisely defined major and/or minor histoincompatibilities. Complete major incompatibilities generally produced strong graft rejection, although it was confirmed that prolonged survival occurs in certain combinations that are incompatible for class I, or class I plus class II, antigens. In addition, however, strong rejection of these grafts was produced in some recipient strains when the donor was incompatible only for minor antigens. This strong effect of minor antigens may reflect their strong stimulation of delayed-type hypersensitivity, whereas the influence of class II antigens seems more related to stimulation of mixed lymphocyte culture generation of lymphocyte-mediated cytotoxicity.

Rejection of murine cardiac allografts. II. Evidence that splenocytes bearing Lyt2 inhibit responsiveness in long-term heart graft recipients.

Mixed lymphocyte culture--lymphocyte-mediated cytotoxicity (MLC-LMC) reactions by recipient spleen cells were strongly and specifically increased shortly after primarily vascularized heart grafts were placed in mice. Subsequently, in strain combinations in which eventual long-term graft survival occurred, the MLC-LMC became markedly suppressed, unlike the case in situations in which the recipients rejected their allografts. Unseparated spleen cells from long-term recipients are unchanged or slightly depressed in their in vitro MLC-LMC capacity. However, when splenocytes from the long-term B10.A recipients of B10.BR heart grafts were depleted of B cells and Lyt-2-positive cells, they were found to significantly enhance responses when added as third-party cells to MLC-LMC cultures. Similarly depleted splenocytes from naive mice were unaffected or depressed in their ability to contribute to this response as third-party cells. It was concluded that non-Ig-bearing, nonadherent Lyt 2+ lymphocytes from long-term heart graft recipient spleens were present that inhibited the responsiveness of other cells in these spleens.

Rejection of primarily vascularized heart grafts. III. Depression of the interleukin 2 mechanism early after grafting.

The immunologic status of B10.A recipients of primarily vascularized B10.BR heart grafts, which undergo temporary rejection but then generally survive long term, was investigated. Generation of cytotoxicity in mixed lymphocyte culture was moderately increased soon after grafting, as compared with naive mice, while mixed lymphocyte cultures did not reveal greater lymphocyte activation after grafting. Unexpectedly, the interleukin 2 mechanism was nonspecifically depressed 1 week after placement of the heart graft. This depressed interleukin 2 activity was restored essentially to normal in splenocytes, but it remained in effect in mesenteric lymph nodes, of long-term recipients with active heart allografts.

Measurement of mycophenolate mofetil effect in transplant recipients.

BACKGROUND: Immunosuppression involves the nature of the immunosuppressive agents and individual differences in patient factors. We investigated whether the effect of mycophenolate mofetil (MMF) is measurable using an in vitro measure of immunocompetence and related its effects to cyclosporine (CsA) in vitro. METHODS: Liver or kidney transplant recipients receiving prednisone; CsA or tacrolimus; and MMF, azathioprine (AZA), or neither, were studied. Immunocompetence was assessed by one-way mixed lymphocyte culture using patients' peripheral blood leukocytes (PBL) and three validated stimulators. The effect of immunosuppressive agents added in vitro on normal PBL stimulation by Staphylococcus enterotoxin B was determined by the carboxyfluorescein diacetate succinimidyl ester measurement of division. RESULTS: Patients receiving MMF had an average immunocompetence level of 12+/-23, compared with 39.7+/-65 and 25.5+/-42 for those receiving AZA or neither AZA nor MMF, respectively. Thus, there was an approximately 80% suppression of the response in the MMF group. Assessment of normal cell division revealed that CsA allowed multiple cell generations but suppressed the numbers of cells in each, whereas MMF blocked proliferation into subsequent generations. Addition of clinically relevant levels of mycophenolic acid, the active agent for MMF, added to more moderate levels of CsA, was required to achieve greater than 80% suppression, consistent with the degree of immunocompetence depression measured in patients. CONCLUSIONS: These data provide the novel finding that the effect of MMF treatment on patients is measurable in their PBL as decreased immunocompetence in vitro. The effect of MMF on normal PBL approximates the 80% inhibition that we found in patients. Moreover, the effect of MMF on cell division provides a rationale for the superior effectiveness of regimens including MMF.

National impact of pulsatile perfusion on cadaveric kidney transplantation.

BACKGROUND: The simplicity and success of cold storage of cadaveric kidneys have led to the infrequent use of pulsatile perfusion. However, there may be advantages to pulsatile perfusion for less optimal donors. METHODS: United Network for Organ Sharing data were analyzed retrospectively to determine the impact of pulsatile perfusion on initial function and 1-year graft survival. The analysis included 60,827 cadaveric kidney transplants performed between 1988 and 1995. Multivariate logistic regression analyses were used to determine the effect of preservation method on both early kidney function (need for first-week dialysis after transplant) and 1-year graft survival, after adjusting for other known risk factors. RESULTS: The preservation method exhibited a highly significant impact on the need for first-week dialysis. Ice-preserved kidneys were associated with a 2.13-fold increase in the odds of requiring dialysis compared with perfused kidneys. If the donor age was > or =55 years, the odds were 2.33-fold higher for ice-preserved as compared with perfused. If cold ischemic time was > or =24 hr, there was a 2.19-fold increase in the odds of dialysis for ice-preserved kidneys. African-American recipients of cold-stored kidneys had a 2.29-fold greater odds of first-week dialysis. CONCLUSIONS: Based on these findings, it was estimated that the increased cost of perfusing kidneys from all donors > or =55 years of age would be balanced by the decreased need for posttransplant dialysis if the cost related to dialysis were $14,700 or greater per patient. These facts, coupled with the ability to assess an older donor kidney before transplant, could make pulsatile perfusion for the expanded donor financially as well as medically desirable.

Plasmapheresis: an effective therapy for primary allograft nonfunction after liver transplantation.

BACKGROUND: The role of plasmapheresis in liver failure and hepatic coma remains controversial. Also, its use as a salvage strategy for patients with severe allograft dysfunction after liver transplantation has not been defined. This report reviews the use of plasmapheresis in primary hepatic allograft nonfunction (PNF). METHODS: From May of 1997 to October of 1998, five patients underwent plasmapheresis for PNF after other causes of immediate allograft dysfunction were excluded. These patients underwent two to five plasmapheresis procedures during which one plasma volume was removed and replaced with fresh frozen plasma (FFP) or with 50% FFP and 50% albumin. RESULTS: All recipients who underwent plasmapheresis had restoration of liver function. There was one death from pulmonary embolism, for an overall survival rate of 80%. The four surviving patients all had functioning allografts 1 year after liver transplantation. In contrast, during the same period, there were two patients in whom PNF was treated by retransplantation, and both died within 3 months after surgery with functioning allografts. CONCLUSIONS: Plasmapheresis provides an effective treatment option for PNF immediately after liver transplantation and may obviate the need for retransplantation.

Characteristics of early routine renal allograft biopsies.

The assumption that renal allograft histology should be perfectly normal during quiescence in the absence of rejection or nephrotoxic insults has not been adequately investigated. To study this, routine renal allograft biopsies were performed at approximately 1 and 4 weeks, when patients often had normal function or stable acute tubular necrosis (ATN). These were compared with biopsies from other patients during autologous ATN or clinically evident allograft rejection. There were two new findings: (1) Almost all biopsies contained an interstitial infiltrate, so that only the presence of vasculitis provided a clear distinction between rejection and quiescence. Most of the biopsies with infiltrates were from patients who had never received cyclosporine, so that an infiltrate does not necessarily signify toxicity due to this drug. (2) A major proportion of the cells in some biopsies appeared to express both the helper/inducer and the cytotoxic/suppressor phenotype, and a similar finding after in vitro stimulation suggests that this represents a cell population that is activated in some way.

Donor horseshoe kidneys for transplantation.

BACKGROUND: Experience with donor horseshoe kidneys for transplantation is very limited. Currently, horseshoe kidneys may be underutilized for transplantation because of the greater incidence of vascular anomalies, associated renal anomalies, and predisposition to renal disease. METHODS: In this report, we review five transplantations using horseshoe kidneys: the largest reported institutional experience. In addition, a review of all published cases in the English literature is performed. RESULTS: All five patients underwent successful renal transplantations with a median follow-up of 35 months. One patient lost his kidney from recurrent disease soon after transplantation. CONCLUSION: With appropriate reconstruction of the vessels, careful division of the isthmus, and avoidance of ureteral obstruction, long-term data revealed good graft survival of donor horseshoe kidneys in renal transplantation.

Human monocytic ehrlichiosis: an emerging pathogen in transplantation.

BACKGROUND: The spectrum of disease caused by Ehrlichia spp. ranges from asymptomatic to fatal. Awareness and early diagnosis of the infection is paramount because appropriate therapy leads to rapid defervescence and cure. If left untreated, particularly in immunosuppressed patients, ehrlichioses may result in multi-system organ failure and death. METHODS: We report the second case of human monocytic ehrlichiosis (HME) in a liver transplant recipient, and review the literature. RESULTS: The patient presented with fever and headache, had negative cultures, and despite broad-spectrum antimicrobial coverage appeared progressively septic. After eliciting a history of tick exposure we treated the patient empirically with doxycycline. The diagnosis of HME was confirmed by 1) polymerase chain reaction (PCR) for Ehrlichia chaffeensis, 2) acute and convalescent serum titers, and 3) in vitro cultivation of E chaffeensis from peripheral blood. CONCLUSION: Although human ehrlichioses are relatively uncommon, they are emerging as clinically significant arthropod-borne infections. Although epidemiological exposure is responsible for infection, immunosuppression makes patients more likely to succumb to disease. A high index of suspicion and early treatment results in a favorable outcome.

Cardiopulmonary bypass with profound hypothermia. An optimal preservation method for multiorgan procurement.

Numerous techniques have been devised for the harvesting of individual organs during a multiorgan procurement operation. Cardiopulmonary bypass with profound hypothermia (PH) has been employed in successful harvesting of heart-lung, kidney, pancreas, and liver grafts. This report summarizes our experience using CPB-PH for the harvesting of multiple organs from 10 brain-dead donors during the period from July 1983 to January 1988. Organs harvested included 10 heart-lungs, 17 kidneys (3 kidneys were not harvested due to anatomy and elevated creatinine), 1 liver, and 1 pancreas. Mean ischemic time for the distantly procured heart-lung grafts was 281 +/- 10 min. Adequate pulmonary function, as assessed by arterial blood gases, was observed in each heart-lung recipient (mean PO2 was 119 +/- 46 mmHg, 164 +/- 47 mmHg, 130 +/- 30 mmHg, 114 +/- 26 mmHg at immediate post-CPB, 6 hr postop, 24 hr postop, and postextubation, respectively). Mean length of intubation was 34 +/- 8 hr. Mean creatinines of kidney recipients at days 2, 7, and current creatinine were 7.4 +/- 3.6 mg%, 3.6 +/- 2.4 mg%, and 1.6 +/- 0.66 mg%, respectively. Eight kidney recipients (47%) required dialysis, (2 patients required only a single dialysis). Ninety-four percent of the kidney transplant patients are alive, and 88% (15/17) have functioning kidneys. One liver and 1 pancreas were harvested during this time period. Preservation was satisfactory in both the pancreas (Johns Hopkins Hospital) and liver (Dr. Thomas Starzl, personal communication). The technique of CPB-PH has resulted in excellent function of heart-lung grafts. Follow-up of the transplanted kidneys, liver, and pancreas utilizing this technique shows equal or better function compared with standard techniques. This technique offers other advantages in addition to satisfactory multiorgan preservation. Placement of an unstable patient on CPB ensures adequate organ perfusion and allows for a gradual yet uniform cooling of all organ systems. Cooling to a core temperature of 10-15 degrees C requires 30 min, during which time preliminary intraabdominal and mediastinal dissection can be carried out. Following cessation of CPB and subsequent exsanguination, organs can be more easily dissected in a near-bloodless field. This technique does not preclude additional crystalloid organ flushing. Since multiorgan procurement occurs with virtually every donor, this technique may be the optimal method providing excellent preservation, ease of dissection, and better control of hemodynamics during the operation.

Effects of tacrolimus on hyperlipidemia after successful renal transplantation: a Southeastern Organ Procurement Foundation multicenter clinical study.

BACKGROUND: Tacrolimus has been shown to have a less adverse effect on the lipid profiles of transplant patients when the drug is started as induction therapy. In order to determine the effect tacrolimus has on lipid profiles in stable cyclosporine-treated renal transplant patients with established hyperlipidemia, a randomized prospective study was undertaken by the Southeastern Organ Procurement Foundation. METHODS: Patients of the 13 transplant centers, with cholesterol of 240 mg/dl or greater, who were at least 1 year posttransplant with stable renal function, were randomly assigned to remain on cyclosporine (control) or converted to tacrolimus. Patients converted to tacrolimus were maintained at a level of 5-15 ng/ml, and control patients remained at their previous levels of cyclosporine. Concurrent immunosuppressants were not changed. Levels of total cholesterol, triglycerides, total high-density lipoprotein, low-density lipoprotein (LDL), very-low-density lipoprotein, and apoproteins A and B were monitored before conversion and at months 1, 3, and 6. Renal function and glucose control were evaluated at the beginning and end of the study (month 6). RESULTS: A total of 65 patients were enrolled; 12 patients failed to complete the study. None were removed as a result of acute rejection or graft failure. Fifty-three patients were available for analysis (27 in the tacrolimus group and 26 controls). Demographics were not different between groups. In patients converted to tacrolimus treatment, there was a -55 mg/dl (-16%) (P=0.0031) change in cholesterol, a -48 mg/dl (-25%) (P=0.0014) change in LDL cholesterol, and a -36 mg/dl (-23%) (P=0.034) change in apolipoprotein B. There was no change in renal function, glycemic control, or incidence of new onset diabetes mellitus in the tacrolimus group. CONCLUSION: Conversion from cyclosporine to tacrolimus can be safely done after successful transplantation. Introduction of tacrolimus to a stable renal patient does not effect renal function or glycemic control. Tacrolimus can lower cholesterol, LDL, and apolipoprotein B. Conversion to tacrolimus from cyclosporine should be considered in the treatment of posttransplant hyperlipidemia.

Efficacy of tacrolimus as rescue therapy for chronic rejection in orthotopic liver transplantation: a report of the U.S. Multicenter Liver Study Group.

BACKGROUND: A study was performed by 17 different U.S. liver transplantation centers to determine the safety and efficacy of conversion from cyclosporine to tacrolimus for chronic allograft rejection. METHODS: Ninety-one patients were converted to tacrolimus a mean of 319 days after liver transplantation. The indication for conversion was ongoing chronic rejection confirmed by biochemical and histologic criteria. Patients were followed for a mean of 251 days until the end of the study. RESULTS: Sixty-four patients (70.3%) were alive with their initial hepatic allograft at the conclusion of the study period and were defined as the responder group. Twenty-seven patients (29.7%) failed to respond to treatment, and 20 of them required a second liver graft. The actuarial graft survival for the total patient group was 69.9% and 48.5% at 1 and 2 years, respectively. The actuarial patient survival at 1 and 2 years was 84.4% and 81.2%, respectively. Two significant positive prognostic factors were identified. Patients with a total bilirubin of < or = 10 mg/dl at the time of conversion had a significantly better graft and patient survival than patients converted with a total bilirubin > 10 mg/dl (P=0.00002 and P=0.00125, respectively). The time between liver transplantation and conversion also affected graft and patient survival. Patients converted to tacrolimus < or = 90 days after transplantation had a 1-year actuarial graft and patient survival of 51.9% and 65.9%, respectively, compared with 73.2% and 87.7% for those converted > 90 days after transplantation. The mean total bilirubin level for the responder group was 7.1 mg/dl at the time of conversion and decreased significantly to a mean of 3.4 mg/dl at the end of the study (P=0.0018). Thirteen patients (14.3%) died during the study. Sepsis was the major contributing cause of death in most of these patients. CONCLUSIONS: Our results suggest that conversion to tacrolimus for chronic rejection after orthotopic liver transplantation represents an effective therapeutic option. Conversion to tacrolimus before development of elevated total bilirubin levels showed a significant impact on long-term outcome.

A study of the lateral wall pressure exerted by balloon-tipped catheters.

A technique was developed for assessing the lateral wall pressure exerted by balloon-tipped catheters. It was found that (1) high intraballoon pressures do not produce high lateral wall pressures in a minimally constricting lumen; (2) inflation of the balloon by partial compression of a large initial volume of air results in a much more compressible balloon and produces markedly less arterial distortion; and (3) use of the catheters inflated in this way is effective clinically. Therefore, instead of the usual technique, we recommend inflation of the embolectomy balloon by partial compression of a comparatively large initial volume of air in the syringe.

The multiple sequential distal bypass graft: improving nature's alternatives.

Sequential femoral-distal bypass grafts were employed to revascularize 21 ischemic limbs. An end-to-side distal anastomosis plus an additional side-to-side anastomosis to an isolated popliteal segment or an additional distal tibial vessel were employed. If one graft that underwent successful early revision is included, the initial patency rate was 100%. After a median follow-up of 8 months, complete graft failure had occurred in three patients. A previously unreported occurrence in two patients was proximal graft segment occlusion, with patency of th distal segment that was successfully revascularized. One additional patient had occlusion of the distal graft segment. Limb salvage was achieved in 20 of the 21 patients. This approach offers several hemodynamic advantages and may be the procedure of choice in patients with distal runoff that si not ideal for a routine femoropopliteal bypass graft.

Revascularization for bilateral renal artery occlusion after umbilical artery catheterization.

The case of a newborn infant who became hypertensive and oliguric because of bilateral renal artery occlusion following umbilical artery catheterization is presented. Eventual treatment was by microsurgical placement of an aortorenal graft, with subsequent marked improvement in the patient's course. A scan and arteriogram at 1 year showed that the revascularized kidney was responsible for the patient's normal blood urea nitrogen and creatinine concentrations, but the renal artery had recanalized and the graft occluded. The role of umbilical artery catheterization in such catastrophes and the possible future role such microsurgical reconstruction could play in neonatal hypertension are discussed.

Intestinal necrosis due to sodium polystyrene (Kayexalate) in sorbitol enemas: clinical and experimental support for the hypothesis.

Five patients who suffered catastrophic colonic necrosis are presented. All patients were uremic and received sodium polystyrene (Kayexalate) in sorbitol enemas for the treatment of hyperkalemia shortly before the development of signs and symptoms of colonic necrosis. In all specimens extensive ischemic necrosis was present, and Kayexalate crystals were noted in the intestinal lumen. Four of the five patients eventually died. To further investigate the occurrence of colonic necrosis after the administration of Kayexalate in sorbitol enemas, a series of experiments were performed in rats. Two groups of Sprague-Dawley rats were studied. One group was made uremic by performance of bilateral nephrectomy. The other group underwent sham operation. Enemas of saline, Kayexalate alone, sorbitol alone, or Kayexalate in sorbitol were administered. In nonuremic rats, transmural necrosis was noted in seven of 10 rats receiving sorbitol enemas and in six of 10 rats receiving Kayexalate in sorbitol enemas. No significant pathologic changes were noted in the rats receiving other enemas. In uremic rats, extensive transmural necrosis was noted in all rats receiving enemas of sorbitol or Kayexalate in sorbitol. All of these 19 rats died within the period of observation compared with no deaths in 18 rats that received enemas without sorbitol (p less than 0.001).