The prevalence and severity of late effects in normal rat duodenum following intraoperative irradiation.

In humans, a portion of the duodenum is often at risk for radiation-induced complications following intraoperative radiation therapy for pancreatic carcinoma. To determine experimentally the prevalence and severity of late effects in the normal mammalian duodenum, 190 rats received single doses of 0, 15, 20, 25, 30, or 40 Gy orthovoltage X rays to temporarily exteriorized 3 cm circumferential segments of duodenum. The animals were killed 2, 6, 8, or 10 months later. Actuarial survival, change in body weight, and a radiation injury score based on eight histopathologic alterations were used as endpoints. Epithelial atypia, intestinal wall fibrosis, serosal thickening, and vascular sclerosis were the dominant histopathologic alterations at all dose levels throughout the 10-month observation period. The prevalence and severity of histologic radiation injury showed sigmoidal dose-response relationships with the plateaus starting at 20 Gy. Doses of 20 Gy or greater also resulted in a substantial loss of body weight and a high level of early deaths (20-80 days). All endpoints indicate that intraoperative doses of 20 Gy or greater are associated with unacceptable risks of late and irreversible complications.

Intestinal complications following accelerated fractionated x-irradiation. An experimental study in the rat.

Due to paucity of suitable animal models, it has been difficult to study the development of long-term intestinal complications following fractionated irradiation. We recently developed a model which allows multiple radiation exposures of a short segment of rat ileum without the need for repeated surgery. In the present series, this model was used to study the influence of shortening the total treatment time (accelerated fractionation) on development of radiation enteropathy. Male rats were orchiectomized and a short segment of distal ileum was transposed to the scrotum. Starting 3 weeks after surgery, the scrotum containing the intestinal segment was x-irradiated with 20 fractions of 2.8 Gy (total dose 56 Gy). Two fractionation schedules were compared: One fraction per day (total treatment time 26 days) and 3 fractions per day (total treatment time 7 days). Actuarial survival curves were obtained, and the degree of radiation injury was assessed 2, 8, and 26 weeks after the last radiation exposure using a semiquantitative histopathologic scoring system. There was no mortality from acute radiation injury in either treatment group. All animals of the 1-fraction/day group survived the observation period (26 weeks). In the 3-fraction/day group, there was significant mortality due to intestinal obstruction, and cumulative mortality at 26 weeks was 100%. Radiation injury, as assessed by the histopathologic scoring system, was also more pronounced in this group than in the 1-fraction/day group. We conclude that shortening the total treatment time significantly increases the severity of late intestinal complications. Our data are suggestive of an association between acute mucosal damage and chronic radiation injury of the small intestine.

Effects of exocrine pancreatic secretions on hyperthermic injury of rat small intestine.

The influence of the exocrine pancreatic secretions on development of small intestinal injury following localized hyperthermia was studied. In male Holtzman rats the excretory pancreatic ducts were occluded with metal hemostatic clips. An intraperitoneal injection of [3H]thymidine was given 3 weeks later. Three or 48 h after the injection a 10 cm segment of small intestine was exteriorized through a midline abdominal incision and heated at 38.0 degrees C, 42.5 degrees C, or 43.5 degrees C for 45 min. Intestinal damage was assessed 24 h after hyperthermia. The following four endpoints were used: histopathological injury score, the number of villi per intestinal circumference, the number of labelled epithelial cells in fixed areas of autoradiographic specimens, and incorporation of [3H]thymidine as determined by liquid scintillation counting. The correlation of results among the four methods of assessment was highly significant. The autoradiography data showed better correlation with both morphological parameters than the results of liquid scintillation counting. There was significantly less damage in heated segments from pancreatic duct-occluded animals than in segments from sham-operated controls. When hyperthermic injury was assessed morphologically the protection conferred by pancreatic duct occlusion was equivalent to lowering the temperature of heating by 1 degree C. It is concluded that morphological criteria may be superior to endpoints based on [3H]thymidine incorporation for assessment of hyperthermic injury in rat small intestine. Reducing the intraluminal pancreatic secretions appears to confer significant protection from small bowel injury after localized hyperthermia.

Intraoperative irradiation in a rat model: histopathological changes in irradiated segments of duodenum.

During intraoperative radiation therapy for carcinoma of the head of the pancreas in humans, a portion of duodenum is often at risk for radiation-induced complications because of its fixed anatomical position within the treatment field. This study was undertaken to determine the feasibility of using the rat as a model to determine the radiotolerance of normal mammalian duodenum. The procedures used to exteriorize and irradiate a selected segment of duodenum are described. Histopathologic changes in 5-cm segments of midduodenum were studied 14 and 28 days after 0, 30, 40, or 50 Gy X-radiation. Complete denudation of the epithelium and thickening of the muscularis and serosal layers occurred in all irradiated segments by day 14. By day 28, even though crypt and villus architectures were atypical, large areas of epithelial regeneration were seen in rats receiving 30 Gy. In contrast, complete denudation of the epithelium were still evident along most of the length of the irradiated segments in rats receiving 40 or 50 Gy. Serosal fibrosis was prominent in all irradiated animals, regardless of dose. These results indicate that radiation doses above 30 Gy carry high risks of complications. The rat is considered a suitable animal model.

Effects of accelerated fractionation on radiation injury of the small intestine: a new rat model.

The present study assessed the influence of shortening the overall treatment time (accelerated fractionation) on radiation injury of the small intestine. A rat model which allowed repeated irradiation of a localized segment of small intestine was developed. Young adult male Sprague-Dawley rats were orchiectomized, and a loop of the distal ileum was transposed to the left part of the scrotum. The intestinal segment was irradiated with a total dose of 56 Gy, given in 20 fractions, the total treatment time being either 26, 12, or 7 days (i.e. 1, 2, or 3 fractions per day). Radiation injury was assessed by histopathologic examination at 6 hr and at 2 weeks after the last irradiation. The surgical procedure was without complications. Shortening the overall treatment time by giving more than one radiation dose per day resulted in markedly increased injury both at 6 hr and at 2 weeks. It is concluded that accelerated fractionation results in increased radiation injury of the intestine when compared with standard fractionation. Because there may be a relationship between early and late effects in the intestine, our results also indicate that increased late radiation enteropathy may result from accelerated fractionation.

Further probes into the molecular sites of damage to cerebral adenylate cyclase following postischemic reperfusion.

A variety of pharmacological agents were used as experimental probes to determine with greater precision the site(s) of damage to cerebral adenylate cyclase as a consequence of postischemic reperfusion in the gerbil. A paradigm of 60-min bilateral ischemia followed by 40-min reperfusion results in a decreased sensitivity of the catalytic site of adenylate cyclase to Mn2+. Likewise, the GTP-transducer site (guanine nucleotide regulatory or G protein) revealed depressed responses to GTP in the absence or presence of norepinephrine, dopamine agonists, substance P, yohimbine, and cholera and pertussis toxins. Moreover, a crude preparation of GTPase disclosed that damage elicited by postischemic reperfusion was directed to the higher-affinity form of this enzyme, which is associated with the overall function of the guanine nucleotide regulatory protein. Injury to adenylate cyclase was unrelated either to the ability of adrenergic ligands to bind to associated receptor sites or to the capacity of the brain to generate visual evoked potentials in response to visual stimuli.

Regional cyclic AMP systems during secondary ischemia in gerbils: influence of anesthetic agents.

The effect of three modes of anesthesia was evaluated with regard to regional damage to central cyclic nucleotide systems in the gerbil brain as a consequence of bilateral ischemia (clamping the common carotids) followed by various periods of recirculation. The injection of thiopental as much as 90 min before stroke prevented damage to chemical activation [catecholamines, guanosine triphosphate (GTP), or forskolin] of adenylate cyclase. However, the basal enzyme activity was lower in all brain regions whether thiopental was administered to stroke or sham-operated animals. Injection of ketamine drastically shortened the survival times of gerbils undergoing stroke followed by recirculation. About 90% of the animals could tolerate a maximum of only 15 min stroke with 15 min recirculation. At this time frame the patterns of activation of adenylate cyclase in only the olfactory tubercle and hippocampus were altered. When procaine was used as a local anesthetic agent during surgery, damage to catecholamine-, GTP-, or forskolin-activated adenylate cyclase was evident to varying degrees in the frontal cortex, hippocampus or olfactory tubercle, but not in the nucleus accumbens and olfactory bulb of gerbils subjected to 60-min stroke followed by 15 or 150 min of recirculation. The degree of enzyme damage was neither correlated with the fed vs. fasted state of the animal nor with the whole blood concentration of glucose. A depression in the amplitude of visually evoked potentials correlated to neurological signs and to enzyme damage. During anesthesia, ketamine increased steady-state concentrations of cyclic AMP in the frontal cortex and hippocampus from gerbil brains that had been rapidly inactivated by microwave irradiation. Thiopental increased steady-state cyclic AMP in only the olfactory tubercle. Cyclic GMP concentrations were unchanged by any anesthetic agent. In animals completely recovering from anesthesia and occluded for a brief period followed by 10 min of reflow, steady-state concentrations of only cyclic AMP were augmented.

The cell kinetics of human mammary cancers.

The cellular kinetics of human mammary tumors were studied by in vitro methods. These techniques include single 3HTdR labeling to measure the 3HTdR LI, double labeling with 3HTdR and 14CTdR to measure DNA synthesis time, and an estimation of the growth fraction by the PDP index. Calculations of the potential doubling time and cell cycle time were made from these measurements. The 3HTdR LI of primary malignant tumors was greater than that of benign tumors, but only half that of metastatic lesions. There was considerable heterogeneity in the 3HTdR LI of primary tumors, but the DNA synthesis times were relatively invariant. Estimation of the growth fraction by the PDP index also revealed extensive heterogeneity, but the primary tumors were not different from metastases. There appear to be subsets of tumors with high and low proliferative values that correlate with some clinical parameters, such as age and nodal positivity. This material provides a data base for stratification of patients for future protocols and the use of cell kinetics in treatment programs.

Cell kinetics in vivo and in vitro for C3H/He spontaneous mammary tumors.

Cell kinetics in spontaneous C3H/HeJ mammary tumors of retired-breeder mice was studied by in vivo and in vitro techniques. The [3H]TdR labeling index (LI), the DNA synthesis time (TS), and the primer-dependent DNA polymerase assay LI [an in vitro estimate of tumor growth fraction (GF)] were compared to similar measurements made in vivo. These measurements as well as the calculated cell kinetic parameters derived from these data were not different in in vivo and in vitro studies. Furthermore, the cell kinetic parameters in tumors classified histologically as type A or type B mammary tumors were also similar. Although considerable variation in volume doubling times (Td's), [3H]TdR LI's, potential doubling times, cell cycle times (Tc's), and cell loss was found, Ts's were similar in all mammary tumors. No correlation between tumor volume or tumor weight and cell kinetic parameters was seen. However, the most slowly growing tumors (i.e., tumors with the longest Td's) tended to have the lowest [3H]TdR LI's, the longest Tc's, and the highest cell loss factors. No correlation was found between the GF and Td. However, tumors with the most rapidly proliferating cell populations tended to have the highest GF's.

In vitro labeling and gold activation autoradiography for determination of labeling index and DNA synthesis times of solid tumors.

In vitro labeling and gold activation autoradiography were used to determine the [3H]thymidine ([3H]TdR)-labeling indices and DNA synthesis times for C3H/He spontaneous mammary tumors. Three variations of the [3H]TdR, [14C]thymidine ([14C]TdR) double-labeling method, together with double-emulsion autoradiography, were used to determine the DNA synthesis times (TS). Tumors labeled totally in vivo (in vivo-in vivo method) and tumors labeled with [3H]TdR in vivo and subsequently labeled with [14C]TdR in vitro showed similar TS values. DNA synthesis times for tumors determined totally in vitro by double labeling (in vitro-in vitro method) were significantly longer than those observed in vivo; however, identical samples subjected to Hypaque-Ficoll gradient separation after double labeling showed TS's similar to those found in vivo. Furthermore, the interval between [3H]TdR and [14C]TdR administration had no effect on TS estimates in vitro. Gold activation autoradiography was used in the present experiments to reduce autoradiographic exposure times. This method, together with in vitro labeling, permits [3H]TdR labeling index and TS determinations after 6-hr and 7-day exposures, respectively.

Treatment of solid P815x2 murine mastocytoma with adriamycin (NSC-1231127): toxity limitations.

The efficacy of adriamycin (NSC-123127), given as weekly or as 5-day-per-week doses, on the control of solid P815X2 murine mastocytomas was severely limited by hematopoietic and gastrointestinal toxicity. Although daily or weekly drug schedules both elicited dose responsiveness in terms of tumor control, no dose level of drug increased the life-span of tumor bearing animals.

A non-destructive method for measuring intestinal cell transit time.

Villous exfoliation of intestinal epithelial cells, which had been previously labeled in the crypt with 125I-iododeoxy-uridine, can be detected by thyroidal accumulation of the liberated 125I-. The latter can be monitored externally. The interval between labeled precursor injection and the steep portion of the thyroid activity accumulation curve corresponds to the intestinal cell transit time, as measured by destructive techniques, in three animal species. The technique allows estimations of intestinal cell transit time to be made on an individual basis, and can be expeditiously applied to large animals. Very small tracer doses are required for detection.

Control of local tumor growth with combined fractionated radiotherapeutic and chemotherapeutic regimens.

A treatment concept for the control of tumor growth utilized weekday radiotherapy and weekend chemotherapy. Mice were given sc injections of P815X2 mastocytoma cells on the lower back (day 0) and separated into the following treatment groups: 5-day/week X-irradiation, adriamycin alone at either 5 mg/kg body wt (days 6 and 13) or 2 mg/kg (days 5, 12, and 19), and combined radiotherapy and chemotherapy. Untreated controls had a mean tumor volume of 2.77 cm-3 and a mean survival time of 24 days. Adriamycin alone at 5 mg/kg resulted in an eventual tumor of 70 percent of the control value at death, whereas at 2mg/kg the tumor volume was 60 percent of control. After radiotherapy only, tumor size was 52 percent of control. Irradiation plus either 5 or 2 mg drug per kg body wt resulted in tumor volumes of 23 and 30 percent, respectively, of control values. Although no treatment regimen prolonged survival, the marked reduction in local tumor growth with combination therapy indicates that it may be a useful concept in future cancer therapy.

Responses of an experimental solid tumour to irradiation: A comparison of modes of fractionation.

Several radiotherapeutic schedules compatible with continued structural-functional integrity of the gastrointestinal (GI) mucosa were compared utilizing the P815X2 murine mastocytoma grown as a solid subcutaneous tumour. Both the tumour and underlying normal tissues were irradiated during the treatments. The tumour exhibited a Do that increased from 210 rad to 397 rad as the tumour aged and in all instances demonstrated minimal shoulders in survival curves. In spite of a relative radioresistance of cells within the solid tumour, quite effective control of localized disease could be accomplished with radiotherapy schemes compatible with GI tolerance limits. Schedules evaluated utilizing this model included acute exposures to 1122 rad, daily exposure to 187 rad, 5 days/week exposures to 281 rad, twice weekly exposures (561 rad on Mondays and 374 rad on Thursdays) and a high dose, two fractions per day, schedule. Tumours were followed for changes in growth patterns during these schedules. Efficacy of tumour control was determined and schedules were compared on this basis. Aggressive radiotherapy approaching the tolerance limits of any of the fractionation schemes proved most effective.