Estimating the number of animals: a rapid method for unidentified individuals.

A proposed model yields the density of a mobile population from quick, cursory surveys in which the observer identifies none of the animals. When the spaces on which animals were seen are successively removed, the decline in the counts permits estimation of the average probability of seeing a given animal. The method showed promise in initial trials.

Prostaglandins protect against murine hair injury produced by ionizing radiation or doxorubicin.

Several years ago we showed that prostaglandins (PGs) are potent radioprotective agents. To investigate further the potential use of these compounds we employed quantitative measures of murine hair loss and regrowth to assess the effects of PG administration before multi-dose fractionated radiation exposures. We compared these results with findings utilizing the thiol compounds WR-2721 or WR-1065, the "gold standard" laboratory radioprotectors. Three weeks after systemic administration of 16-16 dm PGE2 (Upjohn Company) or WR-2721, given 1 h before each dose of 2-4.5 Gy per fraction for 10-15 fractions, regrowing hair counts increased up to 100% compared to irradiated-only skin sites. The thiol compound effects were slightly superior to the PG effects in these studies. Local applications of 16-16 dm PGE2 or WR-1065 given 15 min before each radiation fraction also enhanced post-radiation hair regrowth, although systemic administration of either agent was more effective than the topical route. We also evaluated possible protective effects of PGs given before doxorubicin, measuring murine hair loss 1 week after parenteral injections of the drug. Five daily doses of doxorubicin, 0.1 mg/25 g animal, reduced the number of hairs in a 4.42 mm2 area of skin from 241 +/- 5 (controls) to 144 +/- 3. Misoprostol (G.D. Searle & Co.), 25 micrograms/mouse, applied locally 2 h before each dose of doxorubicin, resulted in 213 +/- 8 residual hairs. We conclude that clinical use of these compounds may provide significant protection of hair follicles and possibly other normal tissues (skin; oral, rectal, and bladder mucosa) lying within a radiation field or in patients treated with chemotherapeutic agents. Further assessment of possible tumor protection effects are needed, however.

Cerebral glucose metabolism in Wernicke's, Broca's, and conduction aphasia.

Cerebral glucose metabolism was evaluated in patients with either Wernicke's (N = 7), Broca's (N = 11), or conduction (N = 10) aphasia using 18F-2-fluoro-2-deoxy-D-glucose with positron emission tomography. The three aphasic syndromes differed in the degree of left-to-right frontal metabolic asymmetry, with Broca's aphasia showing severe asymmetry and Wernicke's aphasia mild-to-moderate metabolic asymmetry, while patients with conduction aphasia were metabolically symmetric. On the other hand, the three syndromes showed the same degree of metabolic decline in the left temporal region. The parietal region appeared to separate conduction aphasia from both Broca's and Wernicke's aphasias. Common aphasic features in the three syndromes appear to be due to common changes in the temporal region, while unique features were associated with frontal and parietal metabolic differences.

Disconnection and cerebral metabolism. The case of conduction aphasia.

Ten patients with conduction aphasia were studied with computed tomography and 18-F-fluorodeoxyglucose positron emission tomography to examine glucose metabolism. Computed tomographic results identified a postrolandic structural locus for conduction aphasia. All patients demonstrated resting glucose hypometabolism throughout the parietal and temporal regions, and half of the patients also demonstrated reduced metabolic rates in the posterior, inferior, frontal (Broca's) regions. These data suggest that disconnection between posterior and anterior language areas may not be the best anatomical explanation for conduction aphasia.

Subcortical structures in aphasia. An analysis based on (F-18)-fluorodeoxyglucose, positron emission tomography, and computed tomography.

Subcortical structural damage that includes the anterior and posterior internal capsule, caudate, thalamus, lenticular nuclei, and insula has been shown to cause aphasias. A critical question that has not been resolved is whether the role of these structures on behavior is a direct one or whether it is indirect through the cortex. We have used pathway analysis to evaluate computed tomography, glucose metabolic, and language data from 47 aphasic patients to answer this question. For fluency (from the Western Aphasia Battery), subcortical structural damage had direct and indirect (through frontal lobe) effects on the behavior. For a comprehension task (sequential commands), subcortical damage had no direct effect and only a slight indirect effect through the temporal lobe. Thus, both direct and indirect effects of subcortical damage can be demonstrated for specific behavioral measures.

Temporoparietal cortex in aphasia. Evidence from positron emission tomography.

Forty-four aphasic patients were examined with (F18)-fluorodeoxyglucose positron emission tomography in a resting state to determine whether consistent glucose metabolic abnormalities were present. Ninety-seven percent of subjects showed metabolic abnormalities in the angular gyrus, 89% in the supramarginal gyrus, and 87% in the lateral and transverse superior temporal gyrus. Pearson product moment correlations were calculated between regional metabolic measures and performance on the Western Aphasia Battery. No significant correlations were found between the Western Aphasia Battery scores and right hemisphere metabolic measures. Most left hemisphere regions correlated with more than one score from the Western Aphasia Battery. Temporal but not frontal regions had significant correlations to the comprehension score. The left temporoparietal region was consistently affected in these subjects, suggesting that common features in the aphasias were caused by left temporoparietal dysfunction, while behavioral differences resulted from (1) the extent of temporoparietal changes, and (2) dysfunction elsewhere in the brain, particularly the left frontal and subcortical areas.

Cerebellar glucose metabolism in chronic aphasia.

(18F)-Fluorodeoxyglucose PET was used to compare left/right cerebellar hemispheric glucose metabolism in 37 aphasic patients with left hemisphere lesions and 22 age-matched controls. Sixteen aphasic subjects showed cerebellar symmetry. Twenty-one aphasic subjects were found to have cerebellar metabolic asymmetry, which (1) resulted from an absolute reduction in local cerebral metabolic rates of glucose in the right cerebellar hemisphere; (2) was associated with left less than right glucose metabolic asymmetry in the frontal, parietal, caudate, and thalamic regions; (3) was associated with Broca's region and deep hemisphere structural damage to the internal capsule and basal ganglia; (4) related to reduced functional motor performance, spontaneous speech, naming, reading, and writing; and (5) included all Broca's aphasia subjects.

Left hemisphere intracerebral hemorrhages studied by ( (F-18)-fluorodeoxyglucose PET.

We used PET to study patients with intracerebral hemorrhages in the left hemisphere. Three anatomic and physiologic patterns were observed. Patients 1 and 2 had midputamen hemorrhages with diffuse left less than right hemispheric metabolic asymmetry most prominent in temporal and parietal regions. Patients 3 and 4 had posterior putamen-insula-temporal hemorrhages with left less than right metabolic asymmetry in temporoparietal cortex and thalamus. Patients 5, 6, and 7 had smaller posterior hemorrhages. Left cortical metabolism was little affected in these three cases. Persistent aphasia was associated with severe metabolic left less than right asymmetry in posterior middle temporal regions.

Cytosar-U (Ara-C) induced changes in mouse jejunal crypt epithelial kinetics and radiosensitivity to gamma rays and fast neutrons.

Pretreatment with the S phase specific cytotoxic agent Cytosine Arabinoside (Ara-C) protects the intestinal stem cells from gamma radiation injury by nearly tenfold. Studies were undertaken to test whether an altered cell age distribution could account for the reported duration and degree of Ara-C induced protection and to measure the degree of protection from the high energy neutrons of the Fermilab Cancer Treatment Facility. Twelve hours after treatment with Ara-C, B6CF1/ANL mice were exposed to increasing single doses of either 137Cs gamma-rays or neutrons from the Fermilab accelerator, or a split dose of neutrons with intervals of 1, 2, and 3 hours. The number of regenerating microcolonies per jejunal circumference in Ara-C treated and irradiated animals was compared to irradiated controls. Another group of mice was given Ara-C but in the 12-hour interval between Ara-C and irradiation, colcemid was given every 3 hours to continuously block and kill cells in mitosis. The results suggest that Ara-C given 12 hours prior to neutron irradiation protects intestinal stem cells to nearly the same degree as it does from 137Cs gamma-ray damage. Furthermore, the control split-dose recovery ratio to neutron irradiation at 1, 2, or 3 hours was 1.8 and was unchanged 12 hours after Ara-C. Colcemid reduced the crypt cell population to less than half the normal 250 cells per crypt; however, the cell survival curve was unaltered from the survival curve 12 hours after Ara-C. These results suggest that Ara-C recruits intestinal clonogenic stem cells, but inhibits their normal passage through DNA synthesis. These cells, responsible for intestinal mucosal regeneration, appear to be held in a radioresistant portion of the cell cycle for a period of about 10-16 hours after Ara-C.

Misoprostol-induced radioprotection of oncogenic transformation.

PURPOSE: Prostaglandins are associated with a variety of both pathologic and normal physiological effects in mammals. Among this broad array of effects, prostaglandins have been shown to provide protection to tissues from a variety of injurious agents including ionizing radiation. Of the prostaglandins tested to date, an analogue of prostaglandin E1, misoprostol (cytotec) was found to be a very effective radioprotector. The purpose of this study was to assess the ability of misoprostol to protect cells from the cytotoxic and oncogenic effects of ionizing radiation. METHODS AND MATERIALS: Pregnant Syrian hamsters were injected subcutaneously with 125 micrograms misoprostol/100 g body weight 2 h before being exposed to graded doses of X rays. Embryos were excised immediately after irradiation and cells were explanted into culture dishes. Following 14 days of incubation, cells were fixed in formalin and stained with giemsa for examination of cell clonogenicity and morphological transformation. RESULTS: First, misoprostol protected cells from some degree of radiation toxicity. A reduction in cell killing by a factor of 1.5 was seen at 10% cell survival. Second, based on transformation studies, a higher frequency of oncogenic transformation is seen for cells exposed in utero to graded doses of X rays alone than for cells exposed to the combination of misoprostol followed by radiation. In the presence of misoprostol, transformation is reduced by a factor of 20 at the level of 10(-3) transformants per surviving cell. CONCLUSION: Misoprostol may have clinical utility, not only in protecting selected normal tissues during cancer therapy, but it may also be useful in protecting cells from secondary tumors caused by ionizing radiation.

Subcutaneous or topical administration of 16,16 dimethyl prostaglandin E2 protects from radiation-induced alopecia in mice.

Alopecia, a common sequel of radiation treatment of brain tumors, increases patient stress to the extent that refusal of treatment may occur. The expectation that loss of hair will be prevented, or that regrowth will occur, is extremely important to patients. To investigate prostaglandin-induced radiation protection against alopecia, the hair of B6D2F1 male mice was plucked from the right thigh and surrounding area to induce anagen. Fourteen days later, mice were injected subcutaneously in the neck with 10 micrograms 16,16 dm PGE2 in 0.2 ml of vehicle, or with the vehicle alone. In another group of previously plucked mice, 16,16 dm PGE2 in the same concentration, or the vehicle was applied topically. One hour later, graded single doses from 6.5 to 12.5 Gy 137Cs gamma irradiation were given to groups of six animals. On day 21 post-plucking, all animals were killed and a portion of the irradiated site was excised. The average hair counts per field in irradiated animals were 85 +/- 4 (6.5 Gy), 25 +/- 5 (8.5 Gy), and 5.5 +/- 0.7 (10 Gy). Animals receiving the prostaglandin systemically had values of 60 +/- 10 (6.5 Gy), 54 +/- 3 (8.5 Gy), 66 +/- 6 (10 Gy), and 30.1 +/- 8 (12.5 Gy). Topical application of the prostaglandin resulted in protection that yielded 52 +/- 3 (8.5 Gy), 34 +/- 4 (10 Gy), and 3.2 +/- 0.9 (12.5 Gy) hairs per field. Both systemic and topical application of 16,16 dm PGE2 protected from some degree of radiation-induced alopecia, which supports the conclusion that prostaglandins may be useful in the protection of hair follicles in patients treated with radiation for brain tumors.

Radiation protection of murine intestine by WR-2721, 16,16-dimethyl prostaglandin E2, and the combination of both agents.

The survival of murine intestinal clonogenic cells (ICC) and the survival of mice after whole-body exposure to 137Cs irradiation were used to measure radiation protection by ethiophos (WR-2721), 16,16-dimethyl prostaglandin E2, and the combination of the two. Doses from 2 to 12.5 mg/mouse of WR-2721 increased cell survival linearly from 3.2 +/- 0.3 in controls given 15.0 Gy to 93.1 +/- 5.2 per jejunal circumference. In contrast, 16,16-dm PGE2 increased ICC survival at 15.0 Gy rapidly from 1 to 10 micrograms/mouse, followed by a plateau up to 100 micrograms/mouse. Animal survival at 6 days (LD50/6) increased from 16.3 +/- 0.4 Gy (95% confidence limits) in controls to 20.3 +/- 0.6 Gy in the PG-treated animals. WR-2721 increased the LD50/6 to 26.1 +/- 1.4 Gy. The dose modification factors were 1.25 and 1.60, respectively. The combination of agents increased ICC survival above that seen with each agent alone up to 8 mg WR-2721, above which no additional protection was seen. Animals given 10 micrograms PG plus 10 mg WR-2721 survived longer than with either agent given alone. The LD50/6 was 36.3 +/- 1.8 Gy for a dose modification factor (DMF) of 2.23. In addition, the slope of the probit curve was reduced from those of each agent alone. PG-induced changes in villus epithelial cell morphology and survival may account, in part, for these observations. The results suggest that either the mechanisms for these two types of radiation protectors are different or they act on separate subcellular targets which are critical to survival from radiation injury.

Misoprostol, a PGE1 analog, protects mice from fission-neutron injury.

The eicosanoids are associated with pathophysiological events of tissue injury linked to a large number of diseases. In contrast, prostaglandins have been found to protect tissues from injuries sustained by exposure to a variety of physical and chemical agents including radiation. Little is known about the mechanism of protection by prostaglandins; however, some evidence suggests that the eicosanoids may influence the repair of DNA lesions that would influence cell or animal survival after irradiation. To investigate an association between repair of sublethal radiation damage and eicosanoid-induced radioprotection, experiments were designed to study similarities and differences in protection by misoprostol (a radioprotective PGE1 analog), WR-2721, or the combination of both, before photon or neutron injury. Misoprostol alone, WR-2721 alone, or the combination of the two increased survival of intestinal clonogenic cells and animal survival following exposure to JANUS fission-spectrum neutrons in a way similar qualitatively to protection by these same treatments from the effects of 137Cs gamma radiation. The split-dose survival ratio for JANUS neutrons is 1, whereas the ratio for 137Cs gamma radiation is about 6. Since misoprostol, alone or with WR-2721, both protected intestinal clonogenic cells and increased animal longevity following JANUS neutron irradiation, it is unlikely that a prostaglandin-induced increase in sublethal damage repair can explain the observed eicosanoid-induced radioprotection.

16,16-Dimethyl prostaglandin E2 induces radioprotection in murine intestinal and hematopoietic stem cells.

Exogenous prostaglandins (PGs) have been shown to protect gastrointestinal mucosa, liver, and pancreas from several injurious agents, including the PG inhibitor, indomethacin. Previous studies from this laboratory showed exogenous administration of 16,16-dimethyl (dm) PGE2 also protected mouse intestinal stem cells from radiation injury. The present study extended that observation and demonstrated that PGs given to B6D2F1 mice 1 hr before irradiation increased the shoulder of the intestinal clonogenic cell survival curve. The D0 increased from 1.10 + 0.09 to 1.58 + 0.10 Gy. PGs increased the LD50/6 from 16.3 + 0.41 (95% confidence limits) in controls to 20.25 + 0.55 Gy. The 16,16-dm PGE2 increased the hematopoietic CFU-S survival in a qualitatively similar way; the extrapolation number (n) was increased from 1.03 (0.89-1.20) to 1.40 (1.27-1.54) and the D0 increased from 0.92 (0.87-0.98) to 1.14 (1.10-1.19) Gy. A large number of human tumors secrete a variety of PGs. Our results suggest that those tumors may be, in part, protected from radiation injury.

16, 16-dimethyl prostaglandin E2 increases survival of murine intestinal stem cells when given before photon radiation.

A variety of prostaglandins (PG) protect the gastric and intestinal mucosa when given before damaging agents such as absolute ethanol, acidified taurocholate, boiling water, or nonsteroidal anti-inflammatory agents (NSAI). A synthetic prostaglandin, 16, 16-dimethyl PGE2, shown to be cytoprotective at physiologic levels to the above agents was given to mice 1 hr before or 15 min after 137Cs gamma (gamma) whole-body irradiation. The survival of intestinal stem cells measured by their ability to form in situ colonies of regenerating epithelium was increased when 16, 16-dimethyl PGE2 was given before but not after 137Cs gamma irradiation. The maximum degree of 16, 16-dimethyl PGE2-induced radioprotection was seen when the drug was given 1 hr before irradiation. No radioprotection was seen when the interval between drug and irradiation was 3 hr or longer. When the time between 16, 16-dimethyl PGE2 and irradiation was kept at 1 hr, the degree of radioprotection was dependent on the PG drug dose. There was a steep rise in the number of surviving cells at low doses of PG. These results imply that tumors which secrete PGE2 may in part be protected from the lethal effects of ionizing photon radiation.

Misoprostol-induced radioprotection of Syrian hamster embryo cells in utero from cell death and oncogenic transformation.

Misoprostol, a PGE1 analog, is an effective radioprotector of murine intestine and hematopoietic and hair cell renewal systems. The radioprotective nature of misoprostol was extended to examine its ability to influence clonogenic cell survival and induction of oncogenic transformation in Syrian hamster embryo cells exposed to X rays in utero and assayed in vitro. Hamsters in their 12th day of pregnancy were injected subcutaneously with misoprostol, and 2 h later the pregnant hamsters were exposed to graded doses of X rays. Immediately after irradiation, hamsters were euthanized and embryonic tissue was explanted into culture dishes containing complete growth medium. After a 2-week incubation period, clonogenic cell survival and morphologically transformed foci were determined. Survival of misoprostol-treated SHE cells was increased and yielded a dose reduction factor of 1.5 compared to SHE cells treated with X rays alone. In contrast, radiation-induced oncogenic transformation of misoprostol-treated cells was reduced by a factor of 20 compared to cells treated with X rays alone. These studies suggest that misoprostol not only protects normal tissues in vivo from acute radiation injury, but also protects cells, to a large extent, from injury leading to transforming events.

The prostaglandin E1 analog, misoprostol, a normal tissue protector, does not protect four murine tumors in vivo from radiation injury.

The clinical development of radioprotectors, such as misoprostol, to protect normal tissue during cancer treatment must proceed with the assurance that tumors are not protected similarly or significantly. To provide data on this critical question, radiation-induced growth delay with or without the presence of misoprostol was measured in four murine tumors grown in the flanks of mice: the Lewis lung carcinoma, M-5076 ovarian sarcoma, FSA and NFSA. The effect of misoprostol on the tumor control dose (TCD50) of radiation was measured in FSA-bearing mice with or without prior treatment with the nonsteroidal anti-inflammatory agent, indomethacin. Misoprostol did not influence the in vivo growth of any of the four tumors, nor did it protect any of the tumors from radiation-induced growth delay. Likewise, there was no increase in the radiation TCD50 to treat the FSA in vivo in control or indomethacin-treated tumor-bearing mice. To measure any possible influence of tumor burden on the protective effect of misoprostol on normal tissue in mice, the protective effect of misoprostol on the survival of intestinal clonogenic cells was measured in M-5076-bearing mice and found to be the same as in non-tumor-bearing mice. These data suggest that misoprostol protects normal tissue in mice without protecting at least four experimental murine tumors. The data support the contention that misoprostol can achieve therapeutic gain by protecting normal tissues without protecting tumors.

Ionizing radiation induces early, sustained increases in collagen biosynthesis: a 48-week study in mouse skin and skin fibroblast cultures.

Groups of 10 CF1 female mice, irradiated to the thorax with a dual-head 137Cs gamma-RAY source, received single doses of 0, 5, 10, 15, or 25 Gy. One to forty-eight weeks later collagen synthesis was measured in minced skin specimens incubated in medium containing [3H]proline and then assayed for radioactive hydroxyproline. A progressive, generally dose-dependent increase in collagen biosynthesis, up to 50% above control sites, was found 1, 4, and 12 weeks after radiation exposure. These changes showed further small fluctuations at 12-36 weeks, increasing again at the 48-week interval. At the same times throughout the study fibroblasts were cultured from skin explants. Following the second subculture, these cells were also incubated in medium containing [3H]proline, and collagen synthesis was again determined by [3H]hydroxyproline assay. At all radiation dose levels studied, collagen production increased threefold by 12 weeks postradiation and remained elevated for the 48-week duration of the study. In vitro radiation dose response differences were not observed.

A comparison of radioprotection from three neutron sources and 60Co by WR-2721 and WR-151327.

Two thiophosphoroate radiation protectors (WR-2721 and WR-151327) were assessed for their ability to modify the effects of neutron or gamma irradiation on the gastrointestinal tract. Three neutron sources (DOSAR, JANUS, and FERMILAB) were compared to the response obtained after 60Co irradiation. The end points studied were intestinal stem cell survival and LD50(6). DOSAR and JANUS, both fission-spectrum neutrons, showed somewhat different gut sensitivities [LD50(6)] of about 240 and 400 cGy respectively. The intestinal LD50 obtained with FERMILAB neutrons (25 meV) was closer (875 cGy) to that obtained after 60Co (1068 cGy) irradiation. WR-151327 protected against the lethal effects of fission neutron (DOSAR and JANUS) to a greater degree (DMF = 2.2) than with lower LET sources such as FERMILAB neutrons (DMF = 1.7) or 60Co (DMF = 1.7). The results did not correlate with the intestinal stem cell assays where WR-2721 when compared to WR-151327 showed either similar (DOSAR; fission spectrum neutrons) or somewhat better (60Co and FERMILAB neutrons) protection. Possible explanations for the differing results are discussed.

Comparison of intestine and bone marrow radiosensitivity of the BALB/c and the C57BL/6 mouse strains and their B6CF1 offspring.

The radiosensitivity as measured by LD50/6 or LD50/30 of the F1 hybrid B6CF1 (C57BL/6 X BALB/c) is similar to that of C57BL/6 mice but markedly different from BALB/c. The LD50/6 for BALB/c mice was about 8.8 Gy compared to 16.4 Gy for the B6CF1. The difference in LD50/6 between the parent strains or between BALB/c and the F1 hybrid could not be explained by any differences in crypt cell number, cell cycle time, or transit time. Likewise, the observed differences in the LD50/6 do not appear to result from marked differences in the radiosensitivity of marrow stem cells (CFU-S) since the D0's for the three genotypes of mice were similar. Also, there were no apparent differences in the red blood cell contents of several enzymes associated with antioxidant defenses. The microcolony assay was used to determine the D0 for the crypt clonogenic cells and the D0 values for 60Co gamma rays were about 0.8 Gy for BALB/c mice and 1.4 Gy for B6CF1 mice. However, the D0 values for JANUS fission neutrons were similar; 0.6 Gy for the BALB/c mice and 0.5 for the B6CF1 mice. A comparison of clonogenic cell kinetics, using prolonged colcemid block to distinguish between slowly and rapidly cycling cells suggest that, normally, the stem cells are slowly cycling in both the BALB/c and the B6CF1 hybrid. However, the stem cells of the B6CF1 appear to go into rapid cell cycle more rapidly than those of the BALB/c following irradiation or prolonged colcemid treatment. The more rapid recovery in intestinal epihelial cell production in the B6CF1 hybrid after irradiation may provide an increased mucosal barrier and may, in part, explain the difference in the response to radiation compared to that in the BALB/c.