Biomedical subjects
W Dörr
Publications and source records attributed to W Dörr.
Three A's of repopulation during fractionated irradiation of squamous epithelia: Asymmetry loss, Acceleration of stem-cell divisions and Abortive divisions.
PURPOSE: To analyse the time-course and efficacy of repopulation in squamous epithelia, and to describe possible mechanisms of this regeneration response. MATERIAL AND METHODS: Experimental and clinical studies of repopulation in squamous epithelia have been reviewed to outline general features of repopulation during fractionated radiotherapy. RESULTS: Repopulation processes result in a relative increase in re-irradiation tolerance, which can be quantified as the dose equivalent compensated. CONCLUSIONS: If the stem-cell concept is accepted, changes in residual tissue tolerance must be based on net production of stem cells. For this, normal asymmetrical stem-cell divisions that render equal numbers of stem cells and differentiating daughters, must turn into symmetrical divisions with the production of two stem cells. Furthermore, the rate of change in residual tolerance indicates that the stem-cell proliferation rate is increased. In addition to these changes in the stem-cell proliferation pattern, a limited number of divisions by sterilized cells contributes to overall cell production and maintenance of tissue function. A valid model of repopulation in squamous epithelia hence must be based on three distinct mechanisms summarized as the three A's: Asymmetry loss, Acceleration of stem-cell divisions, and Abortive divisions.
Effects of ageing and X-irradiation on the diurnal rhythm of mouse urinary bladder capacity.
The effect of single-dose irradiation (19 Gy) or sham treatment on circadian variations in urinary bladder storage capacity was assessed during a 250-day period following treatment using transurethral cystometry under anesthesia. Changes in bladder function were quantified as the deviation from individual pretreatment values of bladder volume at defined intravesical pressures. A marked diurnal rhythm with a peak at 19.00 h and an extended nadir between 1.00 h and 12.00 h was found in 11- to 12-week-old untreated mice. No systematic changes in bladder function with age could be observed in repeated cystometries after sham treatment. X-Irradiation resulted in a triphasic response. After an early reversible decrease in bladder capacity (acute phase) complete recovery was observed within 30 days after irradiation. Following a symptom-free latent period of about 15 weeks, chronic progressive impairment of bladder storage function developed (chronic phase). During the acute and latent period, no changes in the diurnal pattern were observed. In the late phase, not only a decrease in the absolute capacity values but also in the amplitude of diurnal fluctuations was seen. The daily minimum values, however, were still found between 1.00 and 12.00 h. The data indicate that cystometry values obtained between 8.00 and 12.00 h provide suitable parameters for the quantification of radiation effects in the urinary bladder in longitudinal studies.
Acute response of pig skin to irradiation with 12C-ions or 200 kV X-rays.
The acute response of pig skin to treatment with high energy carbon ions (plateau region) at the Gesellschaft für Schwerionenforschung (GSI, Darmstadt, Germany) was compared with changes after 200 kV x-irradiation. Carbon doses isoeffective to the x-ray doses were computed with a recently established model for calculation of the biological effect of heavy ions. Clinical changes and physiological symptoms (blood flow, erythema, trans-epidermal water loss, skin hydration) were scored. The parameters analyzed were maximum and mean values of each symptom during days 24 to 70 after irradiation, and the quantal endpoints for the establishment of dose effect curves were the median values of these. With exception of the maximum change in the red blood cell concentration (p < 0.02) no significant differences could be found in the response to x-rays and RBE-corrected heavy ions. These results indicate that the model is valid for the calculation of biological effects of 12C-ions (plateau region) and may at least for epidermis be applied to treatment planning.
Complications of combined intraoperative radiation (IORT) and external radiation (ERT) of the upper abdomen: an experimental model.
An experimental model in the rabbit is presented which is suitable for analysis of clinically relevant, early side-effects of combined upper abdominal IORT and ERT. Fractionated ERT alone given through an upper abdominal a.-p. field including the entire stomach caused gastric ulcerations within < or = 58 days. Latent times decreased with increasing dose and the ED50 for occurrence of ulcers was 39 +/- 3.3 Gy. Single doses of IORT of 20-40 Gy alone administered through a 2-cm diameter field localized on the coeliac axis and carefully excluding any intestinal mucosa caused neither gastric ulcerations nor other clinical symptoms. When ERT with 40 Gy was preceded by IORT with 20-40 Gy or by sham IORT, 13 out of 15 animals developed ulcers after latent times which in a life-table analysis were shown to be significantly shorter than after ERT alone. However, a statistically significant IORT dose-dependence of latent time or incidence of ulcers could not be demonstrated in the present experiment. The most significant histological changes were observed in the areas of gastric ulcers. Already during ERT, the mucosal epithelium was depleted and regenerative activity was evident in spite of ongoing fractionated irradiation. However, profound irregularities in glandular structure and distribution, as well as number of proliferating epithelial cells were still present in healed ulcers at 80 days. In summary, IORT to the coeliac artery did precipitate the development of gastric ulcers induced by subsequent ERT. On the one hand, the data indicate that the surgical procedure of IORT did contribute to this effect. On the other hand, IORT to the coeliac artery could cause transient, functional alterations in blood supply to the depending organs, i.e. the stomach, and could thus precipitate the development of radiation-induced ulcers.
Tissue kinetics in mouse tongue mucosa during daily fractionated radiotherapy.
The purpose of the present investigation was to quantify cell flux between the distinct layers of the epithelial lining of the ventral surface of mouse tongue during daily fractionated radiotherapy. A fraction of DNA-synthesizing cells in control epithelium, or at various days through a course of daily fractionated radiotherapy with 3 or 4 Gy per day, was labelled with [3H]dT or BrdUrd, respectively. The labelling indices (LI) in the different epithelial layers were defined histologically after autoradiography, or immunohistochemistry, at intervals between 1 and 10 days after label administration. In tongue epithelium of untreated mice, the minimum residence time of cells in the germinal layer is 2-3 days. Migration through the functional layers requires an additional 2-3 days before labelled cells are observed in the most superficial layer of nucleated cells. A plateau in LI is observed for several days post-labelling in control epithelium, indicating an equilibrium between loss and proliferation of labelled cells. During fractionated radiotherapy, the minimum time from division to occurrence of labelled cells in the stratum lucidum is less than 2 days, and hence significantly shorter than in control epithelium. In contrast to untreated epithelium, no plateau in the germinal layer LI is seen, indicating that frequently both labelled daughters from dividing labelled cells are being lost from this compartment. In conclusion, the present data support a recently described model of radiation-induced accelerated repopulation in squamous epithelia, which postulates that the majority of damaged cells undergoes abortive' divisions resulting in two differentiating daughters.
[Nomenclature of modified fractionation protocols in radiotherapy].
BACKGROUND/AIM: During the last few years a number of new, unconventional fractionation protocols have been established for various biological reasons. Recently specific terms, established for these schedules, have frequently been applied inappropriately in oral presentations as well as in publications. Hence, the present work was initiated in order to clarify definitions and, in addition, to illustrate the respective biological basis for the various fractionation designs.
Radiation-induced changes in transforming growth factor beta and collagen expression in the murine bladder wall and its correlation with bladder function.
Late radiation-induced changes in transforming growth factor beta (TGF-beta), collagen I and collagen III content of the bladder wall, as well as morphological alterations of the uroepithelium, were analyzed quantitatively in an immunohistochemical study. An interlaboratory, i.e. interstrain, comparison of two mouse strains (Amsterdam C3H/Hen Af-nu+ and Munich C3H Neu) with different dose-effect relationships for late bladder damage was made, choosing radiation doses producing equivalent functional alterations in both strains (ED80 of 25 Gy and 19 Gy, respectively, 40 weeks after irradiation). In one strain of mouse, cystometry was also performed in the same animals at different times after irradiation. The TGF-beta staining intensity showed a progressive increase between 90 and 360 days after irradiation. This increase was similar in both strains of mouse treated with functionally equivalent doses (ED80) and was less pronounced after a lower, ED40, dose in the Munich mice. In both strains, there was a radiation-induced increase in both collagen subtypes from 180 days after irradiation with the ED80. The ratio of collagen type I/III, however, decreased in the Amsterdam mice and increased in the Munich mice. The relative radioresistance of the Amsterdam mice may therefore be partly due to a greater contribution of the elastic collagen type III, affording greater bladder compliance after irradiation. The extent of radiation-induced uroepithelial denudations or papillomatous outgrowths, the TGF-beta staining intensity and collagen I/III ratio were each correlated to bladder function determined by cystometry for the Munich mice. This correlation was statistically significant for all three parameters for group mean responses and, with the exception of the collagen I/III ratio, also for individual mice. These experiments indicate that chronic radiation-induced alterations in TGF-beta expression and connective tissue metabolism in the bladder wall are possibly important factors determining reduced bladder function after irradiation.
[Combined intraoperative and external irradiation of the celiac artery in the rabbit: the effects on the blood flow of the gastric mucosa].
AIM: To demonstrate changes in gastric mucosal blood flow caused by intraoperative radiotherapy of the celiac artery combined with external radiotherapy of the upper abdomen in a rabbit model. The study was designed to identify a possible correlation between a radiation-induced reduction in mucosal blood flow and the induction of gastric ulcer. MATERIAL AND METHOD: Intraoperative radiation doses of 0 or 30 Gy were given to the celiac artery in rabbits. After a delay of 14 days external radiotherapy of the upper abdomen with 3 x 4 Gy/week to a maximum total dose of 40 Gy was initiated. Gastric mucosal blood flow was assessed by intraventricular injection of radioactively-labelled microspheres (15 microns) followed by measurement of radioactivity in the mucosa. The injections were performed at various time intervals between 2 and 63 days after intraoperative radiation. RESULTS: Intraoperative radiotherapy, including sham-intraoperative radiation, resulted in a transitory reduction of mucosal blood flow by about 50% of the control value on day 7 (Figure 3). After a temporary recovery by day 14, a marked and permanent reduction in blood flow was assessed after week 6. This time corresponds to the time of development of gastric ulcer. CONCLUSIONS: A relationship between the time of ulcer development and of reduced gastric mucosal blood flow was observed after combined intraoperative and external radiotherapy. The mechanical component of intraoperative treatment has to be emphasized. Reduced blood flow was also seen after intraoperative radiotherapy alone, without an induction of ulcer by this treatment. Hence additional mucosal damage by external radiation must be present for the induction of gastric ulcer.
Effects of stimulated repopulation on oral mucositis during conventional radiotherapy.
The effect of local conditioning of human oral mucosa by silver nitrate solution (3%) on epithelial proliferation rates was tested in 11 healthy volunteers by in vitro labelling of biopsies with tritiated thymidine. Compared to control biopsies from 13 volunteers, stimulation over 3 days, 3 times per day, yielded a significant (p = 0.006) increase in the epithelial labelling index (LI) from 4.75 +/- 0.32% to 6.85 +/- 0.65%, i.e., by 44%. The increase in the absolute number of labelled cells per mm epithelial length was dependent on the overall cell density at the various intraoral sites and varied between 45% in the maxillary vestibule and 91% at the floor of the mouth. In an analysis of variance, stimulation turned out to be the most important source causing the effect (p = 0.011 for LI and 0.015 for labelled cells per mm). In a radiotherapy trial with conventional postoperative treatment with 5 x 2 Gy/week to a total dose of 60 Gy in 6 weeks, the left buccal mucosa in 10 patients with squamous cell carcinomas of the head and neck was conditioned (3% silver nitrate, 3 times per day, 5 days before and the first 2 days of radiotherapy) while the contralateral mucosa, receiving an identical dose, served as individual control. Mucositis scores according to the EORTC/RTOG or the Dische system showed that the time course and severity of the mucosal response was almost identical in both cheeks, which is in clear contrast to a previous clinical study (Maciejewski et al. Radiother. Oncol. 22, 7-11, 1991). Differences in radiation dose intensity, i.e., weekly dose, in these studies are discussed as a tentative explanation for the different clinical findings.
Repopulation response of mouse oral mucosa during unconventional radiotherapy protocols.
Repopulation in mouse tongue epithelium was determined during unconventional fractionation schedules, i.e., hyperfractionation (2 x 1.5 and 2 x 1.75 Gy/day) and accelerated treatment (2 x 3 Gy/day). The residual tolerance of the epithelium at defined days of the fractionated treatment was tested by graded single test doses (top-up design). The dose required to induce complete epithelial denudation in 50% of the animals (ED50) was used to calculate the number of fractions repopulated during the preceding treatment. After the first week of hyperfractionation, tolerance was reduced compared to untreated epithelium. However, subsequently no further change was observed, indicating complete compensation of the weekly dose with all doses per fraction used. Epithelial cell density, defined by histological examination in additional experiments, in all fractionation arms decreased similarly by approximately 40% during the first week and remained constant at 60-80% in the subsequent 2 weeks. During accelerated fractionation, the residual mucosal tolerance decreased continuously with treatment time and resulted in epithelial denudation after 12 fractions. However, a substantial repopulation effect was observed, compensating 1.5 fractions by day 2, and 5 fractions by day 5, respectively. After cessation of the therapy the repopulation rate clearly decelerated to compensate a dose equivalent to about 0.5 fractions per day. Cell density decreased linearly during the treatment with 5, 10 or 12 fractions at a rate close to normal cell loss. Marked cell production, dependent on the total fractionated dose, was seen from one day after the last fraction in each experimental arm. These results indicate that maximum stem cell repopulation occurs predominantly during treatment, while major production of differentiating cells take place in treatment splits.
Effect of changing weekly dose on accelerated repopulation during fractionated irradiation of mouse tongue mucosa.
The effect on accelerated repopulation of fractionated radiotherapy with changing weekly dose was studied in mouse oral mucosa. The basic conventional treatment protocol comprised five daily fractions per week, 3 Gy per fraction, given over 3 weeks. In dynamic treatment schedules the same total dose was delivered by giving 5 x 2, 5 x 3 and 5 x 4 Gy, or the reverse sequence. In concomitant boost experiments five additional daily doses of 1 or 2 Gy respectively were applied either during the first or last week of the conventional treatment. Graded terminating test doses were given in order to establish dose-effect curves for complete mucosal denudation. Higher initial doses consistently resulted in longer latent times between top-up irradiation and complete epithelial denudation, corresponding to significantly higher cell densities at the time of test irradiation. The top-up ED50's served as a measure of repopulation efficacy. The difference in repopulated dose between schedules with initial or terminal dose concentration was 10% for dynamic fractionation (p = 0.02) and about 7% in the 2-Gy boost protocol (p = 0.13), and no difference was observed with boost doses of 5 x 1 Gy. In conclusion, a clear though moderate increase in both cell density and residual tissue tolerance can be achieved by protocols with substantially higher doses in the first treatment week.
[The documentation and publishing of radiotherapy side effects. A workshop report].
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Impact of time between X-ray fractions on acute changes in the function of the urinary bladder in the mouse.
Noninvasive transurethral cystometry was used in mice anesthetized with pentobarbitone to quantify the effect of the interval between X-ray fractions on acute radiation-induced changes in the function of the urinary bladder. Bladder capacity was defined at intravesical pressures of 10 and 20 mm Hg. A reduction in the storage capacity of the bladder by > or = 50% of the individual preirradiation control value, occurring at any time during the initial 30 days after radiotherapy, was the quantal end point used for dose-response analysis. Four equal-sized dose fractions were applied with intervals of 0, 0.5, 1, 1.5, 2, 4 and 8 h, respectively. The ED50 values increased from 18.2 Gy (95% confidence interval 16.8-20.6) for single-dose treatment to 28.1 Gy (26.2-29.9) in the 8-h protocol. Linear-quadratic analysis of the results, using an indirect repair model and the direct analysis approach, yielded a half-time of repair of 1.2 h (0.9-1.9) with an alpha/beta ratio of 10.4 Gy. If combined with previous data for split doses where 1 to 10 fractions were given within < or = 7 days (Dörr and Schultz-Hector, Radiat. Res. 131, 35-42, 1992), an even longer repair half-time of 1.7 h (1.3-2.7) was determined.
Repopulation in mouse oral mucosa: treatment splits.
The efficacy of repopulation during treatment splits in conventional radiotherapy (5 fractions/week, 2.5, 3.5, 4.5 Gy/fraction) was studied by delayed top-up treatment of mouse tongue epithelium. Splits of 6 h to 13 days were introduced after 1 or 2 weeks of fractionated irradiation. Following 5 fractions, compensation of about 3 dose fractions was assessed after the first weekend and original tissue tolerance was restored after a split of 4-10 days. About 4.6 dose fractions were repopulated during the second treatment week, followed by a further 1.5 fractions during the first 3 split days; restoration of the initial tolerance required 3-8 days. These results indicate that repopulation was more efficient during fractionated radiotherapy than during a subsequent treatment split. Latent times to complete denudation after the top-up treatment decreased to a dose-dependent minimum after 5 fractions and remained at the decreased level when a second treatment week was added. Original values were restored within 5-8 days after 5 fractions and 6 days after 10 fractions. Epithelial cell density during treatment with 3 or 4 Gy/fraction decreased to a dose-dependent nadir of 63% and 52% of the original number after 5 fractions, and original cell counts were then restored after 5 days. Cellularity remained at 60-70% during the second treatment week and subsequently reached normal values within 4 days. In conclusion, reconstitution of epithelial cellularity precedes restoration of radiation tolerance during treatment splits.
Response of mouse oral mucosa to repeated doses of bleomycin.
Bleomycin (BLM) applied at systemically tolerable doses induces denudation of tongue mucosa in the C3H-Neuherberg mouse strain. The dose-incidence curve after single injections has a sigmoid shape with an ED50 of 17.5 mg/kg. In contrast, the dose-response curves to repeated (two, five and 10) drug injections follow triphasic shapes and show dose-effect inversions. The effect initially increases with dose to a maximum of 70-100% at 2 x 7, 5 x 2, and 10 x 0.9 mg/kg. A marked decrease in response is observed at higher doses with a nadir of 10-30% after 2 x 11 mg/kg, 5 x 4 to 5 x 5 mg/kg and 10 x 2 mg/kg, followed by a second rise when dose is further increased. These clinical results were confirmed in a histological study. Variation of the time interval between two drug injections caused marked fluctuations in the treatment efficacy. A clear increase in drug response was induced by splitting total drug doses of 6, 14 or 22 mg/kg, the maximum effect (100%) was seen at intervals of 2 h, 0.5-1 h and 0.25 h between two injections of 3, 7 or 11 mg/kg, respectively. At longer intervals of up to 6 h, a dose-dependent decrease in drug efficacy resulted in an inverse dose-effect. Original tissue tolerance to BLM was restored only in the 2 x 3 mg/kg arm but was still elevated in the other arms after 96 h. The results can be plausibly explained by the dose-dependent induction of detoxifying processes.
Proliferation equivalent of 'accelerated repopulation' in mouse oral mucosa.
The proliferation response and changes in cellularity of mouse tongue epithelium were studied after single doses of X-rays and during 3 weeks of daily irradiation. A single dose of 13 Gy resulted in minimum cellularity (70% of control values) on days 3-5 and complete restoration on day 7. Mitotic activity ceased for 1 day followed by normal-to-supranormal values until day 15. A wave of abnormal mitoses was observed with a peak at days 4-7. Daily irradiation with 3 or 4 Gy induced neither major structural nor visible cellular damage. Cellularity decreased to approximately 60% during week 1 and subsequently remained at 60-70%. The proliferation activity was reduced to approximately 8% by day 2. Mitotic activity during weeks 2 and 3 was subnormal-to-normal, with a dose-dependent increase to normal counts during the first weekend and a distinct overshoot over the second weekend respectively. A proliferation model is presented to explain the present findings and previous functional measurements of changes in tissue tolerance. Its major features are accelerated symmetrical stem cell divisions and abortive divisions of sterilized cells.
Cell kinetic studies in mouse tongue mucosa by autoradiographic, immunohistochemical and flow cytometric techniques.
A number of techniques, including autoradiography after in vivo administration of tritiated thymidine ([3H]dT), immunohistochemistry after in vivo administration of bromodeoxyuridine (BrdUrd), and flow cytometry (FCM) with and without BrdUrd detection were compared in the epithelium of ventral mouse tongue. Investigation of the diurnal proliferative rhythm by immunohistochemical detection of incorporated BrdUrd with different primary antibodies in combination with the alkaline-phosphatase anti-alkaline-phosphatase technique, the peroxidase-anti-peroxidase method, and an indirect method with a polyclonal peroxidase-conjugated secondary antibody yielded results similar to standard autoradiography. Preparation of single cell suspensions for flow cytometry was not successful. A maximum yield of about 8.5% of the original cell number was achieved by ultrasound disintegration in combination with trypsin and dithioerythrol treatment, but neither a G0/G1 peak nor a G2 + M peak was observed in DNA histograms. A better yield of about 38% of the original nuclei number was obtained by preparation of suspensions of nuclei using citric acid and the detergent Tween 20 in combination with magnetic stirring. Both S-phase index and BrdUrd labelling index could be determined by FCM and showed the normal diurnal variations. However, the BrdUrd labelling index in suspensions of nuclei was significantly higher than the labelling index determined after immunohistochemistry. The FCM S-phase index at times of day with low DNA synthesizing activity was higher than the BrdUrd index, indicating a fraction of unlabelled S-phase cells. In conclusion, detection of incorporated BrdUrd in oral mucosa by immunohistochemical techniques or flow cytometry is feasible and provides a useful tool for fast measurements of proliferation.