The radioprotectors amifostine and sodium selenite do not modify the radiosensitivity of rat rhabdomyosarcomas.

BACKGROUND: Radiotherapy of head and neck tumors often leads to acute reactions of the adjacent normal tissues resulting e.g. in mucositis and xerostomia. Therefore, radioprotective drugs have been developed to reduce these effects. Studies on a tumor model using amifostine and sodium selenite adjuvant to fractionated irradiation should show whether the radioprotective effect on normal tissue leads to an increase of radioresistance in the tumor and its metastatic potential. METHODS: Rhabdomyosarcomas R1H of the rat growing subcutaneously in the right flank of male adult WAG/RijH rats were irradiated with 60Co-gamma rays (60 Gy/30 fractions/6 weeks). Amifostine (375 mg/m(2)), sodium selenite (15 microg/kg), and equivalent volumes of 0.9% saline were administered intraperitoneally 30 min before each irradiation. Tumor response was determined. Statistical analysis was performed using the post-hoc-test. RESULTS: Irradiation alone inhibited R1H tumor growth (AUC 86.8+/-18.3). The efficacy of irradiation during radiotherapy was significantly improved by amifostine (AUC 63.1+/-15.8) in comparison to the irradiated controls. The radiosensitizing effect of sodium selenite (AUC 73.6+/-21.3) as well as irradiation and amifostine plus sodium selenite (AUC 68.3+/-7.8) was less compared to the irradiated controls and not statistically significant. However, tumor growth delay and metastasis rate were not changed by the radioprotective drugs. Further, the administration of amifostine and amifostine plus sodium selenite induced an enhanced decrease of animal body weight except for sodium selenite in comparison to the controls. CONCLUSIONS: The application of amifostine and sodium selenite to conventionally fractionated irradiation have no influence on the radiosensitivity of the rhabdomyosarcoma R1H. The systemic toxicity of amifostine might be of importance for the radiation treatment of a patient.

[Histomorphologic and salivary gland scintigraphic findings in radiation-induced sialadenitis due to fractionated irradiation of the head and neck region of rats. A model for evaluating potentially radioprotective substances]. / Histomorphologische und sialoszintigraphische Befunde zur Radiosialadenitis unter fraktionierter Bestrahlung der Kopf-Hals-Region von Ratten. Ein Modell zur Evaluierung potenziell radioprotektiver Substanzen.

INTRODUCTION: The aim of this study was to correlate structural, histomorphological damage of the salivary gland with scintigraphic findings during fractioned radiotherapy. METHODS: The head and neck area of 27 WAG/RijH rats was irradiated with (60)Co-gamma-rays (60 Gy/30f/6 weeks). A port-system was implanted and (99m)Tc-pertechnetat applied at different stages of irradiation (0, 16, 30, 46, 60 Gy and 6 months post irradiation). RESULTS: After the application of 16 Gy an intra- and extra-cellular oedema developed in the salivary glands. The progressive vacuolisation (30 Gy) passed over into lipomatosis (46 Gy) and necrosis (60 Gy) in the parotid and mandibular glands. Six months after irradiation treatment, the chronic histomorphological damage corresponded to stage II according to Seifert. The corresponding loss in gland function was 13% (16 Gy); 26% (30 Gy); 57% (46 Gy); 75% (60 Gy) and 66.5% (6 months post irradiation). CONCLUSION: This animal model demonstrates the correlation between histomorphological and scintigraphic findings.

[Reducing late toxicity with amifostine in fractionated irradiation of the rat salivary glands]. / Reduktion der Spättoxizität durch Amifostin bei fraktionierter Bestrahlung der Rattenspeicheldrüsen.

Clinical studies show that amifostine can reduce xerostomia and mucositis during radiotherapy of head and neck cancers. The aim of this study was to evaluate the radioprotective potency of amifostine with respect to late toxicity of salivary glands of rats. The head-neck-area of 8 male WAG/RijH rats (295 +/- 7 g) were irradiated with 60Co-gamma-rays (60 Gy/30 f/6 weeks). Amifostine (250 mg/m2 body surface) was applied via a venous port 15 min before each irradiation. Rats of a control group were irradiated with the same schedule with equal volumes of physiological saline. The morphological and sialoscintigraphical findings clearly demonstrate that amifostine has a remarkable cytoprotective effect on the late toxicity of irradiated salivary glands.

[Radioprotective effectiveness of amifostine on the salivary glands of the rat during fractionated irradiation]. / Radioprotektive Wirksamkeit von Amifostin auf die Speicheldrüsen der Ratte unter fraktionierter Bestrahlung.

BACKGROUND: The aim of this study was to evaluate the cytoprotective potency of amifostine during a fractioned irradiation of salivary glands but also on the tumor response of rhabdomyosarcomas R1H in rats. METHOD: The head-neck-area of male WAG/RijH rats and the tumor in the flank were irradiated with 60Co-gamma-rays (60 Gy/30 f/6 weeks). Amifostine (250 mg/m2) was applicated 15 min before irradiation. The control group was treated with an equal volume of physiologic sodium chloride. The salivary glands were exstirpated and examined histopathologically. Tumour volume was measured, the body-weight of rats determined. RESULTS: A dose dependent radiosialadenitis developed in all salivary glands. Amifostine had no influence on the response of R1H tumours to radiation. The animal weight loss in the amifostine group was higher as compared to control group. CONCLUSIONS: No cytoprotective effects on acute toxicity of salivary glands of rats could be detected under irradiation with synchronous application of amifostine. In addition, no effects on tumor response were observed. The general animal constitution decreased by additional medication of amifostine.

Multiple application of drugs in rats--a new port system.

The implantation of a small port system for repeated intravenous applications of drugs in rats is described. The system basically consists of a Teflon catheter which is inserted into the right internal jugular vein. The open end of the catheter under the right foreleg is subcutaneously carried through to the back and closed by a small port. The port then is implanted into a skin pocket on the back of the rat. The advantage of this method is that repeated intravenous injections of drugs into rats can easily be applied with high accuracy. Complications were rarely observed (7%) and could be mastered successfully in all cases.

[Comparison between the Eppendorf histograph and the Licox system for determination oxygen partial pressure in tumor tissue]. / Vergleich zwischen dem Eppendorf-Histographen und dem Licox-System bei der Bestimmung des Sauerstoffpartialdruckes im Tumorgewebe.

BACKGROUND: The oxygenation of a tumor is certainly one of the major facts influencing the response to treatment. pO2 measurement in tumor tissue can be done by using the Eppendorf-histograph. In recent years the Licox-system (GMS) was developed for oxygen tension measurement in tissues and fluids under clinical routine conditions and for research work. Aim of the present study was to assess the suitability of the Licox-system in drawing up histograms of pO2 values in tumor tissues. METHOD: Histograms of the distribution of oxygen tension in twelve tumors were prepared with both measuring systems in parallel. Investigations were performed on rats with isotransplanted R1H rhabdomyosarcomas in continuous intravenous anesthesia with Fentanyl and Dormicum. Ventilation with a mixture of air and oxygen was applied through a tracheostoma. Arterial pressure and body temperature were analysed and balanced continuously. RESULTS: No correspondence between data of both measuring methods could be found. CONCLUSIONS: Because of constructional advantages the Eppendorf-histograph seems to be comparatively more suitable in drawing up histograms of oxygen tension tumor tissue.

Quantitative changes of metabolic and bioenergetic parameters in experimental tumors during fractionated irradiation.

PURPOSE: Previous studies with rat rhabdomyosarcomas indicate that during fractionated irradiation profound alterations of the tumor microvasculature and the oxygenation status occur when the total dose exceeds 45 Gy. At this dose a destruction which included all structures of the vessels and a significant worsening in tumor oxygenation were found. The aim of the present study was to analyze whether these effects of fractionated irradiation on the microvasculature and on tumor oxygenation also induce changes in the bioenergetic and metabolic status in the tumors during radiation treatment. METHODS AND MATERIALS: R1H rhabdomyosarcomas of the rat implanted into the flank were irradiated with 60Co-gamma-rays using 5 fractions of 3 Gy per week over 5 weeks. During this irradiation schedule, tumors were investigated each week for the microregional distributions of glucose, lactate, and ATP concentrations. For this, tumors were rapidly excised, shock-frozen and quantitative bioluminescence measurements were performed on tumor tissue sections. RESULTS: ATP concentrations remained unchanged during fractionated irradiation up to a total dose of 45 Gy. Above this dose, a significant decrease in ATP levels was observed. Lactate concentrations changed only slightly during irradiation whereas glucose levels increased continuously over the whole irradiation period. CONCLUSIONS: During fractionated irradiation of R1H tumors with a total dose of 75 Gy, the bioenergetic and metabolic status of the tumors changed considerably. This became most obvious once a dose of 45 Gy had been achieved. The severe energy depletion and worsening of tumor oxygenation might be the result of destruction of tumor blood vessels as has been described previously in the same tumor model. The modification of the tumor micromilieu appears to be an important parameter in the responsiveness of tumor cells to radiation and for local tumor control.

Microvascular and tumor cell alterations during continuous hyperfractionated irradiation: an electron microscopic investigation on the rat R1H rhabdomyosarcoma.

PURPOSE: Conventionally fractionated y-irradiation results in severe damage of tumor capillaries associated with decreasing oxygen partial pressure within the tumor. The present study was undertaken to assess whether vasculo-connective changes are less pronounced after continuous hyperfractionated irradiation, implying better tumor oxygenation and improved radiosensitivity. MATERIALS AND METHODS: Twenty rats with an isotransplanted R1H rhabdomyosarcoma were irradiated for 12 days with 2 daily fractions of 2.5 Gy (delta t = 6 h). After 0, 15, 30, 45, and 60 Gy, tumor tissue of 4 rats each was analyzed histologically and electron-microscopically. RESULTS: Untreated rhabdomyosarcomas were composed of spindle-shaped tumor cells with numerous mitoses. There were many apoptotic nuclei and a large central necrosis. Tumor capillaries showed a continuous lining of flattened endothelial cells with broad overlapping cell contacts overlying a delicate continuous basal lamina. During irradiation, mean tumor volume declined from 1.9 cm3 to 1.2 cm3. The number of atypical mitoses and apoptoses increased and numerous giant tumor cells appeared. The proportion occupied by necrotic tumor tissue rose from 30% to 60%. After 15 Gy (3 days), a marked vasodilatation was apparent accompanied by an interstitial edema. Occasionally, endothelial cells were rounded up and showed indented nuclei, with the underlying basal lamina disintegrated. These changes progressed with increasing radiation doses. After 30 Gy (6 days), leukocytes started to adhere to the endothelial wall. Electron-dense fine fibrillar and basal lamina-like deposits appeared in the perivascular space. Endothelial cell edema was only observed after 60 Gy (12 days). Cell contact areas were shortened, however, the endothelial lining was not interrupted. No signs of radiation fibrosis were observed. CONCLUSION: Continuous hyperfractionated irradiation induces relatively discrete alterations of the vasculo-connective tumor tissue as compared to conventional irradiation. This may be an advantage with respect to tumor blood flow, oxygenation, and thus, radiosensitivity.

Changes in tumor oxygenation during a combined treatment with fractionated irradiation and hyperthermia: an experimental study.

PURPOSE: To determine the influence of adjuvant hyperthermia on the oxygenation status of fractionated irradiated tumors. METHODS AND MATERIALS: Oxygen partial pressure (pO2) in rat rhabdomyosarcomas (R1H) was measured sequentially at weekly intervals during a fractionated irradiation with 60Co-gamma-rays (60 Gy/20f/4 weeks) in combination with local hyperthermia (8 f(HT) at 43 degrees C, 1 h/4 weeks). Tumors were heated twice weekly with a 2450 MHz microwave device at 43 degrees C, 1 h starting 10 min after irradiation. The pO2 measurements (pO2-histograph, Eppendorf, Germany) were performed in anesthetized animals during mechanical ventilation and in hemodynamic steady state. All tumor pO2 measurements were correlated to measurements of the arterial oxygen partial pressure (paO2) determined by a blood gas analyzer. RESULTS: The oxygenation status of R1H tumors decreased continuously from the start of the combined treatment, with increasing radiation dose and number of heat fractions. In untreated controls a median tumor pO2 of 23 +/- 2 mmHg (mean +/- SEM) was measured. Tumor pO2 decreased to 11 +/- 2 mmHg after 30 Gy + 4 HT (2 weeks), and to 6 +/- 2 mmHg after 60 Gy + 8HT (4 weeks). The increase in the frequency of pO2-values below 5 mmHg and the decrease in the range of the pO2 histograms [delta p(10/90)] further indicated that tumor hypoxia increased relatively rapidly from the start of combined treatment. After 60 Gy + 8HT 48 +/- 5% (mean +/- SEM) of the pO2-values recorded were below 5 mmHg. CONCLUSIONS: These findings suggest that adjuvant hyperthermia to radiotherapy induces greater changes in tumor oxygenation than radiation alone [cf. (39)]. This might be of importance for the temporary application of hyperthermia in the course of a conventional radiation treatment.

Simultaneous treatment of an experimental tumour with fractionated radiation and infrared-A-hyperthermia.

Experiments on rat rhabdomyosarcomas R1H were performed to evaluate the therapeutic potential of a simultaneous radiation-hyperthermia treatment of superficially growing tumours. Tumours were heated locally with infrared-A radiation and irradiated in the middle of the heating period. After a treatment with 32 Gy in 8 fractions combined with 8 heat fractions of 43 degrees C, 1hr, during 4 weeks, tumours showed an enhanced regression and growth delay in comparison to radiation alone. A thermal enhancement ratio (TER) of 1.4 was determined. Acute skin reactions were of minor importance. In conclusion, a simultaneous treatment of superficial tumours with infrared-A-hyperthermia and radiation at a therapy unit is relatively easy to perform and enhances considerably the therapeutic efficacy of a radiation treatment.

Tumor oxygenation in a transplanted rat rhabdomyosarcoma during fractionated irradiation.

PURPOSE: To quantify the changes in tumor oxygenation in the course of a fractionated radiation treatment extending over 4 weeks. METHODS AND MATERIALS: Rhabdomyosarcomas R1H of the rat were irradiated with 60Co-gamma-rays with a total dose of 60 Gy, given in 20 fractions over 4 weeks. Oxygen partial pressure (pO2) in tumors was measured at weekly intervals using polarographic needle probes in combination with a microprocessor-controlled device (pO2-Histograph/KIMOC). The pO2 measurements were carried out in anesthetized animals under mechanical ventilation and in respiratory and hemodynamic steady state. Tumor pO2 values were correlated to the arterial oxygen pressure paO2, arterial pCO2, and pH determined with a blood gas analyzer. RESULTS: Tumor oxygenation did not change significantly during the 3 weeks of irradiation (up to 45 Gy), from a median pO2 of 23 +/- 2 mmHg in untreated controls to 19 +/- 4 mmHg after the third week. The decrease of the number of pO2 values between 0 and 5 mmHg indicated that an improved oxygenation in the tumors occurred. However, with increasing radiation dose (fourth week, 60 Gy) a significant decrease in tumor oxygenation to a median pO2 of 8 +/- 2 mmHg and a rapid increase in the frequency of pO2 values (35 +/- 4%) between 0 and 5 mmHg was found. CONCLUSION: Improved oxygenation in rhabdomyosarcomas R1H was only present in the early phase of the fractionated irradiation. Radiation does above 45 Gy led to a considerable decrease of tumor oxygenation in the later phase of irradiation.

Ultrastructural studies on tumor capillaries of a rat rhabdomyosarcoma during fractionated radiotherapy.

Sequential morphological changes in tumor capillaries of istransplanted R1H rat rhabdomyosarcoma were observed weekly by transmission electron microscopy during fractionated radiotherapy (75 Gy/25 fractions/5 weeks). During the first 2 weeks of irradiation (up to 30 Gy), edema of the tumor capillary wall was induced. Swollen endothelial cells bulged into the vascular lumen and were surrounded by a widened subendothelial space with increase amounts of collagen fibrils (subendothelial edema). The endothelial lining was preserved up to the 3rd week of irradiation (45 Gy). Prolonged irradiation was associated with progressive destruction of the vascular wall including shrinkage, gradual loss of cell contacts, disappearance of the normal chromatin pattern, and increase of cytoplasmic vacuoles in endothelial cells as well as disruption of basal laminae. One week after the end of radiotherapy (75 Gy), the tumor capillaries showed complete necrosis. Progressive damage to tumor capillaries in the course of fractionated radiotherapy might have adverse effects on blood supply and thus on tumor oxygenation.

Kinetics of depopulation, repopulation and host cell infiltration in the rhabdomyosarcoma R1H after 14 MeV neutron irradiation.

The kinetics of depopulation and repopulation of the solid transplantable rhabdomyosarcoma R1H in the rat was studied following irradiation with 5 Gy of 14 MeV neutrons. Several parameters were sequentially measured over a time period of 4 weeks after irradiation: the tumour volume was assessed by in situ caliper measurements; the numerical density of tumour cells was obtained by morphometry; the clonogenic fraction of tumour cells was derived from in vitro colony assay; and the numerical ratio of host to tumour cells was determined by flow cytometry. From these primary parameters the number of clonogenic tumour cells, non-clonogenic tumour cells, and nucleated host cells per tumour, as well as their variation with time, were derived. The results were compared with two sets of data obtained previously for the same tumour exposed to 15 Gy of 200 kVp X-rays. Survival of tumour cells was reduced to 5.5 +/- 0.5% by 5 Gy neutrons and to 4.5 +/- 0.5% by 15 Gy X-rays, i.e. an RBE of close to 3. There was a lag period before the onset of repopulation (4.9 +/- 0.4 days and 4.9 +/- 0.5 days, respectively), followed by a high initial rate of repopulation corresponding to a doubling time of 2.0 +/- 0.2 days for neutrons and 2.1 +/- 0.2 days for X-rays. The rate of depopulation was significantly different for the two treatment modalities; the halving time for the number of non-clonogenic tumour cells was 11 +/- 4 days for neutrons and 2.8 +/- 0.5 days for X-rays.(ABSTRACT TRUNCATED AT 250 WORDS)

A new method for multiple applications of cytostatic drugs in rats.

The implantation of a small silicon catheter for repeated intravenous applications of drugs in laboratory animals (rats) is described. Under an operation-microscope, the catheter is inserted into the external jugular vein and remains in situ in a skin pocket during the treatment. By dissecting the wound, multiple fractions of drugs can be administered via the catheter at intervals of days or weeks. The method has the advantage that in long-term studies with rats, intravenous injections or infusions of cytostatic drugs are possible with high accuracy.

Vascular and cellular damage in a murine tumour during fractionated treatment with radiation and hyperthermia.

Tissue reactions and vascular damage in rat tumours (rhabdomyosarcoma R1H) were studied with morphological methods during fractionated treatments with 60Co-gamma-irradiation of 75 Gy alone and in combination with ten fractions of microwave hyperthermia at 43 degrees C, 60 min for five weeks. The nature and time course of tissue and vascular damage change individually with the treatment applied. During radiation-heat treatment congestion, dilation and rupture of tumour blood vessels were observed. The cellular and vascular damage, especially in the central parts of the tumours, occurred earlier and were generally more intense during the combined treatment than found after irradiation alone. The enhanced tissue damage can be related to the local temperatures in the R1H tumours during the microwave heat treatments.

Cell population kinetics of the rhabdomyosarcoma R1H of the rat after single doses of X-rays.

The kinetics of depopulation and repopulation of the solid transplantable rhabdomyosarcoma R1H of the rat following local irradiation with single subcurative X-ray doses of 7.5, 15 and 30 Gy was studied. Several parameters were sequentially measured over a time interval of 4 weeks after irradiation: the ratio of the number of tumour to host cells, and the cellular DNA content of tumour and host cells, were determined by flow cytometry; the amount of DNA per gram of tumour tissue was determined biochemically; the clonogenic fraction of tumour cells was obtained from in vitro colony assay; and the tumour volume was assessed by in situ caliper measurements. From the amount of DNA per gram and the average DNA content per cell, the total number of cells per gram of tumour tissue was obtained. From this and the other parameters measured, the number of clonogenic tumour cells, non-clonogenic tumour cells and nucleated host cells per tumour, as well as their variation with time and dose, could be derived. The results showed that there was a lag period prior to depopulation amounting to 3.8 +/- 1.4, 1.4 +/- 0.8 or 0 +/- 0.7 days for 7.5, 15 or 30 Gy, respectively. The rate of depopulation of non-clonogenic tumour cells increased with dose; the halving times of non-clonogens were 4.7 +/- 1.8, 2.6 +/- 0.7 or 2.1 +/- 0.4 days for the three doses applied. There were no indications that proliferation of doomed cells contributed significantly to tumour growth after irradiation. After lag periods that were similar in length to those prior to depopulation, a massive immigration of host cells was observed. Under certain conditions more than 97 per cent of the cells present in irradiated tumours were found to be of host origin. There was a lag period before the onset of repopulation by clonogenic tumour cells, the length of which increased from 2.7 +/- 0.7 to 5.0 +/- 0.8 or 6.3 +/- 1.0 days for 7.5, 15 or 30 Gy, respectively. The initial rate of repopulation increased with radiation dose; after the end of the lag period the doubling time of clonogenic tumour cells (in controls amounting to 3.7 +/- 0.2 days) was 3.1 +/- 0.1, 2.1 +/- 0.1 and 1.1 +/- 0.1 days for the three doses applied.(ABSTRACT TRUNCATED AT 400 WORDS)

No acquisition of metastatic capacity of R1H rhabdomyosarcoma upon transfection with c-Ha-ras oncogene.

The effect of the activated c-Ha-ras oncogene on invasiveness and formation of spontaneous metastases was studied using the rhabdomyosarcoma R1H of the rat. R1H tumor cells which are able to grow in vitro and produce tumors upon subcutaneous injection in syngeneic WAG/Rij rats were transfected with the c-Ha-ras (EJ) oncogene and the neomycin gene for selection. Two R1H cell lines harboring and expressing the human c-Ha-ras oncogene, one cell line containing the neomycin gene only, and the parent R1H cell line were compared. The expression of the transfected c-Ha-ras oncogene was assessed by Northern blot analysis and by flow cytometry using antibodies against ras p21. No difference in tumor growth rate and morphology was observed for the transfected and untransfected cell lines. Tumor volume doubling time was about 2 days in R1H-ras as well as in R1H parent tumors. Formation of spontaneous metastases was tested by excising the tumors when they had reached a volume of 2 cm3; after that the animals were observed up to 12 months. The excised tumors still contained and expressed the transfected ras oncogene as proved by Southern blot analysis and antibody staining using anti-ras p21. In contrast to most previous work on ras-transfected tumorigenic cells the R1H-ras tumors did not acquire invasive growth potential or increased metastatic capacity.

Thermotolerance kinetics and growth rate changes in the R1H tumour heated at 43 degrees C.

R1H rhabdomyosarcomas implanted into the foot of the right hind leg of female WAG/Rij rats were exposed to fractionated hyperthermia at 43 degrees C and the kinetics of thermotolerance and heat-induced growth rate changes were studied. Tumours of anaesthetized animals were exposed to heat by immersing the leg up to the thigh in a water bath. Tumour growth delay (TGD) and tumour volume doubling time were calculated from individual growth curves. After single heating, TGD increased with increasing heating time, the increase being linear for heating times exceeding 60 min. Thermotolerance was induced by a priming heat treatment at 43 degrees C for 60 min and the kinetics of development and decay was studied for fractionation intervals ranging from 4 to 144 h. After 4 h the thermal sensitivity of the tumours was enhanced by about 30 per cent, probably due to the sensitizing effect of heat-induced physiological alterations in the tumour tissue such as suboptimal environmental conditions caused by depressed blood flow. For longer time intervals thermotolerance developed and reached a maximum at 24 h where the thermotolerance ratio was 4.5 +/- 1.5. From 24 to 144 h thermotolerance decayed exponentially with a half-time of 28 +/- 8 h. Heat also affected the growth rate of the treated tumours. After single heat treatments at 43 degrees C for 15-60 min the tumours grew faster than untreated control tumours. This change was statistically significant. After prolonged single heating, growth rate was found to be reduced. Tumour volume doubling time was not detectably changed after fractionated heat treatments.

[Histological and morphometric research on rhabdomyosarcoma in the rat after irradiation and microwave hyperthermia]. / Histologische und morphometrische Untersuchungen am Rhabdomyosarkom der Ratte nach Bestrahlung und Mikrowellen-Hyperthermie.

Cellular effects of a combined treatment with X-rays (15 Gy) and local microwave hyperthermia (43 degrees C, 60 min) on the tumor tissue of the rhabdomyosarcoma R1H of the rat were studied with light and electron microscopy. The results were compared with the cell reactions obtained after X-rays (15 Gy) alone. The histological results and the morphometric data show that the time course of cell damage changes individually with the treatment applied. After radiation alone a cell oedema occurs within some days. After a combined treatment, however, a marked cell oedema and cell lysis is already observed after 24 hours. An intensive shrinking of the cell nuclei is found at day 6th, attended with pyknosis, karyolysis and cell fragments in the extended extracellular area. In comparison with radiation alone the processes of repair and repopulation in the R1H tissue are remarkably delayed after a combined treatment.