Acta Radiologica: Oncology, Radiation, Physics, Biology
ISSN: 0348-5196 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/ionc18
Protective Effect of Hypoxia Against Radiation Induced Fibrosis in the Rat Gut J. O. Forsberg, H. Jiborn & B. Jung To cite this article: J. O. Forsberg, H. Jiborn & B. Jung (1979) Protective Effect of Hypoxia Against Radiation Induced Fibrosis in the Rat Gut, Acta Radiologica: Oncology, Radiation, Physics, Biology, 18:1, 65-75, DOI: 10.3109/02841867909128191 To link to this article: https://doi.org/10.3109/02841867909128191
Published online: 08 Jul 2009.
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Citing articles: 11 View citing articles
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FROM THE DEPARTMENTS OF SURGERY AND RADIATION PHYSICS, UNIVERSITY HOSPITAL, S-750 14 UPPSALA, THE DEPARTEMENT OF BIOPHYSICS, THE GUSTAF WERNER INSTITUTE, UNIVERSITY OF UPPSALA, S-751 21 UPPSALA, AND THE DEPARTMENT OF SURGERY, MALMO , ALLMANNA SJUKHUS, s-214 01 M A L M ~SWEDEN.
PROTECTIVE EFFECT OF HYPOXIA AGAINST RADIATION INDUCED FIBROSIS IN THE RAT GUT J. 0. FORSBERG, H. JIBORNand B. JUNG Tissue fibrosis is one of the effects of irradiation (RUBIN & CASARETT 1968). Generally, some tissues are more liable to develop fibrosis than others, but neither the occurrence, nor the extent of this reaction can be predicted with accuracy. Previously the use of hypoxia for protection of the rat ileum against acute radiation injury was reported by FORSBERG et coll. (1978). Acute hypoxia was achieved by intraarterial injection of degradable starch microspheres and the hypoxia modified the radiation injury corresponding to a change in dose by a factor of 0.44.The fibrotic reaction in non-protected segments of rat ileum and in segments protected by injection of starch microspheres has later been compared and the results are now reported. Material and Methods
Sprague-Dawley male rats weighing about 300 g, fed on a standardized diet and with free access to food and water were used. After catheterization of a branch to the superior mesenteric artery three 4 cm long segments of the distal ileum with 5 cm interspaces were marked with silk ligatures. The procedure is described in detail previously (FORSBERG et coll.). Irradiation was performed with the segment placed in a saline bath outside the abdomen. In 23 animals, 3 segments of the distal part of the -
Submitted for publication 22 February 1978. Acta Radiologica Oncology 18 (1979) F a x . 1
5 - 795845
65
66
J. 0. FORSBERG, H. JIBORN AND B. JUNG
Fig. 1. Sampling from a removed, sagitally incised ileal segment (schematic). Half of the irradiated segment (shadowed) was compared microscopically with the continuous, non-irradiated segment. The other half of the irradiated segment was removed for hydroxyprolineassay.
exteriorized distal ileum were irradiated. Two of the three segments were nonprotected and the third protected with induced ischemia. The dose to each segment was randomly chosen in a predetermined set, for the non-protected segmentscin a lower dose range and for the protected segments in a higher dose range. As control a non-irradiated ileal segment was used in each animal (Table 1). Degradable microspheres (Pharmacia AB) with a diameter of 44 7 ,am were administered at a concentration of 80 mg/ml - 6.08 x lo6 spheres/ml; 0.7 x loo of the spheres suspended in a 20 ml buffered NaC1-solution with 240 or 1 500 IU standard amylase gave (in vitro) a degrading half time of 40 and 20 min, respectively (NYGREN 1976). Irradiation was started with the non-protected segments and with one segment at a time. The degradable microspheres were then injected in the catheter to induce gut ischemia and after 5 min the third segment, located between the other two segments, was irradiated. Each animal was anaesthetized by intraperitoneal mebumal-sodium (ACO, 40 mg/ kg) administered 30 min before irradiation, which was given with 8 M V roentgen rays from a linear accelerator (MEL SL Super, 600 pulses/s, pulse-length 2 ,as, souice-toskin-distance 73 cm and mean dose rate 13 Gy/min). The irradiation dose was controlled with radiation sensitive diodes (Scanditronix DPDS, Uppsala, Sweden). The scatter dose to the center of the animal was 2.5 per cent of each segment dose given, which means that the range of the total scatter doses in this material was between 0.8 and 1.55 Gy. Table 1 Number of non-protected and protected ileal segments referred to radiation a h ? (GY) 7.5
Non-protected n= Protected n=
8
10.0
12.5
15.0
17.5
20.0
7
7
7
7
6
6
6
25.0
6
RADIATION INDUCED FIBROSIS
67
Fig. 2. Segments of rat ileum 4 weeks after irradiation. Nonprotected segment irradiated with 20 Gy (*). The segment heavily fibrotized. Protected segment irradiated with 25 Gy with a small nodulus of fibrous tissue not encircling the gut wall (+), Non-protected segment irradiated with 7.5 Gy (*).
After irradiation the segments werc replaced into the abdomen and the abdominal incision was stitched. Following irradiation the animals were placed in separate cages. Microscopic grading of fibrosis. The animals w x e killed after 4 weeks. The gut was removed and the segments identified. The irradiated segments were cut out together with a non-irradiated, contiguous segment. The specimens were cut sagitally into two congruent pieces. If, as s e n macroscopically, only one part of the circumference of the gut was fibrotic, the incision was made through the middle of that part. One of the pieces was fixed in 6 per cent formaldehyde for microscopy. The irradiated part of the other half was taken for hydroxyproline analysis (Fig. 1). The control segments were similarly treated. The sections for microscopy were stained with haematoxylin-eosin and van Gieson stain. Microscopically the specimens were divided into two groups. To the first group were referred specimens with no increase in fibrous tissue and to the second group specimens with a distinct augmentation of the fibrous tissue. Extra-serosal fibrosis, referable to omental adhesions was not considered. The extent of the fibrosis was not subjected to further microscopic gradation. Hydroxyproline assay. The specimens analysed for hydroxyproline were dried in an oven at 100°C until a constant weight was reached (3 days), and the dry weight was then recorded. Hydroxyproline was determined after hydrolysis in 6N HC1 for 3 h at 130°C using the method described by PIKKARAINEN (1968). The hydroxyproline concentration was calculated as mg per g dry tissue.
Results Of the 23 animals, one died after 11 days due to unknown causes and one died after 3 weeks of ileus due to fibrotic constriction of a non-protected segment. Of 21 segments
68
J. 0. FORSBERG, H. JIBORN AND B. JUNG
Fig. 3. Irradiated, protected segment (20 Gy) with 8 small fibrotic nodulus and the continuous, non-irradiated segment (left). ( x 30)
Fig. 4. Non-protected segment 4 weeks after irradiation with 7.5 Gy. The normal appearances of villi and crypts destructed and villi not fully epithelialized. ( x 130)
which should have been protected in the experiments, 3 were excluded immediately due to catheter failure resulting in incomplete blockage. When killed, two of the rats had chronic mechanical ileus due to fibrotic constriction of the non-protected segments and with a thick gut wall in the dilated gut proximal to the stenoses. Macroscopically, fibrosis was easily observed in several of the irradiated segkents, which were most often covered by omental tissue. In the heavily fibrotic non-protzcted segments, the gut had shrunk to about 1/3 of. its original length and the gut wall was fibrotized around the gut circumference. In protected segments such abnormalities were found in only two segments, and in a few segments there were one or several
RADIATION INDUCED FIBROSIS
69
Fig. 5. Heavily destructed mucosa in a non-protected segment 4 weeks after irradiation with 20 Gy. Cystlike crypts, marked inflammatory reaction and an abnormal abundance of goblet cells. ( x 160)
noduli of fibrotic tissue with a radius of 2 mm or less, but not circumscribing the gut wall (Fig. 2). The lengths of these segments were not reduced. Micrcscopic evaluation. As the sections included irradiated as well as non-irradiated segments, a direct comparison with unaffected mucosa was possible in each section (Fig. 3). In the non-protected segments irradiated with the lowest doses, epithelial recovery was usually almost complete, but sometimes recovery was incomplete even after these doses (Fig. 4). In the segments irradiated with higher doses the mucosa was either abnormal (Fig. 5 ) or absent (Fig. 6). Compared to the non-protected segments, the mucosal recovery in the protected segments was obvious (Fig. 7). The findings concerning mucosal recovery are summarized in Fig. 8. Fibrosis increased in frequency at the higher doses, less for the protected than for the non-protected segments (Fig. 9). The fibrous tissue almost invariably included all the different layers of the gut wall (Fig. 10) and in most fibrotic segments the mucosal lining was more or less absent. In the non-protected segments, the fibrous tissue always included the entire extent of the segment; whereas in the protected segments (but for 2) it was restricted to one or several small noduli-the remaining part of the segment being unaffected (Fig. 10).
70
J. 0. FORSBERG, H. JIBORN AND B. JUNG
Fig. 6. Heavily fibrotized non-psotected segment 4 weeks after irradiation with 17.5 Gy. No mucosal regeneration. ( x 45)
Fig. 7. Protected segment irradiated with 20 Gy. No fibrosis. Appearances of villi and crypts almost entirely normal. Few goblet cells compared to Fig. 5, indicating a higher survival proportion of the regenerating, columnar epithelium. ( x 50)
Hydroxyproline evaluation. The hydroxyproline content in mg per g of dry tissue in the segments is given in Table 2.zThe hydroxyproline content in per cent relative to the control segments is given in Fig. 11. The values show a constant elevation of hydroxyproline in the non-protected segment from 12.5 Gy and a significant difference to the control value at 15.0 Gy. The protected segments show no such increase relative to the control values. Discussion
The aim was to determine whether deep hypoxia during irradiation would protect against development of fibrosis.
71
RADIATION INDUCED FIBROSIS
Fig. 8. Appearance of the mucosa 4 weeks after irradiation of non-protected (NP) and rotected (P) ileal segments. -non-recovery of epithelial lining over a fibrotic ulcer. epithelial lining unbroken, but normal structure of the mucosa considerably destructed. Abundance of goblet cells. = slight destruction of the mucosal structure. Less abundant goblet cells. Incidence in per cent of total number of segments in each group.
e
5
50
n Z5
0.0
125
M
175
150
xx) #
I
NP n = 0
I
I
MD
250 Gy
P
7
7
6
6
6
6
YO
'""1 Fig. 9. Incidence of fibrosis in per cent of total number of segments irradiated with different =fibrosis including the whole irradiated segment. = fibrosis of a small nodulus, a few mm in diimeter, but with no fibrosis in t'ie rest of the segment. NP = non-protected, P = protected. L
1
L
NP n.8
7
7
P
I
7
6
6
6
6
The fibrotic process is an inevitable mechanism of tissue repair. It is well known that the extent of trauma is a modifying factor in the fibrotic development. Thus, in surgery, a gentle handling of the tissues and an active regimen against infection prevent excessive scar formation. In radiation therapy, a low dose-rate (multiple fractions) will give a similar result. Experiments aiming at reducing already established fibrosis following trauma, has been performed with Ca EDTA. This increased the urinary excretion of hydroxyproline, but did not decrease the observable fibrosis (TOBINet coll. 1974). The exact mechanism of fibrosis development after irradiation is still obscure, but generally the degree of trauma (dose), the trauma mechanism and the specific sen-
72
J. 0. FORSBERG, H. JIBORN AND B. JUNG
Fig. 10. Fibrotic tissue in all layers of the gut wall. The muscle layer is completely broken up. ( x 70)
Fig. 11. Hydroxyproline content of each irradiated segment relative to nonirradiated segments of the same animal. Filled circles = non-protected(NP) segments. Open circles = protected (P) segments. Dose (Gy). Mean and
SD. 7.5
10.0 7
125 7
15.0 125 20.0 7 7 6 6 6
250 Gy 6
73
RADIATION INDUCED FIBROSIS
Table 2 Hydroxyproline content (mglg dry tissue) of non-protected and protected ileal segments 4 weeks after irradiation. Mean and SD, n=number of segments, * or indicate significant difference ( p 0.05) between values with the same sign. The high value indicated by *, is attributable to one segment with an extremely high control value. Exclusion of this segment will give figures in brackets Dose (Gy)
Mean
SD
n
Non-protected 0 (control) 7.50 10.0 12.5 15.0 17.5 20.0
7.56' 8.18 8.07 9.18 13.28' 21.47O 25.89
2.46 2.31 1.91 2.60 6.70 8.76 8.99
20 8 7 7 7 7 6
Protected 15.0 20.0 25.0
13.40O (9.10)' 9.09 9.02
4.40 (2.35)* 3.33 2.05
6 6 6
sitivity of the tissue concerned determine when and to what extent fibrosis will occur (RUBIN& CASARETT). In differentiated tissue, fibrosis may start as a primary stimulation of the interstitial fibrocytes or the fibrocytes in the vicinity of a vessel, or it may be secondary to the inflammatory reaction induced by tissue necrosis (RUBIN& CASARETT). In the gut, another mechanism must also be considered, namely inflammation induced by bacteria from the gut lumen invading the injured mucosal wall. In experimental work on irradiated granuloma tissue, RANTANEN (1973) found that the mature fibrocytes reacted with a fibrotic repair in spite of decreased number of cells. He suggested, that radiation induced fibrosis was an effect of collagenolytic impairment which resulted in excess accumulation of collagen into the irradiated tissues. In older granuloma tissue irradiated with 10 Gy he found already after a few days an increase in the hydroxyproline. Hydroxyproline is a suitable substance for detecting fibrosis since essentially all of the hydroxyproline in animal tissue is found in the collagen (ADAMS1970). The present histologic evaluation indicates and the hydroxyproline assay shows significantly a development of fibrosis in the non-protected segments following a dose of 15 Gy. At higher dose levels the hydroxyproline increased further. However, a considerable variation in the dose response occurred between specimens within a certain group, as indicated by the variance. The protected segments did not show a tendency toward a hydroxyproline increase at the chosen dose levels.
74
J. 0. FORSBERG, H. JIBORN AND B. JUNG
The fibrosis at the higher dose levels in non-protected segments is in good agreement with the experiencesfrom radiation therapy, where it is well known that fibrosis following irradiation is dose dependent. Thus, a high dose will give more and earlier fibrosis than will a low dose. Also a dose deliveredin a few fractions will cause the fibrosis to come more rapidly than if the same dose is given in several fractions (RUBIN & CASAREXT). After a single dose of 30 Gy to exteriorized rat ileum OSBORNE et coll. (1970) found a massive fibrosis already on the tenth day. They found a higher survival rate of animals in which the superior mesenteric artery was clamped during irradiation, indicating protection against the acute injury, but they 'did not mention that this protection resulted in less fibrosis. In clinical conditions irradiation fibrosis usually occurs late. The interval between irradiation and manifest fibrosis may often be several years, but the initiation of the fibrosis is immediate (RANTANEN). The modifying effect of hypoxia on fibrotic development is obvious. As most fibrotic segments showed a non-repaired mucosal lining, irradiation injury to the mucosa might be one primary mechanism for the achieved fibrosis. This would explain the absence of fibrosis in the protected segments, in 50 per cent having preserved et coll.). A commucosal lining on the fourth day after a dose of 26 Gy (cf. FORSBERG plementary mechanism could be hypoxic protection of the fibrocytes and the collagenolytic processes, as suggested by RANTANEN. It cannot be excluded, however, that the protected segments, with no fibrosis after 4 weeks, would not have developed fibrosis later, due to a delayed repair mechanism. Acknowledgements This investigation was supported by the Swedish Cancer Society. The irradiations were performed at the Department of Oncology, University Hospital, Uppsala.
SUMMARY The protective effect of hypoxia, induced by degradable microspheres, against the development of fibrosis was investigatedafter single-dose irradiation of exteriorized rat ileum. Evaluation, based upon microscopy and analysis of hydroxyproline in protected and nonprotected gut segments, demonstrated an evident protective effect at doses of 15 to 25 Gy.
ZUSAMMENFASSUNG Der Schutzeffekt von Hypoxie, der durch abbaubare Mikrosphiirenhervorgerufen worden war, gegen die Entwicklungeiner Fibrose nach einer Einzel-Strahlendosisdes herausgelegten Ratten-Ileums wurde untersucht. Die Untersuchung, die sich auf Mikroskopie und Analyse von Hydroxyprolin in geschiitzten und nicht geschiitzten Darm-Segmenten basiert, zeigte einen klaren Schutzeffekt bei Dosen von 15 bis 25 Gy.
RADIATION INDUCED FIBROSIS
75
RESUME L‘effet radioprotecteur de I’hypoxie produite par des microsphkres dkgradables contre le dkveloppement d’une fibrose a CtB etudiC aprbs ]’irradiation par une dose uniqued‘ileon de rat exttriorisk. L’Ctude des segments intestinaux protkgts et non proteges, basee sur la microscopie et I’analyse de I’hydroxyproline, a mis en evidence un effet protecteur net a des doses de 15 A 25 Gy.
REFERENCES ADAMS E. : Metabolism of proline and hydroxyproline. Zn: International review of connective tissue research, Vol. 5, p. 1. Edited by D. A. Hall and D. S. Jackson. Academic Press Inc., London 1970. FORSBERG J. O., JUNGB. and LARSSON B.: Mucosal protection during irradiation of exteriorized rat ileum. Effect of hypoxia induced by starch microspheres. Acta radio]. Oncology 17 (1978), 485. NYGREN A.: Personal communication. Pharmacia Laboratories. 1976. OSBORNE J. W., PRASAD K. M. and ZIMMERMAN G. R.: Changes in the rat intestine after Xirradiation of exteriorized short segments of ileum. Radiat. Res. 43 (1970), 131. PIKKARAINEN J. : The molecular structures of vertebrate skin collagens. Acta physiol. scand. (1968) Suppl. No. 309. RANTANEN J.: Radiation injury of connective tissue. A biochemical study with experimental granuloma. Acta radio]. (1973) Suppl. No. 330. RUBINP. and CASARETT G. W.: Clinical radiation pathology. W. B. Saunders Company, Philadelphia 1968. TOBING., ARONSON A. and CHVAPILM.: Effect of CaEDTA administration on urinary hydroxyproline excretion and skin wound healing in the rat. J. surg. Res. 17 (1974), 346.
ISSN: 0348-5196 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/ionc18
Protective Effect of Hypoxia Against Radiation Induced Fibrosis in the Rat Gut J. O. Forsberg, H. Jiborn & B. Jung To cite this article: J. O. Forsberg, H. Jiborn & B. Jung (1979) Protective Effect of Hypoxia Against Radiation Induced Fibrosis in the Rat Gut, Acta Radiologica: Oncology, Radiation, Physics, Biology, 18:1, 65-75, DOI: 10.3109/02841867909128191 To link to this article: https://doi.org/10.3109/02841867909128191
Published online: 08 Jul 2009.
Submit your article to this journal
Article views: 52
View related articles
Citing articles: 11 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ionc18
FROM THE DEPARTMENTS OF SURGERY AND RADIATION PHYSICS, UNIVERSITY HOSPITAL, S-750 14 UPPSALA, THE DEPARTEMENT OF BIOPHYSICS, THE GUSTAF WERNER INSTITUTE, UNIVERSITY OF UPPSALA, S-751 21 UPPSALA, AND THE DEPARTMENT OF SURGERY, MALMO , ALLMANNA SJUKHUS, s-214 01 M A L M ~SWEDEN.
PROTECTIVE EFFECT OF HYPOXIA AGAINST RADIATION INDUCED FIBROSIS IN THE RAT GUT J. 0. FORSBERG, H. JIBORNand B. JUNG Tissue fibrosis is one of the effects of irradiation (RUBIN & CASARETT 1968). Generally, some tissues are more liable to develop fibrosis than others, but neither the occurrence, nor the extent of this reaction can be predicted with accuracy. Previously the use of hypoxia for protection of the rat ileum against acute radiation injury was reported by FORSBERG et coll. (1978). Acute hypoxia was achieved by intraarterial injection of degradable starch microspheres and the hypoxia modified the radiation injury corresponding to a change in dose by a factor of 0.44.The fibrotic reaction in non-protected segments of rat ileum and in segments protected by injection of starch microspheres has later been compared and the results are now reported. Material and Methods
Sprague-Dawley male rats weighing about 300 g, fed on a standardized diet and with free access to food and water were used. After catheterization of a branch to the superior mesenteric artery three 4 cm long segments of the distal ileum with 5 cm interspaces were marked with silk ligatures. The procedure is described in detail previously (FORSBERG et coll.). Irradiation was performed with the segment placed in a saline bath outside the abdomen. In 23 animals, 3 segments of the distal part of the -
Submitted for publication 22 February 1978. Acta Radiologica Oncology 18 (1979) F a x . 1
5 - 795845
65
66
J. 0. FORSBERG, H. JIBORN AND B. JUNG
Fig. 1. Sampling from a removed, sagitally incised ileal segment (schematic). Half of the irradiated segment (shadowed) was compared microscopically with the continuous, non-irradiated segment. The other half of the irradiated segment was removed for hydroxyprolineassay.
exteriorized distal ileum were irradiated. Two of the three segments were nonprotected and the third protected with induced ischemia. The dose to each segment was randomly chosen in a predetermined set, for the non-protected segmentscin a lower dose range and for the protected segments in a higher dose range. As control a non-irradiated ileal segment was used in each animal (Table 1). Degradable microspheres (Pharmacia AB) with a diameter of 44 7 ,am were administered at a concentration of 80 mg/ml - 6.08 x lo6 spheres/ml; 0.7 x loo of the spheres suspended in a 20 ml buffered NaC1-solution with 240 or 1 500 IU standard amylase gave (in vitro) a degrading half time of 40 and 20 min, respectively (NYGREN 1976). Irradiation was started with the non-protected segments and with one segment at a time. The degradable microspheres were then injected in the catheter to induce gut ischemia and after 5 min the third segment, located between the other two segments, was irradiated. Each animal was anaesthetized by intraperitoneal mebumal-sodium (ACO, 40 mg/ kg) administered 30 min before irradiation, which was given with 8 M V roentgen rays from a linear accelerator (MEL SL Super, 600 pulses/s, pulse-length 2 ,as, souice-toskin-distance 73 cm and mean dose rate 13 Gy/min). The irradiation dose was controlled with radiation sensitive diodes (Scanditronix DPDS, Uppsala, Sweden). The scatter dose to the center of the animal was 2.5 per cent of each segment dose given, which means that the range of the total scatter doses in this material was between 0.8 and 1.55 Gy. Table 1 Number of non-protected and protected ileal segments referred to radiation a h ? (GY) 7.5
Non-protected n= Protected n=
8
10.0
12.5
15.0
17.5
20.0
7
7
7
7
6
6
6
25.0
6
RADIATION INDUCED FIBROSIS
67
Fig. 2. Segments of rat ileum 4 weeks after irradiation. Nonprotected segment irradiated with 20 Gy (*). The segment heavily fibrotized. Protected segment irradiated with 25 Gy with a small nodulus of fibrous tissue not encircling the gut wall (+), Non-protected segment irradiated with 7.5 Gy (*).
After irradiation the segments werc replaced into the abdomen and the abdominal incision was stitched. Following irradiation the animals were placed in separate cages. Microscopic grading of fibrosis. The animals w x e killed after 4 weeks. The gut was removed and the segments identified. The irradiated segments were cut out together with a non-irradiated, contiguous segment. The specimens were cut sagitally into two congruent pieces. If, as s e n macroscopically, only one part of the circumference of the gut was fibrotic, the incision was made through the middle of that part. One of the pieces was fixed in 6 per cent formaldehyde for microscopy. The irradiated part of the other half was taken for hydroxyproline analysis (Fig. 1). The control segments were similarly treated. The sections for microscopy were stained with haematoxylin-eosin and van Gieson stain. Microscopically the specimens were divided into two groups. To the first group were referred specimens with no increase in fibrous tissue and to the second group specimens with a distinct augmentation of the fibrous tissue. Extra-serosal fibrosis, referable to omental adhesions was not considered. The extent of the fibrosis was not subjected to further microscopic gradation. Hydroxyproline assay. The specimens analysed for hydroxyproline were dried in an oven at 100°C until a constant weight was reached (3 days), and the dry weight was then recorded. Hydroxyproline was determined after hydrolysis in 6N HC1 for 3 h at 130°C using the method described by PIKKARAINEN (1968). The hydroxyproline concentration was calculated as mg per g dry tissue.
Results Of the 23 animals, one died after 11 days due to unknown causes and one died after 3 weeks of ileus due to fibrotic constriction of a non-protected segment. Of 21 segments
68
J. 0. FORSBERG, H. JIBORN AND B. JUNG
Fig. 3. Irradiated, protected segment (20 Gy) with 8 small fibrotic nodulus and the continuous, non-irradiated segment (left). ( x 30)
Fig. 4. Non-protected segment 4 weeks after irradiation with 7.5 Gy. The normal appearances of villi and crypts destructed and villi not fully epithelialized. ( x 130)
which should have been protected in the experiments, 3 were excluded immediately due to catheter failure resulting in incomplete blockage. When killed, two of the rats had chronic mechanical ileus due to fibrotic constriction of the non-protected segments and with a thick gut wall in the dilated gut proximal to the stenoses. Macroscopically, fibrosis was easily observed in several of the irradiated segkents, which were most often covered by omental tissue. In the heavily fibrotic non-protzcted segments, the gut had shrunk to about 1/3 of. its original length and the gut wall was fibrotized around the gut circumference. In protected segments such abnormalities were found in only two segments, and in a few segments there were one or several
RADIATION INDUCED FIBROSIS
69
Fig. 5. Heavily destructed mucosa in a non-protected segment 4 weeks after irradiation with 20 Gy. Cystlike crypts, marked inflammatory reaction and an abnormal abundance of goblet cells. ( x 160)
noduli of fibrotic tissue with a radius of 2 mm or less, but not circumscribing the gut wall (Fig. 2). The lengths of these segments were not reduced. Micrcscopic evaluation. As the sections included irradiated as well as non-irradiated segments, a direct comparison with unaffected mucosa was possible in each section (Fig. 3). In the non-protected segments irradiated with the lowest doses, epithelial recovery was usually almost complete, but sometimes recovery was incomplete even after these doses (Fig. 4). In the segments irradiated with higher doses the mucosa was either abnormal (Fig. 5 ) or absent (Fig. 6). Compared to the non-protected segments, the mucosal recovery in the protected segments was obvious (Fig. 7). The findings concerning mucosal recovery are summarized in Fig. 8. Fibrosis increased in frequency at the higher doses, less for the protected than for the non-protected segments (Fig. 9). The fibrous tissue almost invariably included all the different layers of the gut wall (Fig. 10) and in most fibrotic segments the mucosal lining was more or less absent. In the non-protected segments, the fibrous tissue always included the entire extent of the segment; whereas in the protected segments (but for 2) it was restricted to one or several small noduli-the remaining part of the segment being unaffected (Fig. 10).
70
J. 0. FORSBERG, H. JIBORN AND B. JUNG
Fig. 6. Heavily fibrotized non-psotected segment 4 weeks after irradiation with 17.5 Gy. No mucosal regeneration. ( x 45)
Fig. 7. Protected segment irradiated with 20 Gy. No fibrosis. Appearances of villi and crypts almost entirely normal. Few goblet cells compared to Fig. 5, indicating a higher survival proportion of the regenerating, columnar epithelium. ( x 50)
Hydroxyproline evaluation. The hydroxyproline content in mg per g of dry tissue in the segments is given in Table 2.zThe hydroxyproline content in per cent relative to the control segments is given in Fig. 11. The values show a constant elevation of hydroxyproline in the non-protected segment from 12.5 Gy and a significant difference to the control value at 15.0 Gy. The protected segments show no such increase relative to the control values. Discussion
The aim was to determine whether deep hypoxia during irradiation would protect against development of fibrosis.
71
RADIATION INDUCED FIBROSIS
Fig. 8. Appearance of the mucosa 4 weeks after irradiation of non-protected (NP) and rotected (P) ileal segments. -non-recovery of epithelial lining over a fibrotic ulcer. epithelial lining unbroken, but normal structure of the mucosa considerably destructed. Abundance of goblet cells. = slight destruction of the mucosal structure. Less abundant goblet cells. Incidence in per cent of total number of segments in each group.
e
5
50
n Z5
0.0
125
M
175
150
xx) #
I
NP n = 0
I
I
MD
250 Gy
P
7
7
6
6
6
6
YO
'""1 Fig. 9. Incidence of fibrosis in per cent of total number of segments irradiated with different =fibrosis including the whole irradiated segment. = fibrosis of a small nodulus, a few mm in diimeter, but with no fibrosis in t'ie rest of the segment. NP = non-protected, P = protected. L
1
L
NP n.8
7
7
P
I
7
6
6
6
6
The fibrotic process is an inevitable mechanism of tissue repair. It is well known that the extent of trauma is a modifying factor in the fibrotic development. Thus, in surgery, a gentle handling of the tissues and an active regimen against infection prevent excessive scar formation. In radiation therapy, a low dose-rate (multiple fractions) will give a similar result. Experiments aiming at reducing already established fibrosis following trauma, has been performed with Ca EDTA. This increased the urinary excretion of hydroxyproline, but did not decrease the observable fibrosis (TOBINet coll. 1974). The exact mechanism of fibrosis development after irradiation is still obscure, but generally the degree of trauma (dose), the trauma mechanism and the specific sen-
72
J. 0. FORSBERG, H. JIBORN AND B. JUNG
Fig. 10. Fibrotic tissue in all layers of the gut wall. The muscle layer is completely broken up. ( x 70)
Fig. 11. Hydroxyproline content of each irradiated segment relative to nonirradiated segments of the same animal. Filled circles = non-protected(NP) segments. Open circles = protected (P) segments. Dose (Gy). Mean and
SD. 7.5
10.0 7
125 7
15.0 125 20.0 7 7 6 6 6
250 Gy 6
73
RADIATION INDUCED FIBROSIS
Table 2 Hydroxyproline content (mglg dry tissue) of non-protected and protected ileal segments 4 weeks after irradiation. Mean and SD, n=number of segments, * or indicate significant difference ( p 0.05) between values with the same sign. The high value indicated by *, is attributable to one segment with an extremely high control value. Exclusion of this segment will give figures in brackets Dose (Gy)
Mean
SD
n
Non-protected 0 (control) 7.50 10.0 12.5 15.0 17.5 20.0
7.56' 8.18 8.07 9.18 13.28' 21.47O 25.89
2.46 2.31 1.91 2.60 6.70 8.76 8.99
20 8 7 7 7 7 6
Protected 15.0 20.0 25.0
13.40O (9.10)' 9.09 9.02
4.40 (2.35)* 3.33 2.05
6 6 6
sitivity of the tissue concerned determine when and to what extent fibrosis will occur (RUBIN& CASARETT). In differentiated tissue, fibrosis may start as a primary stimulation of the interstitial fibrocytes or the fibrocytes in the vicinity of a vessel, or it may be secondary to the inflammatory reaction induced by tissue necrosis (RUBIN& CASARETT). In the gut, another mechanism must also be considered, namely inflammation induced by bacteria from the gut lumen invading the injured mucosal wall. In experimental work on irradiated granuloma tissue, RANTANEN (1973) found that the mature fibrocytes reacted with a fibrotic repair in spite of decreased number of cells. He suggested, that radiation induced fibrosis was an effect of collagenolytic impairment which resulted in excess accumulation of collagen into the irradiated tissues. In older granuloma tissue irradiated with 10 Gy he found already after a few days an increase in the hydroxyproline. Hydroxyproline is a suitable substance for detecting fibrosis since essentially all of the hydroxyproline in animal tissue is found in the collagen (ADAMS1970). The present histologic evaluation indicates and the hydroxyproline assay shows significantly a development of fibrosis in the non-protected segments following a dose of 15 Gy. At higher dose levels the hydroxyproline increased further. However, a considerable variation in the dose response occurred between specimens within a certain group, as indicated by the variance. The protected segments did not show a tendency toward a hydroxyproline increase at the chosen dose levels.
74
J. 0. FORSBERG, H. JIBORN AND B. JUNG
The fibrosis at the higher dose levels in non-protected segments is in good agreement with the experiencesfrom radiation therapy, where it is well known that fibrosis following irradiation is dose dependent. Thus, a high dose will give more and earlier fibrosis than will a low dose. Also a dose deliveredin a few fractions will cause the fibrosis to come more rapidly than if the same dose is given in several fractions (RUBIN & CASAREXT). After a single dose of 30 Gy to exteriorized rat ileum OSBORNE et coll. (1970) found a massive fibrosis already on the tenth day. They found a higher survival rate of animals in which the superior mesenteric artery was clamped during irradiation, indicating protection against the acute injury, but they 'did not mention that this protection resulted in less fibrosis. In clinical conditions irradiation fibrosis usually occurs late. The interval between irradiation and manifest fibrosis may often be several years, but the initiation of the fibrosis is immediate (RANTANEN). The modifying effect of hypoxia on fibrotic development is obvious. As most fibrotic segments showed a non-repaired mucosal lining, irradiation injury to the mucosa might be one primary mechanism for the achieved fibrosis. This would explain the absence of fibrosis in the protected segments, in 50 per cent having preserved et coll.). A commucosal lining on the fourth day after a dose of 26 Gy (cf. FORSBERG plementary mechanism could be hypoxic protection of the fibrocytes and the collagenolytic processes, as suggested by RANTANEN. It cannot be excluded, however, that the protected segments, with no fibrosis after 4 weeks, would not have developed fibrosis later, due to a delayed repair mechanism. Acknowledgements This investigation was supported by the Swedish Cancer Society. The irradiations were performed at the Department of Oncology, University Hospital, Uppsala.
SUMMARY The protective effect of hypoxia, induced by degradable microspheres, against the development of fibrosis was investigatedafter single-dose irradiation of exteriorized rat ileum. Evaluation, based upon microscopy and analysis of hydroxyproline in protected and nonprotected gut segments, demonstrated an evident protective effect at doses of 15 to 25 Gy.
ZUSAMMENFASSUNG Der Schutzeffekt von Hypoxie, der durch abbaubare Mikrosphiirenhervorgerufen worden war, gegen die Entwicklungeiner Fibrose nach einer Einzel-Strahlendosisdes herausgelegten Ratten-Ileums wurde untersucht. Die Untersuchung, die sich auf Mikroskopie und Analyse von Hydroxyprolin in geschiitzten und nicht geschiitzten Darm-Segmenten basiert, zeigte einen klaren Schutzeffekt bei Dosen von 15 bis 25 Gy.
RADIATION INDUCED FIBROSIS
75
RESUME L‘effet radioprotecteur de I’hypoxie produite par des microsphkres dkgradables contre le dkveloppement d’une fibrose a CtB etudiC aprbs ]’irradiation par une dose uniqued‘ileon de rat exttriorisk. L’Ctude des segments intestinaux protkgts et non proteges, basee sur la microscopie et I’analyse de I’hydroxyproline, a mis en evidence un effet protecteur net a des doses de 15 A 25 Gy.
REFERENCES ADAMS E. : Metabolism of proline and hydroxyproline. Zn: International review of connective tissue research, Vol. 5, p. 1. Edited by D. A. Hall and D. S. Jackson. Academic Press Inc., London 1970. FORSBERG J. O., JUNGB. and LARSSON B.: Mucosal protection during irradiation of exteriorized rat ileum. Effect of hypoxia induced by starch microspheres. Acta radio]. Oncology 17 (1978), 485. NYGREN A.: Personal communication. Pharmacia Laboratories. 1976. OSBORNE J. W., PRASAD K. M. and ZIMMERMAN G. R.: Changes in the rat intestine after Xirradiation of exteriorized short segments of ileum. Radiat. Res. 43 (1970), 131. PIKKARAINEN J. : The molecular structures of vertebrate skin collagens. Acta physiol. scand. (1968) Suppl. No. 309. RANTANEN J.: Radiation injury of connective tissue. A biochemical study with experimental granuloma. Acta radio]. (1973) Suppl. No. 330. RUBINP. and CASARETT G. W.: Clinical radiation pathology. W. B. Saunders Company, Philadelphia 1968. TOBING., ARONSON A. and CHVAPILM.: Effect of CaEDTA administration on urinary hydroxyproline excretion and skin wound healing in the rat. J. surg. Res. 17 (1974), 346.
