Late behavioural and neuropathological effects of local brain irradiation in the rat.

The delayed consequences of radiation damage on learning and memory in rats were assessed over a period of 44 weeks, commencing 26 weeks after local irradiation of the brain with single doses of X-rays. Doses were set at levels known to produce vascular changes alone (20 Gy) or vascular changes followed by necrosis (25 Gy). Following T-maze training, 29 weeks after irradiation, irradiated and sham control groups performed equally well on the forced choice alternation task. When tested 35 weeks after irradiation, treated rats achieved a much lower percentage of correct choices than controls in T-maze alternation, with no difference between the two irradiated groups. At 38-40 weeks after irradiation, rats receiving both doses showed marked deficits in water maze place learning compared with age-matched controls; performance was more adversely affected by the higher dose. The extent of impairment was equivalent in the two groups of rats irradiated with 25 Gy, those trained or not previously trained in the T-maze, suggesting that water maze acquisition deficits were not influenced by prior experience in a different spatial task. In contrast to water maze acquisition, rats irradiated with 20 Gy showed no deficits in working memory assessed in the water maze 44 weeks after irradiation, whereas rats receiving 25 Gy showed substantial impairment. Rats receiving 25 Gy irradiation showed marked necrosis of the fimbria and degeneration of the corpus callosum, damage to the callosum occurring in animals examined histologically 46 weeks after irradiation, but in only a third of the animals examined at 41 weeks. However, there was no evidence of white matter necrosis in rats irradiated with 20 Gy, examined 46 weeks after irradiation. These findings demonstrated that local cranial irradiation with single doses of 20 and 25 Gy of X-rays produced delayed impairment of spatial learning and working memory in the rat. The extent of these deficits appears to be task- and dose-related, since rats treated with 25 Gy showed marked impairments in all measures, whereas rats treated with the lower dose showed less impairment in water maze learning and no deficits water maze working memory, despite significant disruption of working memory in the T-maze. The findings further suggest that although high dose irradiation-induced white matter necrosis is associated with substantial impairment, cognitive deficits may also be detected after a lower dose, not associated with the development of necrosis.

Radiation-induced changes in the kinetics of glomerular and tubular cells in the pig kidney.

Both kidneys of 13 mature female Large White pigs were irradiated with a single dose of 9.8 Gy 60Co gamma rays. The pigs were killed serially between 2 to 24 weeks after irradiation. One hour prior to sacrifice bromodeoxyuridine (BrdU) (500 mg/pig) was injected intravenously. At postmortem the kidneys were removed and tissue was taken to prepare cell suspensions. The labeling index (LI) of these suspensions was determined using flow cytometry. In vivo BrdU incorporation in tubular and glomerular cells was determined immunohistochemically. The kinetics of glomerular and tubular cells was evaluated by counting the number of labeled cells/glomerulus and the number of labeled tubular cells/field of view. An average of 1200 glomeruli and 1500 fields of view/time were counted. Similar analyses were performed on renal tissue from unirradiated control animals. Flow cytometry revealed rapid and significant increases in the LI of kidney cells; 2 weeks after irradiation the LI increased from a control value of 0.18 +/- 0.01 to 1.23 +/- 0.22% (P < 0.001). By 4 weeks the maximal value of 2.45 +/- 0.36% was seen; the LI then declined progressively but at 24 weeks after irradiation still remained significantly above control values (P < 0.001). A similar pattern of response was determined by counting the labeled glomerular and tubular cells identified immunohistochemically. However, the increase in labeled glomerular cells occurred 2 weeks after irradiation, whereas that for the tubules occurred 4 weeks after irradiation. These findings indicate that irradiation of the kidney, classically regarded as a "late-responding" organ, is associated with rapid and significant changes in the kinetics of both tubular and glomerular cells.

Influence of a low protein diet on radiation nephropathy in the pig.

Nine mature, approximately 45-week-old pigs, were fed a standard diet (SD) containing 16% protein; 10 pigs were fed an isocaloric low protein (LP) pig feed containing 4% protein 2 weeks prior to and 16 weeks after renal or sham-irradiation. The pigs then received the SD for a further 4 weeks. Both kidneys of seven pigs fed the LP diet, and six pigs fed the SD, were irradiated with a single dose of 9.8 Gy of 60Co gamma-rays. The remaining pigs received sham-irradiation. The individual kidney glomerular filtration rate (GFR), effective renal plasma flow (ERPF), and the haematocrit were serially measured before and up to 20 weeks after irradiation. The radiation-induced reduction in mean individual kidney GFR, expressed as either absolute values or as a percentage of the respective sham-irradiated controls, was significantly greater in pigs fed the SD compared with pigs fed the LP diet (p < 0.05). In contrast, the radiation-induced reduction in mean individual kidney ERPF, expressed as absolute values, observed in pigs fed the SD was not significantly different from that seen in pigs fed the LP diet. However, if these data were expressed as a percentage of the respective sham-irradiated controls, then a significantly greater reduction in ERPF was seen in pigs fed the SD compared with pigs fed the LP diet (p < 0.001). Morphological analysis of renal tissue removed 20 weeks after irradiation showed that the extent and severity of the glomerular lesions seen in pigs fed the SD were significantly reduced in pigs fed the LP diet. Thus dietary protein restriction appears to reduce some of the signs of radiation nephropathy in the pig.

Sequential evaluation of radiation-induced glomerular ultrastructural changes in the pig kidney.

Both kidneys of 12 mature female pigs received either a single dose of 9.8 Gy 60Co gamma rays or sham irradiation. At intervals of 1-4 weeks serial renal biopsies were obtained, followed by sacrifice at 24 weeks after irradiation. Individual kidney glomerular filtration rate (GFR), effective renal plasma flow (ERPF), and the hematocrit (Hct) were measured routinely. Renal irradiation resulted in a progressive decline in GFR, ERPF, and Hct, with minimal values being observed within 12 weeks of irradiation. No change in any of these parameters was noted in the sham-irradiated pigs. The initial morphological change in irradiated glomeruli was leukocyte attachment to capillary endothelial cells 3-6 weeks after irradiation followed by activation and swelling of the endothelial cells. This was followed by pronounced increases in capillary permeability with fluid and erythrocyte, leukocyte, and platelet exudation into the subendothelial/mesangial space. This resulted in compression of glomerular capillary lumina, which occurred concomitantly with the reduction in GFR. By 12 to 15 weeks after irradiation the changes in endothelial cells were less evident. However, mesangial cells exhibited evidence of activation and proliferation accompanied by progressive mesangial expansion and sclerosis. Thus the glomerular capillary endothelial and mesangial cells appear particularly important in the pathogenesis of radiation nephropathy.

Resistance and pulsatility Doppler indices: how accurately do they reflect changes in renal vascular resistance.

Doppler indices such as the resistance (RI) and pulsatility indices (PI) are generally thought to indicate the resistance of the vascular bed supplied by the artery under investigation. However, this association remains uncertain; some studies have demonstrated an excellent correlation between alterations in vascular resistance and changes in Doppler indices, while others have shown no consistent change. In order to assess the validity of these two indices in estimating alterations in renal vascular resistance, 12 female Large White pigs, 45 weeks old, were entered into a double blind study. In nine of the pigs both kidneys received a single dose of 9.8 Gy 60Co gamma-rays; the remaining three served as sham-irradiated controls. Renal haemodynamics were measured using 131I-hippuran renography and Duplex Doppler prior to and at 8, and 12 weeks after kidney (sham-)irradiation. The sham-irradiated animals exhibited no significant changes in any of the parameters. In contrast, the animals in which both kidneys were irradiated exhibited a significant decrease (p < 0.001) in renal blood flow (RBF) 8 and 12 weeks after irradiation. In addition, both PI and RI were increased following kidney irradiation; however, this increase was not significant until 12 weeks after irradiation (p < 0.01). This suggests that the reduction in RBF was not simply a reflection of an increased vascular resistance and/or that PI and RI do not truly represent alterations in vascular resistance. Further studies are required to confirm the applicability of PI and RI in the assessment of renal haemodynamics.

Capillary endothelium. Target site of renal radiation injury.

BACKGROUND: Therapeutic abdominal irradiation may be accompanied by late occurring progressive renal disease associated with glomerular mesangial sclerosis and tubular degeneration. Pathogenesis of this lesion is disputed. EXPERIMENTAL DESIGN: Kidneys of mature pigs were irradiated with a single dose of 9.8 Gy 60Co gamma-rays; serial individual kidney glomerular filtration rate and renal biopsies were obtained at 3-4-week intervals with death 24 weeks after irradiation. RESULTS: Irradiated pigs displayed a progressive reduction in glomerular filtration rate with minimal values observed 12 weeks postirradiation. Morphologic changes in irradiated glomeruli were characterized by leukocytic attachment to capillary endothelial cells 3-6 weeks after irradiation followed by activation and swelling of endothelial cells and occasional microthrombi formation. Similar changes were noted focally in peritubular capillaries with accompanying tubular degeneration and atrophy. In glomeruli these endothelial cell changes were followed by increased capillary permeability and fluid, erythrocyte, platelet, and leukocytic exudation into the subendothelial/mesangial space resulting in compression of glomerular capillary lumina. By 12 weeks postirradiation mesangial cells showed evidence of activation and proliferation accompanied by progressive mesangial expansion and sclerosis with continued reduction of glomerular filtration rate. CONCLUSIONS: It is concluded that endothelial cell injury represents the primary site of radiation damage in the kidney and that the progressive glomerular mesangial sclerosis and reduced renal function of radiation nephropathy may be associated with exposure of capillary elements to various vasoactive and growth promoting factors that stimulate mesangial contraction and proliferation resulting in reduced glomerular filtration area and glomerular filtration rate.

Quantification of radiation-induced epilation in the pig: a biological indicator of radiation dose to the skin.

Epilation in the pig has been quantified following single exposures of X-rays in the range 1.0-25.0 Gy. The number of hairs in each field was determined by counting hairs from photographic negatives weekly for 10 weeks following irradiation, and the percentage hair loss was calculated for each individual field from an initial unirradiated control value. Hair loss was dose-dependent for exposures between 1.0 and 15.0 Gy and this response was linearly related to dose. No further increase in hair loss was observed for doses > or = 15.0 Gy, as 20-30% of the hairs remained. It was assumed that these hairs were not actively growing at the time of irradiation and did, therefore, not express damage. The ED50 for the loss of > or = 30% of hairs was 3.8 Gy whilst that for the loss of > or = 50% of hairs was 6.8 Gy and for the loss of > or = 67% of hairs was 12.5 Gy. There was +/- 7% hair loss per Gy exposure for doses between 0.0 and 15.0 Gy. Quantification of hair loss provided a more sensitive assay than the use of visual scoring systems. Hair loss was detectable within 4 weeks of irradiation. The system is simple, non-invasive and appears to have considerable potential for use as a biological dosimeter.

Long-term studies of cisplatin-induced reductions in porcine renal functional reserve.

Mature Large White female pigs aged approx. 10 months received single intravenous doses of 1.5, 2, or 2.5 mg/kg cisplatin. The glomerular filtration rate (GFR) and the effective renal plasma flow (ERPF) in individual kidneys were measured prior to and at 4-week intervals for up to 24 weeks after cisplatin administration by renography using [99mTc]-diethylenetriamminepentaacetic acid (DTPA) and iodohippurate sodium I 131, respectively. The left kidney of each cisplatin-treated pig and that of three age-matched control pigs was then removed, and GFR and ERPF values were measured in the remaining kidney at 4-week intervals for a further 24 weeks after unilateral nephrectomy (UN). Pigs treated with cisplatin showed no significant reduction in GFR or ERPF for up to 24 weeks after drug infusion. As measured using inductively coupled plasma mass spectrometry, the mean renal platinum concentration in the left kidney removed at UN was 77.5 +/- 9.1 ng/g kidney per mg/kg cisplatin. Histological evaluation of these kidneys revealed narrow interconnecting rays of interstitial fibrosis in the deep cortex and medulla; in these areas, glomeruli exhibited thickened Bowman's capsules and occasionally shrunken sclerotic capillaries. In cisplatin-treated pigs, UN was associated with a marked reduction in the ability of the remaining kidney to increase its function in terms of GFR and, to a lesser extent, of ERPF. The increase seen in GFR following UN in the cisplatin-treated pigs was only ca. 50%-70% of that seen in age-matched UN controls. Histologically, these kidneys revealed resolution of the peritubular fibrosis observed at UN; occasional sclerotic glomeruli were also evident. Platinum remained detectable in these kidneys, the mean levels being 18.8 +/- 4.9 ng/g kidney per mg/kg cisplatin. These findings confirm previous observations and illustrate the need for caution in considering further treatment of patients who have previously received cisplatin along with a second potentially nephrotoxic agent.

Residual radiation-induced damage to the kidney of the pig as assayed by retreatment.

The effect of re-irradiation on the previously irradiated kidney was studied in the Large White female pig. Both kidneys of 14-week-old pigs were initially irradiated with a single dose of 3-7 Gy of 250 kV X-rays. The individual kidney glomerular filtration rate (GFR), effective renal plasma flow (ERPF), and the haematocrit (Hct) were serially measured up to 24 weeks after X-irradiation. Doses of 3 and 5 Gy of X-rays had little effect on the GFR. However, 7 Gy of X-rays caused a marked decline in the GFR up to 12 weeks after irradiation; the GFR then began to recover. A similar pattern of response was seen in the ERPF. In contrast, the Hct was reduced in all pigs 4 weeks after X-irradiation. The extent of the decline and subsequent recovery 24 weeks after irradiation appeared to be related to dose. Twenty-four weeks after X-irradiation both kidneys were re-irradiated with a single dose of 7.9 Gy of 60Co gamma-rays; both kidneys of four age-matched control pigs which had not previously received X-irradiation were similarly treated. Individual kidney GFR, ERPF and Hct were again serially measured up to 24 weeks after gamma-irradiation. Re-irradiation resulted in a significantly greater reduction in the GFR, ERPF and Hct compared with that seen after gamma-irradiation alone. Moreover, the severity of the reduction in the GFR and Hct observed after gamma-irradiation was related, in a dose-dependent manner, to the initial X-ray doses employed. These results indicate that the kidney fails to exhibit complete long-term recovery in function following irradiation. Re-irradiation of the kidney in patients should thus be viewed with extreme caution.

The effect of unilateral nephrectomy on the subsequent radiation response of the pig kidney.

The left kidney of 14 Large White female pigs, approximately 14 weeks of age, was surgically removed. Thirty weeks after unilateral nephrectomy (UN) the remaining kidney was irradiated with a single dose of between 11.9 and 15.6 Gy of 60Co gamma-rays; three pigs received sham irradiation. Following irradiation glomerular filtration rate (GFR), effective renal plasma flow (ERPF) and haematocrit (Hct) were determined for up to 48 weeks after irradiation. Irradiation resulted in a dose-dependent decline in GFR, evident 8 weeks after irradiation. This was followed by a gradual improvement in GFR, although after doses of 14.0 Gy GFR remained below control values throughout the study. A similar pattern of response was seen in terms of ERPF, but this was not dose-related. Doses of greater than or equal to 14.0 Gy also caused a significant reduction (p less than 0.001) in Hct within 4-8 weeks. Minimal levels were evident 16 weeks after irradiation; Hct then increased, but remained below preirradiation values. Dose-effect curves were obtained by determining the percentage of irradiated kidneys which showed a greater than or equal to 50% reduction in GFR and ERPF, fitted by probit analysis, and ED50 values (+/- SE) were calculated for each parameter. The ED50 values for GFR and ERPF were 14.49 +/- 0.27 Gy and 12.56 +/- 0.98 Gy, respectively. These values were not significantly different from those obtained from intact age-matched pigs in which the right kidney alone was irradiated; UN did not compromise or alter the radiation response of the kidney to irradiation. However, the ED50 values obtained for the UN pigs were significantly greater (p less than 0.001) than the values of 9.76 +/- 0.17 Gy and 6.19 +/- 0.93 Gy, seen for GFR and ERPF in intact age-matched pigs in which both kidneys were irradiated. Thus although both experimental situations involved irradiating the entire renal tissue, the relative radiosensitivity of the kidneys varied considerably. These findings show that the radiosensitivity of the pig kidney can vary markedly, depending on the physiological status of the kidney at the time of irradiation.