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Permeability of blood vessels in mice affected with scrapie or fed with cuprizone
J. Cow.
PATH.1973.
VOL.
PERMEABILITY AFFECTED
B. F.
461
83.
OF BLOOD WITH SCRAPIE CUPRIZONE I. H.
SANSOM,
VESSELS IN MICE OR FED WITH
and
PATTISON
JEAN
N.
JEBBETT
Agricultural Research Council, Institute for Research on Animal Diseases, Compton, Newbury, Berkshire
INTRODUCTION
The histopathology of naturally occurring scrapie in sheep and of the experimentally produced disease in sheep, goats, mice, rats and hamsters has been described in detail, but the fundamental nature of the characteristic bilaterally symmetrical spongiform encephalopathy is still obscure. It was suggested by Pattison (1965) that the bilateral symmetry of the lesions might indicate a relationship with the blood supply. A possible basis for involvement of blood vessels in the pathological process might be an increase in their permeability. Moreover, large increases in the permeability of blood vessels supplying nervous tissue have been reported in response to both physical trauma to individual nerves (Mellick and Cavanagh, 1967, 1968) and to the action of a neurotropic virus (Boyse, Morgan, Pearson and Wright, 1956). It was decided, therefore, to measure blood vessel permeability by a double radioactive tracer technique (Mellick and Cavanagh, 1967) in the brains and spleens of mice inoculated with scrapie. Apparent replication of the scrapie agent occurs in both tissues, although its pathological effects are restricted to the brain. In view of histological similarities between scrapie and cuprizone toxicity (Pattison and Jebbett, 1971), the blood vessel permeability of mice showing clinical evidence of cuprizone toxicity was compared with that of mice affected with scrapie. MATERIALS
AND
METHODS
These were females of the BSVS strain bred at Compton. There were 5 to 8 mice in each group in all experiments. Scrapie. Scrapie was produced by the intracerebral (i.c.) inoculation of’ the agent adapted to mice by Chandler (1961). In E x p eriments A, B, C, D and F the animals were showing advanced clinical evidence of the disease and were somewhat smallet than normal mice used as controls. In Exp. E the 2 groups of scrapie mice were in the pre-clinical stage of the disease, either 27 or 34 days after inoculation, and were the same size as the non-inoculated control mice. G&zone. Cuprizone (biscyclohexanone oxaldihydrazone) * was fed at the rate of 0.5 per cent. of a whole-diet meal (an intake of up to 15 mg. of cuprizone per mouse per d.), as described by Pattison and Jebbett (1971). The mice had been on this diet for at least 30 d. and showed clear evidence of cuprizone toxicity. By virtue of the debilitating nature of the disease, cuprizone-affected mice were smaller than either the normal or scrapie-affected mice. Mice.
* Koch-Light
Laboratories
Ltd.,
Colnbrook,
Bucks.
462
B. F. SANSOM
et al.
Double radioactive tracer technique. 1311 and lz51-labelled human serum albumin (HSA) (Codes IBI7P and IMI7P respectively, Radiochemical Centre, Amersham) polyvinylpyrrolidone (PVP) were used for Exps. A and B. 1311 and lz51-labelled (Codes IB33P and IM33P respectively) were used for Exps. C, D and E. [1311]HSA or PVP in O-1 ml. of physiological saline was injected into a tail vein, and between saline was 24 and 25 h. later [lz51]HSA (or PVP) in the same volume of physiological also injected i.v.; between 5 and 10 min. later the mice were killed with ether. Blood was taken immediately by Pasteur pipette from a pool formed by severing the vessels in an axilla, and the whole brain and spleen were removed and chopped finely with scissors. The blood, brain and spleen samples were placed in separate counting tubes, weighed and counted in two channels of a gamma scintillation counter, one channel counting only 13rI and the other mainly 1251. The accuracy of the estimation of extravascular 1311 in individual brains and spleens was such that the coefficient of variation did not exceed 20 per cent. and was usually about 10 per cent. Whole body counting. In Exp. F in which whole body radioactivity was measured over a period of 6 days, mice were inoculated iv. with 1311 PVP in 0.1 ml. physiological saline. For each loo-second count they were held in small plastic tubes attached by rubber bands to one scintillation detector of a large animal whole body counter (Sansom, Taylor, Wheelock and Vagg, 197 1). Counts were made at approximately 4, 24, 48, 120, and 144 h. after injection. Calculations and presentation of data. The i.v. injection of [1311]HSA (or PVP) and [1251]HSA (or PVP) 24 to 25 h. and 5 to 10 min. respectively before samples of blood and tissue were removed made it possible to calculate the amount of extravascular ls11 present in the tissues by means of the formula:is11 in blood Extravascular 1311 c in tissue N 1251 in blood in tissue This is based on the assumption that 5 to COmin. after i.v. injection the 1251 HSA (or PVP) is present only in the blood in the tissue and it makes a simple correction for the 1311 present in the blood. The values for extravascular 1311 in the brain and spleen are presented in the Results in terms of the equivalent volume of blood of concentration equal to the mean [ralI]HSA (or PVP) concentration during the 24-h. period of the experiments. This as follows: mean concentration of [ 1311]HSA (or PVP) in the blood was determined the initial concentration of the [ 1311]HSA (or PVP) was assumed to be the same as the concentration of [issI]HSA (or PVP) and the mean concentration was then calculated by assuming that the blood concentration fell in a simple exponential manner. Summary of experiments. A and B were replicate experiments in which [1311] and [1251]HSA were used to measurevascularpermeability in the brains and spleens of mice showing advanced clinical signs of scrapie or of mice fed cuprizone. C and D were replicate experiments in which [lslI] and [1251]PVP were used to measure vascular permeability in the brains and spleens of mice showing advanced clinical signs of scrapie or of mice fed cuprizone. in In Exp. E, [1311] and [ 1251]PVP were used to measure vascular permeability the brains and spleens of mice in the pre-clinical stage of scrapie. In Exp. F, the retention of radioactivity in the whole body of mice showing advanced clinical signs of scrapie, or of mice fed cuprizone, was measured at intervals after the i.v. inoculation of [lslI]PVP.
1
RESULTS
Advanced Clinical Scrapie Exps. A and B : [I3111 and [125I]HSA. The results of these experiments, in which [1311]HSA and [ 1251]HSA were used, are given in Tables 1 and 3. There was good agreement between the results in the 2 experiments. In all groups of mice from the blood particularly in the normal there was a rapid loss of [ l3lI]HSA
VASCULAR
PERMEABILITY
IN SCRAPIE
mice. The only indication of a difference in leakage from groups of mice was a slightly higher average figure (P < scrapie mice than for either cuprizone or normal animals. were more variable and no significant difference between TABLE MEAN BLOOD Ia11 AND RESPEcT1"EI.Y AFTER I.".
Normal Scrapie Cuprizone
* 5 to 8 mice
7.8kO.3 13.7+ 1.3 21.7k3.2
per
lzsI CONCENTRATION
blood vessels between 0.02) for the brains of The results for spleen groups was detected.
1
dose/g.If:s.~.) AND ADMINISTRATION OF [ ‘S’I]HSA AND CUPRIZONE-TREATEDMICE
47.8k2.0 52.7k3.4 79.Ok5.7
463
MICE
(%
0.163+0.011 0.260 k 0.02 1 0.274 + 0.020
AT 24 h. AND 5 TO 10 min. [‘251]HSA TO NORMAL, SCRAPIE
9.0*0.5 13.4kO.9 17.4+ 1.1
52.5k3.2 54.4 & 4.0 71.6k5.1
0~171+0~015 0.246kO.010 0.243+0.018
group.
Exf,u. C: and D: [1311] and [1251]PVP. B ecause only minor differences had been detected between groups of mice in A and B, the observations were repeated with [1311]PVP and [1251]PVP. PVP was used because of its lowel molecular weight and because there is less free iodide 1311or la51 in the preparation. The lower molecular weight would be expected to increase the extent of leakage and thus possibly to increase the likelihood of detecting differences between groups of mice, and the smaller amount of free 1311would be expec;cd to improve the specificity of the method. The results, given in Tables 2 and 4, were essentially the same as in A and B. An obvious quantitative difference between the 2 pairs of experiments was the greater amount of extravascular [1311]PVP than of [1311]HSA in the spleens, but not the brains, of mice of all groups, presumably due to the smaller molecular size of PVP. Qualitatively the small differences between groups of mice were similar to those in the experiments with HSA, although scrapie and cuprizone mice were not significantly different from normal mice. Preclinical Scrapie Exp. E: [i311] and [1251]PVP. This experiment was carried out to examine the possibility that although no difference in capillary permeability had been detected between normal mice and mice in an advanced clinical stage of scrapie, a difference might be detectable in an early pre-clinical stage of the disease. Groups of 6 normal mice and of mice either 27 or 34 days after i.c. inoculation with scrapie, were injected with [ 1311]PVP and after 24 hours with [lz51]PVP. Five minutes later the animals were killed and 1311 and 1251 were measured in blood, brain and spleen, as in A, B, C and D. No differences between the groups of mice were detected, either in the rate at which [1311]PVP was lost from the blood, or in the amounts of extravascular [ialI]PVP in the brain or spleen. The rate of loss of 1311 from the blood was similar to that in the normal mice in A, B, C and D. B
464
B. F. SANSOM la11
AND AFTER
ias1
CONCENTRATION (% dose/g.+ I.V. ADMINISTRATION OF [lslI]PvP SCRAPIE AND CUPRIZONE-TREATED
Exp.c Group * Normal Scrapie Cuprizone
al.
2
TABLE MEAN BLOOD RESPECTIVELY
et
la11
1251
6.1 kO.2 9.7kO.5 11.8kO.8
35.3+ 1.8 41.1k2.1 45.3k2.4
s.E.) AT 24 h. AND 5 TO 10 min. AND ['*51]PvP TO NORMAL, MICE
Eq. D ------___
-____ 1a11/1B51
0.172+0.010 0.236+0.013 0~260~0.011
1311
1251
7.1 kO.6 10.8 f 0.4 14.1 f 0.9
29.1* 1.1 34.8 f 2.3 466k2.8
131I,l25I
0.244+0.018 0.320 + 0.024 0.308 f 0.022
* 5 to 8 mice per group. 3
TABLE MEANEXTRAVASCULAR la11 (~1. SCRAPIE AND CUPRIZONE-TREATED
blood/g.
tissue+s.~.)
MICE
24b.
AFTER
IN THE BRAINS AND I.V. ADMINISTRATION
SPLEENS OF NORMAL, OF [ialI]HSA
Exfi. A Group* Normal Scrapie Cuprizone
Brain 1.13*0*09 t1.82+0*19 1.26kO.15
* 5 to 8 mice per group.
Exj. B
-
Spleen
Brain
Spleen
11*37+ 1.19 15.09+2.61 9.74* 1.75
1.05 + 0.06 t1.54*0.15 0.75 f 0.09
13.535 1.89 10.94+ 1.62 11.06k1.11
t P < 0.02.
MEAN EXTRAVASCULAR la11 (~1. SCRAPIE AND CUPRIZONE-TREATED
TABLE 4 blood/g. tissueks.~.) IN THE BRAINS AND MICE 24h. AFTER I.V. ADMINISTRATION
SPLEENS OF NORMAL, OF [IalI]PvP
Eq. C
Eq. D
-
Group*
Brain
Spleen
Brain
Spleen
Normal Scrapie Cuprizone
0.86 + 0.16 0.87+0.21 0.76 + 0.07
139.7 * 7.5 115.Ok5.1 129.7 + 5.3
0.86kO.14 1.oo + 0.07 0.62 f 0.06
127.5k2.4 103.Ok5.6 88.6 + 5.8
* 5
to
8 mice per group.
Retention of Radioactivity Exp. F. The results in A to E had shown that in all groups of mice there was more marked in normal animals than a rapid loss of is11 from the circulation, in those showing clinical signs of scrapie or the effects of cuprizone. It was not clear, however, whether this loss was from the blood into extra-vascular tissues, or from the body itself in the urine and faeces. Whole body radioactivity determinations of all groups of mice were therefore made at intervals up to 144 h. after i.v. injection of [ 1311]PVP. The results (Table 5) show that during the first 24 h. normal mice excreted 52 per cent. of the injected 1311,whereas the scrapie and cuprizone treated mice excreted only 38 per cent. Subsequently,
VASCULAR
PERMEABILITY
IN SCRAPIE
465
MICE
however, the normal mice excreted i3rI less rapidly and after 6 days all groups of mice had excreted very similar amounts. TABLE MEAN
PER
CENT
EXCRETION OF Ia11 VARIOUS TIMES AFTER
GOUp* Normal Scrapie Chpnzone
U-24 h. 51.7k4.3 38.4k4.1 38.1k5.4
5
FROM NORMAL, SCRAPIE THE I.“. ADMINISTRATION
AND CUPRIZONE-TREATED OF [‘*lI]PvP
2648 h.
48-120 h.
4.6kO.5 13.7*52 10.8* 1.6
3.1kO.5 10.2kO.5 10.1* 1.2
120-144 h. 1.4kO.5 1.220.8 1.7kO.3
MICE
AT
Total O-144 h. 60.8k3.8 63.5k4.8 60.7k3.4
* 5 to 8 mice per group. DISCUSSION
In order to make the calculated values of extravascular 1311comparable between mice and between groups of mice, and to allow for the variations in radioactive concentrations which occur, for example, because of slight variability in the intravenous dose of i311 or the weight of mouse, it is usual to relate the concentration of extravascular 1311to the concentration of 1311in the blood at the time of death. However, if the reasonable assumption is made that any leakage of 1311into tissues proceeds at a rate proportional to its concensiation throughout the 24 h. after its intravenous injection, the total extravascular 13rI in the tissues could be related more accurately to the mean concentration of the [1311]HSA (or PVP) in the blood during this period. This would make allowance for any variations in the rate at which the [1311]HSA (or PVP) was lost from the blood of different groups of mice. In the experiments reported here the ratio of the concentrations of 1311and lz51 in the blood of normal mice was significantly lower than that in the blood of clinical scrapie or cuprizone mice. This showed that 1311was lost from the blood of normal mice more rapidly than from the other two groups. Consequently the extravascular 1311in the tissues has been related to the mean 1311concentration of the HSA or PVP in blood during the 24-h. period of the experiments. The method of calculation of this mean 1311concentration has been given in Materials and Methods. In Exps. A to D there were consistent differences between the amounts of both 1311and lz51 in the blood of normal, scrapie and cuprizone mice. The differences in lz51 concentration in the blood of the different groups of mice were probably due to differences in their body weight and thus in their blood volumes; because of the debilitating nature of the disease, cuprizone mice were considerably smaller than mice in the other two groups, and the scrapie mice were slightly lighter than the normal mice. The concentrations of Ia11 in the blood were always much lower than those of 1261,indicating that there had been considerable losses of 1311 from the circulation. The 1311/1251ratios showed that this loss was greater in the normal mice than in the clinical scrapie or cuprizone mice, in both of which the relative loss was very similar. A proportion of these lossesof 1311during the first 24 h., and the quantitative difference in this respect between the groups, can be attributed to excretion in the
466
B. F. SANSOM
et
d.
urine and faeces, in accord with the results of whole-body counting. However, during 24 h. these losses accounted for only about 68 per cent. of the total loss in the normal mice and 50 to 55 per cent. in the clinical scrapie and the cuprizone mice. The remaining loss was presumably due principally to the leakage of HSA or PVP into extravascular spaces in tissues. In the two tissues selected, brain and spleen, there was evidence of such leakage, to a very small extent in the brain with both HSA and PVP and to an approximately lo-fold and loo-fold greater extent in the spleen with HSA and PVP respectively. However, the only significant difference between groups of mice was a slightly higher average figure for extravascular [ i311]HSA in the brains of mice with clinical scrapie than in either cuprizone-treated or normal mice. The fact that this finding was not repeated in the experiments with [13’I]PVP suggests that if differences in blood vessel permeability do in fact exist between scrapieaffected or cuprizone-affected mice and normal mice, they must be more subtle than can be consistently detected by molecules as large as HSA or PVP. In view of the histopathological similarities between scrapie and cuprizone toxicity, it was of interest that the rates of loss of radioactive material from the blood and of its excretion from the body were qualitatively similar in these groups of mice and different from those of normal mice. SUMMARY
Blood vessel permeability in normal mice and in mice affected with subclinical or clinical scrapie or fed with cuprizone was measured by a technique using two radioactive tracers ; either 1311 and lz51-labelled human serum (PVP) . No albumin (HSA) or 1311 and 1251-labelled polyvinylpyrrolidone major and constant difference in permeability was detected between the groups. Whole body radioactivity measured at intervals after intravenous injection of [1311]PVP showed that during the first 24 h. normal mice excreted 52 per cent. of the injected [ 1311]PVP, whereas mice with scrapie or fed with cuprizone excreted only 38 per cent.; after 6 days, however, all groups of mice had excreted very similar amounts. ACKNOWLEDGMENTS
We are grateful to Professor J. B. Cavanagh work was initiated.
for a helpful
discussion at the time this
REFERENCES
Boyse, E. A., Morgan, R. S., Pearson, J. D., and Wright, G. Payling (1956). Brit. J. exb. Path.. 37. 333. Chandler, R. ‘L. (1961). Lance& i, 1378. Mellick, R. S., and Cavanagh, J. B. (1967). J. Neural. Neurosurg. and Psychiat., 30, 458; (1968). Brain, 91, 141. Pattison, I. H. (1965). Slow, Latent and Temperate Virus Infections, p. 249. Symp. Natl. Inst. Hlth. ; Washington. Pattison, I. H., and Jebbett, Jean N. (1971). Res. vet. Sci., 12, 378. Sansom, B. F., Taylor, P. J., Wheelock, D., and Vagg, M. J. (1971). Mineral Studies with Isotopes in Domestic Animals, p. 125. I.A.E.A.; Vienna. [Received for publication, .November 18th, 19721
PATH.1973.
VOL.
PERMEABILITY AFFECTED
B. F.
461
83.
OF BLOOD WITH SCRAPIE CUPRIZONE I. H.
SANSOM,
VESSELS IN MICE OR FED WITH
and
PATTISON
JEAN
N.
JEBBETT
Agricultural Research Council, Institute for Research on Animal Diseases, Compton, Newbury, Berkshire
INTRODUCTION
The histopathology of naturally occurring scrapie in sheep and of the experimentally produced disease in sheep, goats, mice, rats and hamsters has been described in detail, but the fundamental nature of the characteristic bilaterally symmetrical spongiform encephalopathy is still obscure. It was suggested by Pattison (1965) that the bilateral symmetry of the lesions might indicate a relationship with the blood supply. A possible basis for involvement of blood vessels in the pathological process might be an increase in their permeability. Moreover, large increases in the permeability of blood vessels supplying nervous tissue have been reported in response to both physical trauma to individual nerves (Mellick and Cavanagh, 1967, 1968) and to the action of a neurotropic virus (Boyse, Morgan, Pearson and Wright, 1956). It was decided, therefore, to measure blood vessel permeability by a double radioactive tracer technique (Mellick and Cavanagh, 1967) in the brains and spleens of mice inoculated with scrapie. Apparent replication of the scrapie agent occurs in both tissues, although its pathological effects are restricted to the brain. In view of histological similarities between scrapie and cuprizone toxicity (Pattison and Jebbett, 1971), the blood vessel permeability of mice showing clinical evidence of cuprizone toxicity was compared with that of mice affected with scrapie. MATERIALS
AND
METHODS
These were females of the BSVS strain bred at Compton. There were 5 to 8 mice in each group in all experiments. Scrapie. Scrapie was produced by the intracerebral (i.c.) inoculation of’ the agent adapted to mice by Chandler (1961). In E x p eriments A, B, C, D and F the animals were showing advanced clinical evidence of the disease and were somewhat smallet than normal mice used as controls. In Exp. E the 2 groups of scrapie mice were in the pre-clinical stage of the disease, either 27 or 34 days after inoculation, and were the same size as the non-inoculated control mice. G&zone. Cuprizone (biscyclohexanone oxaldihydrazone) * was fed at the rate of 0.5 per cent. of a whole-diet meal (an intake of up to 15 mg. of cuprizone per mouse per d.), as described by Pattison and Jebbett (1971). The mice had been on this diet for at least 30 d. and showed clear evidence of cuprizone toxicity. By virtue of the debilitating nature of the disease, cuprizone-affected mice were smaller than either the normal or scrapie-affected mice. Mice.
* Koch-Light
Laboratories
Ltd.,
Colnbrook,
Bucks.
462
B. F. SANSOM
et al.
Double radioactive tracer technique. 1311 and lz51-labelled human serum albumin (HSA) (Codes IBI7P and IMI7P respectively, Radiochemical Centre, Amersham) polyvinylpyrrolidone (PVP) were used for Exps. A and B. 1311 and lz51-labelled (Codes IB33P and IM33P respectively) were used for Exps. C, D and E. [1311]HSA or PVP in O-1 ml. of physiological saline was injected into a tail vein, and between saline was 24 and 25 h. later [lz51]HSA (or PVP) in the same volume of physiological also injected i.v.; between 5 and 10 min. later the mice were killed with ether. Blood was taken immediately by Pasteur pipette from a pool formed by severing the vessels in an axilla, and the whole brain and spleen were removed and chopped finely with scissors. The blood, brain and spleen samples were placed in separate counting tubes, weighed and counted in two channels of a gamma scintillation counter, one channel counting only 13rI and the other mainly 1251. The accuracy of the estimation of extravascular 1311 in individual brains and spleens was such that the coefficient of variation did not exceed 20 per cent. and was usually about 10 per cent. Whole body counting. In Exp. F in which whole body radioactivity was measured over a period of 6 days, mice were inoculated iv. with 1311 PVP in 0.1 ml. physiological saline. For each loo-second count they were held in small plastic tubes attached by rubber bands to one scintillation detector of a large animal whole body counter (Sansom, Taylor, Wheelock and Vagg, 197 1). Counts were made at approximately 4, 24, 48, 120, and 144 h. after injection. Calculations and presentation of data. The i.v. injection of [1311]HSA (or PVP) and [1251]HSA (or PVP) 24 to 25 h. and 5 to 10 min. respectively before samples of blood and tissue were removed made it possible to calculate the amount of extravascular ls11 present in the tissues by means of the formula:is11 in blood Extravascular 1311 c in tissue N 1251 in blood in tissue This is based on the assumption that 5 to COmin. after i.v. injection the 1251 HSA (or PVP) is present only in the blood in the tissue and it makes a simple correction for the 1311 present in the blood. The values for extravascular 1311 in the brain and spleen are presented in the Results in terms of the equivalent volume of blood of concentration equal to the mean [ralI]HSA (or PVP) concentration during the 24-h. period of the experiments. This as follows: mean concentration of [ 1311]HSA (or PVP) in the blood was determined the initial concentration of the [ 1311]HSA (or PVP) was assumed to be the same as the concentration of [issI]HSA (or PVP) and the mean concentration was then calculated by assuming that the blood concentration fell in a simple exponential manner. Summary of experiments. A and B were replicate experiments in which [1311] and [1251]HSA were used to measurevascularpermeability in the brains and spleens of mice showing advanced clinical signs of scrapie or of mice fed cuprizone. C and D were replicate experiments in which [lslI] and [1251]PVP were used to measure vascular permeability in the brains and spleens of mice showing advanced clinical signs of scrapie or of mice fed cuprizone. in In Exp. E, [1311] and [ 1251]PVP were used to measure vascular permeability the brains and spleens of mice in the pre-clinical stage of scrapie. In Exp. F, the retention of radioactivity in the whole body of mice showing advanced clinical signs of scrapie, or of mice fed cuprizone, was measured at intervals after the i.v. inoculation of [lslI]PVP.
1
RESULTS
Advanced Clinical Scrapie Exps. A and B : [I3111 and [125I]HSA. The results of these experiments, in which [1311]HSA and [ 1251]HSA were used, are given in Tables 1 and 3. There was good agreement between the results in the 2 experiments. In all groups of mice from the blood particularly in the normal there was a rapid loss of [ l3lI]HSA
VASCULAR
PERMEABILITY
IN SCRAPIE
mice. The only indication of a difference in leakage from groups of mice was a slightly higher average figure (P < scrapie mice than for either cuprizone or normal animals. were more variable and no significant difference between TABLE MEAN BLOOD Ia11 AND RESPEcT1"EI.Y AFTER I.".
Normal Scrapie Cuprizone
* 5 to 8 mice
7.8kO.3 13.7+ 1.3 21.7k3.2
per
lzsI CONCENTRATION
blood vessels between 0.02) for the brains of The results for spleen groups was detected.
1
dose/g.If:s.~.) AND ADMINISTRATION OF [ ‘S’I]HSA AND CUPRIZONE-TREATEDMICE
47.8k2.0 52.7k3.4 79.Ok5.7
463
MICE
(%
0.163+0.011 0.260 k 0.02 1 0.274 + 0.020
AT 24 h. AND 5 TO 10 min. [‘251]HSA TO NORMAL, SCRAPIE
9.0*0.5 13.4kO.9 17.4+ 1.1
52.5k3.2 54.4 & 4.0 71.6k5.1
0~171+0~015 0.246kO.010 0.243+0.018
group.
Exf,u. C: and D: [1311] and [1251]PVP. B ecause only minor differences had been detected between groups of mice in A and B, the observations were repeated with [1311]PVP and [1251]PVP. PVP was used because of its lowel molecular weight and because there is less free iodide 1311or la51 in the preparation. The lower molecular weight would be expected to increase the extent of leakage and thus possibly to increase the likelihood of detecting differences between groups of mice, and the smaller amount of free 1311would be expec;cd to improve the specificity of the method. The results, given in Tables 2 and 4, were essentially the same as in A and B. An obvious quantitative difference between the 2 pairs of experiments was the greater amount of extravascular [1311]PVP than of [1311]HSA in the spleens, but not the brains, of mice of all groups, presumably due to the smaller molecular size of PVP. Qualitatively the small differences between groups of mice were similar to those in the experiments with HSA, although scrapie and cuprizone mice were not significantly different from normal mice. Preclinical Scrapie Exp. E: [i311] and [1251]PVP. This experiment was carried out to examine the possibility that although no difference in capillary permeability had been detected between normal mice and mice in an advanced clinical stage of scrapie, a difference might be detectable in an early pre-clinical stage of the disease. Groups of 6 normal mice and of mice either 27 or 34 days after i.c. inoculation with scrapie, were injected with [ 1311]PVP and after 24 hours with [lz51]PVP. Five minutes later the animals were killed and 1311 and 1251 were measured in blood, brain and spleen, as in A, B, C and D. No differences between the groups of mice were detected, either in the rate at which [1311]PVP was lost from the blood, or in the amounts of extravascular [ialI]PVP in the brain or spleen. The rate of loss of 1311 from the blood was similar to that in the normal mice in A, B, C and D. B
464
B. F. SANSOM la11
AND AFTER
ias1
CONCENTRATION (% dose/g.+ I.V. ADMINISTRATION OF [lslI]PvP SCRAPIE AND CUPRIZONE-TREATED
Exp.c Group * Normal Scrapie Cuprizone
al.
2
TABLE MEAN BLOOD RESPECTIVELY
et
la11
1251
6.1 kO.2 9.7kO.5 11.8kO.8
35.3+ 1.8 41.1k2.1 45.3k2.4
s.E.) AT 24 h. AND 5 TO 10 min. AND ['*51]PvP TO NORMAL, MICE
Eq. D ------___
-____ 1a11/1B51
0.172+0.010 0.236+0.013 0~260~0.011
1311
1251
7.1 kO.6 10.8 f 0.4 14.1 f 0.9
29.1* 1.1 34.8 f 2.3 466k2.8
131I,l25I
0.244+0.018 0.320 + 0.024 0.308 f 0.022
* 5 to 8 mice per group. 3
TABLE MEANEXTRAVASCULAR la11 (~1. SCRAPIE AND CUPRIZONE-TREATED
blood/g.
tissue+s.~.)
MICE
24b.
AFTER
IN THE BRAINS AND I.V. ADMINISTRATION
SPLEENS OF NORMAL, OF [ialI]HSA
Exfi. A Group* Normal Scrapie Cuprizone
Brain 1.13*0*09 t1.82+0*19 1.26kO.15
* 5 to 8 mice per group.
Exj. B
-
Spleen
Brain
Spleen
11*37+ 1.19 15.09+2.61 9.74* 1.75
1.05 + 0.06 t1.54*0.15 0.75 f 0.09
13.535 1.89 10.94+ 1.62 11.06k1.11
t P < 0.02.
MEAN EXTRAVASCULAR la11 (~1. SCRAPIE AND CUPRIZONE-TREATED
TABLE 4 blood/g. tissueks.~.) IN THE BRAINS AND MICE 24h. AFTER I.V. ADMINISTRATION
SPLEENS OF NORMAL, OF [IalI]PvP
Eq. C
Eq. D
-
Group*
Brain
Spleen
Brain
Spleen
Normal Scrapie Cuprizone
0.86 + 0.16 0.87+0.21 0.76 + 0.07
139.7 * 7.5 115.Ok5.1 129.7 + 5.3
0.86kO.14 1.oo + 0.07 0.62 f 0.06
127.5k2.4 103.Ok5.6 88.6 + 5.8
* 5
to
8 mice per group.
Retention of Radioactivity Exp. F. The results in A to E had shown that in all groups of mice there was more marked in normal animals than a rapid loss of is11 from the circulation, in those showing clinical signs of scrapie or the effects of cuprizone. It was not clear, however, whether this loss was from the blood into extra-vascular tissues, or from the body itself in the urine and faeces. Whole body radioactivity determinations of all groups of mice were therefore made at intervals up to 144 h. after i.v. injection of [ 1311]PVP. The results (Table 5) show that during the first 24 h. normal mice excreted 52 per cent. of the injected 1311,whereas the scrapie and cuprizone treated mice excreted only 38 per cent. Subsequently,
VASCULAR
PERMEABILITY
IN SCRAPIE
465
MICE
however, the normal mice excreted i3rI less rapidly and after 6 days all groups of mice had excreted very similar amounts. TABLE MEAN
PER
CENT
EXCRETION OF Ia11 VARIOUS TIMES AFTER
GOUp* Normal Scrapie Chpnzone
U-24 h. 51.7k4.3 38.4k4.1 38.1k5.4
5
FROM NORMAL, SCRAPIE THE I.“. ADMINISTRATION
AND CUPRIZONE-TREATED OF [‘*lI]PvP
2648 h.
48-120 h.
4.6kO.5 13.7*52 10.8* 1.6
3.1kO.5 10.2kO.5 10.1* 1.2
120-144 h. 1.4kO.5 1.220.8 1.7kO.3
MICE
AT
Total O-144 h. 60.8k3.8 63.5k4.8 60.7k3.4
* 5 to 8 mice per group. DISCUSSION
In order to make the calculated values of extravascular 1311comparable between mice and between groups of mice, and to allow for the variations in radioactive concentrations which occur, for example, because of slight variability in the intravenous dose of i311 or the weight of mouse, it is usual to relate the concentration of extravascular 1311to the concentration of 1311in the blood at the time of death. However, if the reasonable assumption is made that any leakage of 1311into tissues proceeds at a rate proportional to its concensiation throughout the 24 h. after its intravenous injection, the total extravascular 13rI in the tissues could be related more accurately to the mean concentration of the [1311]HSA (or PVP) in the blood during this period. This would make allowance for any variations in the rate at which the [1311]HSA (or PVP) was lost from the blood of different groups of mice. In the experiments reported here the ratio of the concentrations of 1311and lz51 in the blood of normal mice was significantly lower than that in the blood of clinical scrapie or cuprizone mice. This showed that 1311was lost from the blood of normal mice more rapidly than from the other two groups. Consequently the extravascular 1311in the tissues has been related to the mean 1311concentration of the HSA or PVP in blood during the 24-h. period of the experiments. The method of calculation of this mean 1311concentration has been given in Materials and Methods. In Exps. A to D there were consistent differences between the amounts of both 1311and lz51 in the blood of normal, scrapie and cuprizone mice. The differences in lz51 concentration in the blood of the different groups of mice were probably due to differences in their body weight and thus in their blood volumes; because of the debilitating nature of the disease, cuprizone mice were considerably smaller than mice in the other two groups, and the scrapie mice were slightly lighter than the normal mice. The concentrations of Ia11 in the blood were always much lower than those of 1261,indicating that there had been considerable losses of 1311 from the circulation. The 1311/1251ratios showed that this loss was greater in the normal mice than in the clinical scrapie or cuprizone mice, in both of which the relative loss was very similar. A proportion of these lossesof 1311during the first 24 h., and the quantitative difference in this respect between the groups, can be attributed to excretion in the
466
B. F. SANSOM
et
d.
urine and faeces, in accord with the results of whole-body counting. However, during 24 h. these losses accounted for only about 68 per cent. of the total loss in the normal mice and 50 to 55 per cent. in the clinical scrapie and the cuprizone mice. The remaining loss was presumably due principally to the leakage of HSA or PVP into extravascular spaces in tissues. In the two tissues selected, brain and spleen, there was evidence of such leakage, to a very small extent in the brain with both HSA and PVP and to an approximately lo-fold and loo-fold greater extent in the spleen with HSA and PVP respectively. However, the only significant difference between groups of mice was a slightly higher average figure for extravascular [ i311]HSA in the brains of mice with clinical scrapie than in either cuprizone-treated or normal mice. The fact that this finding was not repeated in the experiments with [13’I]PVP suggests that if differences in blood vessel permeability do in fact exist between scrapieaffected or cuprizone-affected mice and normal mice, they must be more subtle than can be consistently detected by molecules as large as HSA or PVP. In view of the histopathological similarities between scrapie and cuprizone toxicity, it was of interest that the rates of loss of radioactive material from the blood and of its excretion from the body were qualitatively similar in these groups of mice and different from those of normal mice. SUMMARY
Blood vessel permeability in normal mice and in mice affected with subclinical or clinical scrapie or fed with cuprizone was measured by a technique using two radioactive tracers ; either 1311 and lz51-labelled human serum (PVP) . No albumin (HSA) or 1311 and 1251-labelled polyvinylpyrrolidone major and constant difference in permeability was detected between the groups. Whole body radioactivity measured at intervals after intravenous injection of [1311]PVP showed that during the first 24 h. normal mice excreted 52 per cent. of the injected [ 1311]PVP, whereas mice with scrapie or fed with cuprizone excreted only 38 per cent.; after 6 days, however, all groups of mice had excreted very similar amounts. ACKNOWLEDGMENTS
We are grateful to Professor J. B. Cavanagh work was initiated.
for a helpful
discussion at the time this
REFERENCES
Boyse, E. A., Morgan, R. S., Pearson, J. D., and Wright, G. Payling (1956). Brit. J. exb. Path.. 37. 333. Chandler, R. ‘L. (1961). Lance& i, 1378. Mellick, R. S., and Cavanagh, J. B. (1967). J. Neural. Neurosurg. and Psychiat., 30, 458; (1968). Brain, 91, 141. Pattison, I. H. (1965). Slow, Latent and Temperate Virus Infections, p. 249. Symp. Natl. Inst. Hlth. ; Washington. Pattison, I. H., and Jebbett, Jean N. (1971). Res. vet. Sci., 12, 378. Sansom, B. F., Taylor, P. J., Wheelock, D., and Vagg, M. J. (1971). Mineral Studies with Isotopes in Domestic Animals, p. 125. I.A.E.A.; Vienna. [Received for publication, .November 18th, 19721