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AMYLOIDOSIS AND LYMPHOID APLASIA IN MOUSE RADIATION CHIMERAS S. BRADBURY, M.A., D.PHIL., AND H. S. MICKLEM, M.A., D.PHIL. From the Department of Human Anatomy, South Parks Road, Oxford, and Medical Research Council, Radiobiological Research Unit, Harwell, Berkshire, Great Britain

It is well known that lethally irradiated mice may be saved from otherwise certain death by the injection of blood-forming cells from normal mouse or rat donors. After initial recovery such mice may develop "secondary disease"-a syndrome of variable, but often fatal outcome-during the second to fourth month after irradiation. This syndrome is characterized clinically by wasting, diarrhea and sometimes skin lesions, and histologically by lymphoid atrophy and aplasia, granulopoietic hyperplasia and, at any rate in the CBA strain, deposits of hyaline material in the spleen.1'2 It commonly develops when the donor of restorative cells is antigenically different from the host and is then attributable to a graft-versus-host reaction. As has recently been shown, however, it may also be found, under certain special conditions, when donor and host are syngeneic (of the same highly inbred strain) and hence presumably unable to react against each other."2 In this latter situation its onset is prevented by an adequate transplant of viable syngeneic lymphoid cells. The present paper describes the incidence of splenic and hepatic hyaline deposits in a series of mice which died with "secondary disease," and also in a smaller series which was killed at various stages before and during the development of the disease. METHODS Male mice of the highly inbred CBA/H strain were exposed to lethal x-irradiation (iooo rads to the whole body). The x-rays, with half-value-layer, I.2 mm Cu, were generated by a Siemens Stabilipan constant potential machine operated at 250 kv and 14 ma. The mice were irradiated in their normal aluminum living cages at a dose rate of 70 to 75 rads per minute. One-half to 4 hours after exposure, the mice were given an intravenous injection of a saline suspension of bone marrow or fetal liver cells from normal donors of the same or another inbred strain. Sometimes lymph node cells were injected as well. The repopulation of the radiation-damaged myeloid and lymphoid tissues by injected cells, the recovery of the recipients from the lethal effects of radiation and the incidence of "secondary disease" have been described in previous publications 1-5 and will not be described in detail here. In several experiments myeloid, lymphoid and other tissues from mice which died or were found moribund and killed between 2 I and I 20 days after irradiation were fixed in phosphateAccepted for publication, July 24, x964. 263

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buffered formalin, Clarke's fluid (i part glacial acetic acid, 3 parts ethanol), or Baker's6 formol-calcium (i part io per cent aqueous CaCl2, i part 40 per cent formaldehyde, 8 parts distilled water). All material, except that fixed in the last-mentioned fluid, was dehydrated in alcohol and embedded in paraffin. Sections were cut at Sim and stained with hematoxylin and eosin. Formol-calcium was used in preparation for histochemical tests for lipid; tissues fixed in it were embedded in gelatin and sectioned at 8 ,. A number of mice were killed at various stages in the development of "secondary disease," and their tissues were fixed and stained as described above. Material fixed in buffered formalin was used for all histochemical reactions apart from those involving lipid. For staining with pyronin and methyl green, fixation in Clarke's fluid was used also. 0 Histochemical and Staining Techniques Staining procedures were carried out as directed in the appendices to Pearse,7 unless indicated to the contrary. General Staining Reactions. Congo red,8 methyl violet,8 phosphotungstic acid hematoxylin,9 methyl green.10 Histochemical Tests. (a) Proteins and aminoacids: mercury bromophenol blue, Millon, dimethylaminobenzaldehyde nitrite (DMAB). (b) Carbohydrates: periodic acid Schiff (PAS), PAS after diastase (i per cent, i hour, 370 C), PAS after hyalase (200 IU per ml, 4 hours, 370 C), toluidine blue, toluidine blue after hyalase (200 IU per ml, 4 hours, 370 C), Hale's reaction with colloidal iron, alcian blue. (c) Nucleic acids: Feulgen, pyronin methyl green. (d) Lipids: Sudan black, acetylated Sudan black, acid hematein, Fettrot 7B, oil red 0, Nile blue. In addition, examination of the sections stained with Congo red was carried out with the polarizing microscope in order to ascertain whether there was any birefringence. For comparison, material of human origin (kindly provided by Mr. J. Jerome of the Sir William Dunn School of Pathology, Oxford), and known to contain amyloid, was subjected to some of the same staining procedures. Eight mice with "secondary disease" of varying severity were given a single injection of sulphur 35 (ioo0 C/mouse Na235SO4, carrier-free, in isotonic saline). One normal control mouse of the same age and strain was similarly injected. Six hours later the mice were killed and the following tissues were fixed in Clarke's fluid or 4 per cent formaldehyde containing o.s per cent weight per volume cetyl pyridinium chloride-a fixative recommended for acid mucopolysaccharides 7"11: spleen, mesenteric and inguinal lymph nodes, liver, kidney, adrenal, ileum and sternum. The duration of fixation was i6 hours in Clarke's fluid and 44 hours in pyridinium-formalin. Paraffin sections were cut at 5,u, taken to distilled water and washed for I hour, the water being changed every io minutes. Stripping film autoradiographs were prepared with Kodak A.R.io film, exposed for 3o days and developed in Kodak D. igb developer. A positive radiographic image would indicate uptake of radioactive sulphur and its incorporation into sulphated compounds currently being synthesized. Inorganic sulphate would not be incorporated into cystine or methionine, although a very small amount might perhaps be incorporated into taurine.12 Some uncombined inorganic sulphate might be expected still to be present in the tissues 6 hours after injection, but most of this would be removed during the washing procedure. Consequently any substantial degree of labelling shown in the autoradiographs could confidently be ascribed to synthesis of sulphated polysaccharides.'s Electron Microscopy Tissues were fixed in one of the following ways: (i) Palade's fluid 14 for i hour at 40 C; (2) Glutaraldehyde 15 for 3 hours at 40 C, followed by washing in 3 changes (i hour each) of o.I M phosphate buffer containiung Io per cent sucrose at pH 7.4

Feb., z965y

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and immersion overnight in phosphate buffered osmium tetroxide, again at 40 C. Material was dehydrated in the usual way and embedded in Araldite. Sections 8o mp thick were cut with a Huxley ultra-microtome, picked up on uncoated 200-mesh grids and stained for 20 minutes with lead citrate, as described by Reynolds.16 The sections were examined in a modified 17 Siemen's Elmiskop I instrument operated at 6o kv.

RESULTS

Incidence of hyaline deposits in the spleen and liver The bone marrow, spleen and lymph nodes and frequently also liver, kidneys, heart, lung, pancreas and adrenals were examined in 59 male CBA mice which died with "secondary disease" between 2I and I20 days after irradiation and treatment with C57BL, A or C3H bone marrow, or CBA fetal liver. Hyaline deposits were present in nearly all the spleens and in a large proportion of the livers (Table I.) They were TABLE I THE INCIDENCE OF VARIOUS HISTOLOGIC FEATURES IN CBA MICE WHICH DIED WITH SECONDARY DISEASE BETWEEN 21 AND I20 DAYS AFTER LETHAL X-IRRADIATION AND INJECTION OF VIABLE HEMATOPOIETIC CELLS

Cells injected

Granulocytopoietic Time of hyperplasia death (days post irrad.) Bone marrow Spleen

Amyloid

Spleen

Liver

Liver necrosis

Lymphoid hypoplasia

2I-60

I8/20

I5/I9

I8/20

3/I7

8/I7

19/19

marrow

6I-I20

I0/I I

5/1I0

10/10

9/9

2/9

I0/I0

C3H bone

2I-60

8/I0

7/10

9/I0

6/7

0/7

I0/l0

marrow

6I-120

5/6

2/5

5/5

4/5

0/5

5/5

CBA

2 I-60

II/II

5/12

I2/I2

8/8

3/8

I2/12

52/58

34/56

54/57

30/46

I3/46

56/56

A or

C57BL bone

fetal

liver

Total

The results are expressed as a fraction; the numerator represents the number of mice with the

lesion; the denominator is the total number examined.

most extensive in those animals which lived longest. Early hyaline deposits appeared in the spleen as narrow bands around the hypocellular Malpighian corpuscles. They gradually extended inwards towards the center of the corpuscle and outwards into the pulp. The arterioles of the corpuscles were not involved. This distribution is similar to that described by Dick and Leiter 18 in the rabbit. No deposits were seen in the kidneys of mice dying within 4 months of irradiation, although they sometimes appeared in the glomeruli after longer intervals. They were occasionally noted in the intestinal villi and the adrenal cortex, and a more systematic survey of these organs might have revealed a high incidence.

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Hepatic necrosis was present in less than one-third of the animals examined. Lymphoid hypoplasia and hyperplastic granulocytopoiesis, on the other hand, were almost invariably seen; the former was usually profound, amounting to virtual aplasia. In the liver the earlier deposits of hyalin formed in the neighborhood of the portal veins and subsequently spread into the sinusoids. They were largely, if not entirely, extracellular. Barnes, Loutit, and Micklem2 described an experiment in which the CBA mice were restored, after lethal irradiation, with CBA bone marrow cells which had been serially transplanted through previous lethally irradiated syngeneic hosts. Simultaneously another group of mice was restored with the same bone marrow and, in addition, lymph node cells from a normal CBA donor. Mice from each group were killed at weekly intervals for ii weeks, and their myeloid and lymphoid tissues were examined histologically. The mice of the first group developed secondary disease and lost weight progressively, while those given injections of lymph node cells in addition remained healthy. The incidence in that series of splenic hyaline deposits and other abnormalities is entered in Table II. The addition of lymph node cells to the restorative inoculum TABLE II THE INCIDENCE OF VARIOUS HISTOLOGIC FEATURES IN CBA MICE KILLED 3 TO II WEEKS AFTER LETHAL X-IRRADIATION AND INJECTION OF THRICE-PASSAGED CBA BONE-MARROW, WITH OR WITHOUT NORMAL LYMPH NODE CELLS

Granulocytopoietic hyperplasia

Splenic Bone marrow Spleen amyloid

PyroninoPlasmatoid hyperplasia, Granulocytes philic cells * Lymphoid mesenteric numerous in numerous hypoplasia Spleen Lymph nodes lymph node lymph nodes in spleen

Without lymphoid

I6/I8

I6/I8

I4/I8

15/I8

15/I7

I2/I7

IO/I7

0/I8

3/I8

3/I8

2/I8

6/i8

7/I8

6/i8

5/I8

0/I8

supplement With lymphoid

supplement x2

i6.i

I6.I

12.66

7.34

7.50

349

2.30

P