INT. J. RAD. BIOL. 1966, VOL. 11, NO. 3, 235-243

Prevention of the bone-marrow syndrome in irradiatedmice. A comparison of the results after bone-marrowshielding and bone-marrow inoculation

F. A. J. DE VRIES and O. VOSMedical Biological Laboratory of the National Defense Research OrganizationTNO, Rijswijk (Z.H.), The Netherlands

(Received 6 April 1966; revision received 18 July 1966)

Shielding of bone-marrow in irradiated mice was compared with intravenousinjection of a bone-marrow cell suspension in its effectiveness to prevent abone-marrow syndrome. Three criteria were used to measure this effectiveness:(a) survival of the irradiated mouse at 30 days after irradiation; (b) re-populationof irradiated haematopoietic tissues, and (c) incidence of spleen colonies.

On the basis of the number of cells involved bone-marrow injection appearedto be about 100 times more effective than bone-marrow shielding.

1. IntroductionShielding a part of the haematopoietic tissues against x-irradiation was

shown to be effective in preventing mortality (cf. Jacobson 1952) even before itwas recognized that transplantation of bone-marrow was capable of preventingdeath due to the bone-marrow syndrome. If a shielded spleen is left intactin the circulation for only one hour after irradiation (Jacobson, Simmons,Marks, Gaston, Robson and Eldredge 1951) the beneficial effect has alreadybeen exerted. This effect has now been explained by a mobilization ofhaematopoietic stem cells, which are transported from the spleen to the bone-marrow and give rise to a re-population. Shielding of bone-marrow had alsobeen shown to enhance survival after x-irradiation (Jacobson et al. 1951),but it was less effective than shielding of the spleen. Since the efficacy ofbone-marrow shielding versus bone-marrow transplantation had not beenstudied in relation to the number of cells involved, we decided to study it.During this investigation two papers appeared, which also dealt with themigration of stem cells from haematopoietic tissues (Hanks 1964, Hellman1965).

2. Materials and methods(CBA/Rij x C57BL/Rij)F x mice (hereafter designated as F), 10-12 weeks old

and weighing 20-26 g, were x-irradiated. During irradiation the mice werenot anaesthetized. They were tied on a layer of hard-board (figure 1), practicallyno movements that might affect the irradiated area were possible. Irradiationconstants were 200 kv (constant potential), 18 mA, filtration 05 mm Cu, h.v.l.1-05 mm Cu, distance to target 50 cm, dose-rate 70 R/min. In the experimentsmentioned in §3.2 (d) the mice were irradiated in a perspex container, inwhich they could move freely. Under the latter circumstances a 15 mm Cufilter was used, resulting in a h.v.l. of 19 mm Cu and a dose-rate of 46 R/min.

t This work was partly performed under contract with Euratom (European AtomicEnergy Community), Brussels, Belgium.

Int J

Rad

iat B

iol D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y Fr

eie

Uni

vers

itaet

Ber

lin o

n 11

/04/

14Fo

r pe

rson

al u

se o

nly.

F. A. J. de Vries and 0. Vos

Figure 1. Position of mice during irradiation, various parts of the animal are shielded(grey area). (A) Mouse received whole-body irradiation; (B) right hind legirradiated, rest of body shielded; (C) hind leg shielded below knee joint or belowhalf of tibia; (D) hind leg completely shielded.

Shielding of the right hind leg or a part of it was established with a 5 mmthick lead sheet, which was applied closely above the skin without touching it(figure 1). In a few experiments the leg was irradiated while the rest of thebody was shielded. Bone-marrow suspensions were prepared, counted andintravenously injected as described earlier (Vos and Weyzen 1962). Suspensionsto be inoculated were prepared from the femur, unless stated otherwise (§3.2 (d)).

Spleen colonies were counted eight days after irradiation and inoculationof bone-marrow. Spleens were fixed in Bouin's fluid and the colonies were

236

Int J

Rad

iat B

iol D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y Fr

eie

Uni

vers

itaet

Ber

lin o

n 11

/04/

14Fo

r pe

rson

al u

se o

nly.

Prevention of the bone-marrow syndrome

examined under a dissection microscope (magnification 20 x). All externallyvisible colonies were counted.

Heart blood samples from mice dying before the eighth day after irradiationwere studied bacteriologically.

3. Results3.1. Mortality after whole-body irradiation

Survival after bone-marrow shielding and bone-marrow transplantationwere both studied in mice that were irradiated while tied on hard-board.

Since the radiation circumstances of these mice were different from thosein previously published experiments, the influence of the altered situation onthe dose-mortality relationship was studied. The LD50 appeared to be about150 R higher for mice tied on hard-board than for mice irradiated in a perspexcage (figure 2). The explanation for this difference is not clear. The alteredgeometry during irradiation may have exerted some influence. On the otherhand there may have occurred some anoxia in the feet, that were tied off bythe string that fixed the mice to the hard-board and this anoxia may have in-fluenced survival of some haematopoietic stem cells. Since 1050 R was chosenas the radiation dose for mice tied down on hard-board and this dose was closeto the LDoo a marked increase in survival in our experiments can always beattributed to treatment.

son _ 1UU

'U

C) so -

80-

-a 60-

D' 40-4)0,'U

c 20Ua)CL

Q1U.

-0-017 20°

270

240 20

340 032O262 20

600 700 800 900 1000 2000

x-ray dose (r)

Figure 2. Percentage survival of mice 30 days after irradiation in a perspex cage (leftcurve) or tied down on hard-board (right curve). The figures indicate the numbersof mice used to estimate each point.

Mice which are irradiated with an LDo10o in a perspex container can beprotected by syngeneic bone-marrow transplantation up to almost 100 per cent.This was not true for mice irradiated in the tied-down position. An earlymortality which was refractory to bone-marrow transplantation was the causeof these results. From 20 mice irradiated with 1050 R in the tied-down position40 per cent died on the fifth day, from 30 mice irradiated in a perspex containeronly 3 per cent died on the same day. The cause of this early mortality wasnot clear. A higher incidence of deaths from the intestinal syndrome, as wellas infections, may have been involved. Heart blood cultures showed no

R.B. P

237

Int J

Rad

iat B

iol D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y Fr

eie

Uni

vers

itaet

Ber

lin o

n 11

/04/

14Fo

r pe

rson

al u

se o

nly.

F. A. J. de Vries and 0. Vos

Pseudomonas aeruginosa infection, but other bacteria-often Proteus vulgaris-were frequently found.

Notwithstanding the difficulties due to early mortality of the irradiatedanimals the radiation procedure was useful in the investigation of effectivenessof bone marrow shielding versus bone marrow inoculation in the treatment ofthe bone marrow syndrome.

3.2. Effects of bone-marrow shielding and bone-marrow inoculation

(a) SurvivalShielding of the hind leg below the hip joint, below the knee joint and below

half of the tibia all protected against irradiation with 1050 R (table). Theremaining mortality was almost completely due to death before the tenth day.Injection of 20 x 103 syngeneic bone-marrow cells gave some protection and40-80 x 103 bone-marrow cells protected at least as well as shielding of a partof the leg.

Shielding of Syngeneic bone-marrow Percentage survivalright hind leg cells injected ( x 103) on thirtieth day

after irradiation

Below hip joint 73 (25)Below knee joint 70 (20)Below half of tibia - 70 (20)

_--~~ ~ 20 20 (20)40-80 90 (10)

Survival of irradiated F mice after bone-marrow shielding or injection of syngeneicbone-marrow cells.

t The number of mice is given between brackets.t Mice were subjected to 1050 R while tied down on hard-board.

The right femur and tibia contained an average of respectively 18 and16 x 106 nucleated bone-marrow cells. Some haematopoietic cells will havebeen present in the fibula and the bones of the foot. Thus when the hind legbelow the hip loint or below the knee joint was shielded, at least 34 x 106 or16 x 106 bone-marrow cells were protected from irradiation. When the legbelow half of the tibia was shielded protection of at least 8 x 106 nucleated cellswill have been involved.

Unfortunately the marginal number of bone-marrow cells that had to beshielded in order to keep some mice alive was not assessed well enough toallow an exact calculation of the relative effectiveness of shielded versus inoculatedcells. If the bone marrow located distally from half of the tibia would be thiscritical amount then the difference in effectiveness between shielded andinoculated marrow would be a factor of about 100-200.

(b) Colony formation in the spleenInjection of syngeneic bone-marrow cells gave about 15 spleen colonies

per 104 injected cells (figure 3). In the case of shielded bone marrow abouttwo colonies were found for one-quarter of a tibia shielded (figure 3). Sincefrom the right tibia of normal mice an average of 16 x 106 nucleated

238

Int J

Rad

iat B

iol D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y Fr

eie

Uni

vers

itaet

Ber

lin o

n 11

/04/

14Fo

r pe

rson

al u

se o

nly.

Prevention of the bone-marrow syndrome

bone-marrow cells could be obtained, it can be estimated that about two spleencolonies are formed per 4x 106 shielded bone-marrow cells. The characterof the spleen colonies in the injected mice differed from that in shielded mice.In the shielded mice a few very small colonies were found among larger coloniesand those of regular size. We attribute the small colonies to stem cells thatwere transported to the spleen at a later time after irradiation. In inoculatedmice these small colonies were missing.

tn

C 80

U

c 6-a)

4 -0

0E 2 -O

0

/

o//o

/7/

/,,,

10 20 30 40 cells injected x101/4 1/2 3/4 1 part of tibia shielded

Figure 3. Number of spleen colonies in mice irradiated with 1050 R and intravenousinjection of syngeneic bone-marrow cells or shielding of a part of the leg.0 --- 0 tibia shielding, * intravenous injection. Each pointrepresents the mean value of 15-20 mice.

An estimation of the difference in relative effectiveness between inoculatedand shielded cells indicates that the intravenously injected cells are about300 times more effective.

(c) Re-population of haematopoietic tissues

In mice that were whole-body irradiated and not inoculated with syngeneicbone-marrow a rapid cellular depletion of bone-marrow and spleen was found(figure 4 (A)). Shielding of the right hind leg or a part of it caused a recoveryin the irradiated bone-marrow and spleen after about the eighth day followingirradiation (figure 4 (A) and (C)). After inoculation of 40 x 103 syngeneicbone-marrow cells recovery began a little later and was slower than after bone-marrow shielding.

A study of the re-population of different parts of the bone-marrow (femur,tibia and humerus, both right and left) indicated no preference for restorationof specific areas of the bone marrow, however the re-population of the spleenpreceded that of the bone-marrow. Playfair and Cole (1964) found an over-shoot in the number of stem cells in the spleen, 2-3 weeks after irradiationwhereas the bone-marrow contained still sub-normal numbers 60 days afterirradiation.

Here again the results are in agreement with a large difference in effectivenessbetween inoculated and shielded bone marrow in their ability to re-populatethe irradiated haematopoietic tissues. This difference may amount to a factorof 100-400.

In shielded bone-marrow the number of nucleated cells decreased to below50 per cent of the normal value. This depletion lasted for at least 8-12 days

239

Int J

Rad

iat B

iol D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y Fr

eie

Uni

vers

itaet

Ber

lin o

n 11

/04/

14Fo

r pe

rson

al u

se o

nly.

F. A. J. de Vries and O. Vos

0too -

80 -

60 -

40o-

20-

a

- a

do

days after irradiation

Figure 4. Recovery after irradiation (1050 R). The number of nucleated bone-marrowcells recovered at different times after irradiation is expressed as a percentage of thecells obtained from non-irradiated controls. Each point represents the averageof 6 mice. (A) Number of nucleated cells in irradiated parts of bone-marrow;* - * whole-body irradiation, cells from both femurs, tibiae and humeriare counted; - -. - * whole body irradiation and intravenous injectionof 40 x 103 syngeneic bone-marrow cells, cells in femurs, tibiae and humeri arecounted; El - 3 right hind leg shielded, cells from left femur and tibiaand both humeri are counted; - 0 right hind leg below knee jointshielded, cells from left tibia and both femurs and humeri are counted;x --- x right hind leg irradiated, rest of body shielded, cells from rightfemur and tibia are counted. (B) Number of nucleated cells in shielded parts ofbone-marrow; [ El right hind leg shielded, cells from right femur andtibia are counted; 0 0 right hind leg shielded below knee joint, cellsfrom right tibia are counted. (C) Number of nucleated cells in spleen; * whole-body irradiation; · - * whole-body irradiation and intravenousinjection of 40 x 10a syngeneic bone-marrow cells; - 0 shielding ofright hind leg below knee joint; E - [] shielding of right hind leg.

240

-6

S

I

2

SC

2

I

.S

.T

�D

eC

Int J

Rad

iat B

iol D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y Fr

eie

Uni

vers

itaet

Ber

lin o

n 11

/04/

14Fo

r pe

rson

al u

se o

nly.

Prevention of the bone-marrow syndrome 241

(figure 4 (B)). It may be partly due to a scattered radiation (compare §3.2 (d))or to migration of cells to the blood and irradiated parts of the haematopoietictissue. Kurnick and Nokay (1965) reported that the number of cells in thebone-marrow from a protected left lower extremity decreased to 65 per cent1 day after irradiation.

If the situation of shielding and irradiation was reversed, so that the righthind leg was irradiated and the rest of the body shielded (figure 1 (D)) there-population of the irradiated bone-marrow began earlier but subsequentlythe increase in cellularity proceeded more slowly (figure 4 (A)).

(d) Effectiveness of shielded bone-marrow in bone-marrow transplantation experiments

The capacity of shielded bone-marrow to restore haematopoiesis wascompared with the capacity of normal bone marrow by injecting it in lethallyirradiated mice and measuring its ability to keep the animals alive for 30 days.The bone marrow suspensions were prepared from marrow of the right tibiaof mice irradiated with 1050 R, while the right hind leg was shielded below theknee joint. The bone-marrow was taken within 15 min after irradiation.Control bone-marrow suspensions were made from corresponding marrowfrom sham-irradiated mice. Normal marrow appeared to be twice as effectiveas marrow from shielded legs (figure 5).

(a90no

ToM 70

dia

.U 50-E

0 30 -

D 10

0 5

U)1 -

(U

CL

0, -

120)X0(0) Q0(20)

. ........ I 10

2 4 6 810 20

number of certs injected (x10 4 )

Figure 5. Survival of lethally irradiated mice (915 R), injected with normal bone-marrow(x) or shielded bone-marrow ( ). Suspensions were prepared from the righttibia of sham irradiated mice or mice irradiated with 1050 R and the right hindleg below the knee joint shielded. The figures between brackets indicate the numberof mice which were used to determine each point.

Int J

Rad

iat B

iol D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y Fr

eie

Uni

vers

itaet

Ber

lin o

n 11

/04/

14Fo

r pe

rson

al u

se o

nly.

F. A. J. de Vries and 0. Vos

4. DiscussionOn the basis of the number of cells involved bone-marrow inoculation

appeared to be more effective than bone-marrow shielding in protecting againstradiation damage. Kurnick (1962) argued that shielding of small volumes ofmarrow in man did not provide re-population of the irradiated marrow. Thusour data obtained with mice are not inconsistent with the data obtained in man.The same number of nucleated cells afforded a better survival, a larger numberof spleen colonies and a more rapid re-population of the haematopoietic tissueswhen inoculated than when shielded in the irradiated animal.

Since the shielded area was sometimes small, irradiation due to side scattercould not be fully excluded. Side-scatter might be responsible for a differencein the effect of shielded and injected bone-marrow. No physical determinationswere made but the biological findings showed that the efficiency of shieldedmarrow was not smaller than half that of normal marrow (figure 5). A decreasein efficiency of the shielded bone-marrow by emigration of stem cells does notseem likely, because the mice were killed within a few minutes after irradiation.Therefore we assume that the difference is caused by scattered radiation.Returning to the possibilities for protection against irradiation, we shouldpoint out that even if we account for the factor of scattered radiation, injectionof bone-marrow is still more than 100 times more effective in protecting againstirradiation than is shielding of bone-marrow.

Thus migration of stem cells to irradiated parts of the haematopoietictissues seems to occur less effectively from intact shielded marrow than frombone-marrow injected into the blood stream.

According to Hanks (1964), almost two colony-forming units (CFU) migratefrom a shielded thigh to the spleen in every hour after irradiation. He investigatedthis migration only for seven hours after irradiation. In our experiments wefound that after shielding of a tibia only eight colonies are formed in the spleenat eight days after irradiation. The possibility for migration is left intact during alleight days. If the migration of cells from the tibia may be compared with themigration from the femur, this would mean that per 1 x 106 shielded bone-marrow cells, Hanks found a migration of 0-6 CFU to the spleen during thefirst seven hours, whereas we found a value of 0:5 CFU during the first eight days.At first sight these results seem to be rather discrepant. However a closerstudy of the findings reveals that our data may not be inconsistent with Hank'sfindings. Migration of stem cells to the spleen is only scored as CFU's if thesize of the colonies they form is great enough to be recognized macroscopically.Cells which migrate late may form colonies that remain unnoticed. The fact thatafter bone-marrow shielding we found a variation between very small andlarger colonies but that after bone-marrow injection only the larger type wasfound may indicate that the time of migration is important. A possibleexplanation for the apparant discrepancy between Hank's and our resultsmay also be that the more easily migratory cells are transported during thefirst hours and that the migration decreases after that period. The data fromHanks are certainly not contradictory to this assumption. The observationof more and less easily migratory cells in the spleen was described by Kurnickand Nokay (1965). If the spleen was ' irrigated ' with saline the first washingcontained more stem cells than the second washing. If such more easilymigratory cells also exist in bone-marrow and are transported during the first

242

Int J

Rad

iat B

iol D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y Fr

eie

Uni

vers

itaet

Ber

lin o

n 11

/04/

14Fo

r pe

rson

al u

se o

nly.

Prevention of the bone-marrow syndrome

hours after irradiation a reduction in the number of stem cells migrating tothe spleen must be expected afterwards. Robinson, Commerford and Bateman(1965) calculated an outflow of per cent per hour of the original stem cellcontent from the shielded marrow of the tail, during the first 35 hours afterirradiation. Our data certainly lead to lower figures for the bone-marrowin the lower extremities.

Finally we would like to discuss a possible relationship between the pro-portion of the shielded area and the speed with which it is re-populated. Aftera local irradiation of human bone-marrow with high doses a hypocellularityof long duration has been described (Goswitz, Andrews and Kniseley 1963).The question arises whether re-population of irradiated bone-marrow occursin man more slowly than in the mouse or whether the speed of the re-populationdepends on the relative magnitude of the area irradiated. We studied thisproblem in mice by irradiating the right hind leg and shielding the rest of thebody. In this situation the re-population appeared to start at an earlier time(figure 4 (A)), but afterwards the increase in cellularity occurred more slowlythan in mice in which a larger proportion of the bone-marrow was irradiated.The explanation for this observation might be the following: the re-populationstarts earlier because more unirradiated stem cells are available for migrationto the irradiated area, however, the stimulus for a rapid proliferation of thesecells may be missing, since the demand for mature cells will be hardly largerthan normal. Thus the finding in man of a prolonged hypoplasia of locallyirradiated bone-marrow may depend on the size of the irradiated area.

L'efficacit6 sauver des souris irradiees de la mort hematopoietique par la protectionde la moelle osseuse avec une lame de plomb et par l'injection des suspensions de celluleshematopoietiques furent compares. Pour valuer cette efficacity, trois criterium furentemployes: (a) la survie des souris irradees a 30 jours apres l'irradiation; (b) la repopulationdes tissus h6matopoietiques irradi6s et (c) la fr6quence des colonies spleniques.

Base sur le nombre des cellules, l'injection de la molle osseuse parait environ 100 foisplus efficace que la protection de la molle osseuse in situ.

Die Wirksamkeit der Abschirmung des Knochenmarkes und intraven6ser Injektionenvon Knochenmarkzellsuspensionen als Schutz gegen das Knochenmarksyndrom beibestrahlten Mausen wurde verglichen. Drei Kriteria wurden zum Vergleich der Wirk-samkeit benitzt: (a) das Oberleben der bestrahlten Miuse 30 Tage nach der Bestrahlung;(b) die Repopulation des bestrahlten hmopoietischen Gewebes; (c) die Zahl von Milz-kolonien.

In Bezug auf die Anzahl der Ben6tigten hamopoietischen Zellen zeigte die Injektionsich etwa 100 x effektiever wie die Abschirmung des Knochenmarkes.

REFERENCES

GOSWITZ, F. A., ANDREWS, G. A., and KNISELEY, R. M., 1963, Blood, 21, 605.HANKS, G. A., 1964, Nature, Lond., 203, 1393.HELLMAN, S., 1965, Nature, Lond., 205, 100.JACOBSON, L. O., 1952, Cancer Res., 12, 315.JACOBSON, L. O., SIMMONS, E. L., MARKS, E. K., GASTON, E. O., ROBSON, M. J., and

ELDREDGE, J. H., 1951, . Lab. dclin. Med., 37, 683.KURNICK, N. B., 1962, Transfusion, 2, 178.KuRNICK, N. B., and NOKAY, M., 1964, Ann. N.Y. Acad. Sci., 114, 528; 1965, Radiat.

Res., 25, 53.PLAYFAIR, J. J. L., and COLE, L. J., 1964, Blood, 24, 655.ROBINSON, C. V., COMMERFORD, S. L., and BATEMAN, J. L., 1965, Radiat. Res., 25, 234.Vos, O., and WEYZEN, W. W. H., 1962, Transplant. Bull., 30, 117.

243

Int J

Rad

iat B

iol D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y Fr

eie

Uni

vers

itaet

Ber

lin o

n 11

/04/

14Fo

r pe

rson

al u

se o

nly.