Hematological effects in dogs after sequential irradiation of the upper and lower part of the body with single myeloablative doses

Radiotherapy Elsevier

and Oncology,

14 (1989) 247-259

247

RTO 005491

Hematological effects in dogs after sequential irradiation of the upper and lower part of the body with single myeloablative doses * Wilhelm Nothdurft,

Klaus Baltschukat

and Theodor

M. Fliedner

Institute of Occupational and Social Medicine, University of Ulm. Ulm, F.R.G.

(Received 9 December

Key words: Sequential

1987, revision received 3 May 1988, accepted

hemibody irradiation;

15 September

Dog; Single doses; Hematological

1988)

effects

Summary

The compensating mechanisms determining the tolerance of the hemopoietic system to sequential hemibody irradiation (HBI) with large single doses, the regeneration of the irradiated bone marrow and the long-term effects of such treatment were studied in dogs. The main emphasis was laid on the determination of the granulocyte/macrophage progenitor cells (GM-CFC) in the bone marrow and blood. The general pattern of events in the GM-CFC compartment after each exposure was similar. Irradiation with a dose of 11.7 Gy of the upper body (UBI), that involved the abrogation of approximately 70% of the total active marrow, was followed by an immediate increase in the proliferation and differentiation of GM-CFC in the protected bone marrow. Repopulation of the GM-CFC in the irradiated sites most probably due to seeding of hemopoietic cells from the protected marrow already became evident at day 7 after UBI. At day 56 after UBI, when the irradiation of the lower body (LBI) was performed, the GM-CFC had recovered to between 30 and 40% of their pre-treatment values. Despite this incomplete regeneration, the GM-CFC compartment responded to LB1 in a similar way as the GM-CFC had in the protected (normal) marrow after UBI, i.e. by an increased proliferation for at least 21 days. Already at day 7, the bone marrow of the iliac crest that had been exposed to LB1 showed a considerable number of GM-CFC. Within no more than 370 days all the bone marrow sites irradiated during either the first or the second treatment had regained their normal GM-CFC values.

* Part of a paper presented at the European Symposium 1987, Half Body and Total Body Irradiation, Dresden, G.D.R., September 30-October 3, 1987. Address for correspondence: Dr. Wilhelm Nothdurft, PhD, Institute of Occupational and Social Medicine, University of Ulm, D-7900 Ulm, F.R.G. 0167-8140/89/$03.50

0 1989 Elsevier Science Publishers

Introduction

Hemibody irradiation (HBI) applied as a “systemic” therapy to the treatment of disseminated cancer [ 10,11,27,3 1, for review see [26]] neces-

B.V. (Biomedical

Division)

248 sarily involves considerable damage to large fractions of the bone marrow organ, depending upon which half of the body is treated, what the radiation dose is and, what fractionation schedule is used, if any. Irradiation of the upper half of the body (UHBI) comprising a field from the level of the umbilicus to above the scalp with single doses from 6 to 8 Gy or even 10 Gy, has been shown in most cases to cause only a moderate and transient depression of the blood cell counts (with the exception of the lymphocytes), i.e. proved to be hematologically safe [ 10,14,26,27]. The changes observed in the blood cell counts after treatment of the lower halfof the body (LHBI) with the same doses, including a field from the level of the iliac crest to the ankles, generally are not much different from those reported for UHBI [ 14,271. Sequential HBI allows the treatment of the whole body with total doses exceeding by far those that can be tolerated hematologically if given as total body irradiation in single or multiple fractions. However, the changes in the blood cell (granulocytes and/or thrombocytes) counts caused by the second irradiation (generally the LHBI) are critical if the interval between the two exposures is too short. The depression of the blood cell concentration after sequential HBI has been found in most cases to be clinically subcritical when the treatment of the second half of the body is not performed until the counts have reattained at least the lower ranges of their pretreatment levels, i.e. between 4 and 6 weeks or up to 8 weeks after the first exposure [6,10,15,26]. However, these empirical findings as well as the hematological complications after HBI in combination with chemotherapy [26] can only be understood and explained on the basis of detailed knowledge about the response of the bone marrow organ to such intensive radiotherapy. The data available so far from patients treated with HBI are quite fragmentary [27]. Therefore, we have performed experiments with dogs in which the response of the hemopoietic system to sequential irradiation of large parts of the body (i.e. “quasi” hemibody irradiation) with single doses of 11.7 Gy was

studied in detail. In these experiments, the main emphasis was laid upon a comprehensive analysis of the radiation-induced alterations in the granulocyte/macrophage progenitor cell compartment in the bone marrow and blood. The studies are based on previous experiments in which the hematological effects of the irradiation of the upper body (UBI) or lower body (LBI) were examined up to one year after exposure [ 1,251. In the present studies UBI and LB1 were given sequentially, separated by an interval of 56 days.

Material and methods Dogs The experiments were performed with three beagles between 12 and 17 months of age and weighing between 13 and 16 kg at the time of irradiation. Experimental design Sequential UBI and LB1 were performed with an interval of 56 days between the two treatments. This interval was applied because 56 days after UBI alone, the hemopoietic function was quite stable [ 251. The hematological examinations were performed up to one year after LB1 and included determinations of the blood cell counts (3 x per week), the assessment of the GM-CFC concentration in the blood and bone marrow and their S-phase fraction, and cell size distribution profiles in the regenerating bone marrow. Irradiations UBI and LBI The conditions for UBI and LB1 were the same as described in detail in the preceding papers [ 1,251. In brief, the irradiations were performed under the standard conditions of our laboratory [24] with 300 kV X-rays (12 mA, HVL = 4 mm Cu) at a dose rate of 6.5 cGy/min measured at the midline of the body. UBI included the larger field from the tip of the nose to a level at the caudal end of the fourth lumbar vertebra including the forelegs; LB1 involved the remaining smaller part

249 UBI

LBI

f

f 56 days

L

Fig. 1. Exposure conditions for sequential irradiation of the upper body (UBI) and the lower body (LBI). The lead box used for protection is represented by the thick black lines.

of the body, including the hind legs and the tail (Fig. 1). The total dose of 11.7 Gy was given to each part of the body by sequential bilateral exposure within 3 h.

Collection of blood and bone marrow

A 7-ml sample of peripheral blood (p.b.) anticoagulated with 20 U/ml heparin was obtained for performing the blood cell counts including the smears for the determinations of the differentials and for the assay of the GM-CFC. Bone marrow (b.m.) was aspirated from the iliac crests, the scapulae, the anterior head of the humeri and the sternal bones representing different irradiated or/and protected sites.

Rib biopsies

The techniques applied to obtain defined b.m. specimens from biopsies of the rib for the determinations of the b.m. cellularity and absolute GM-CFC numbers have been described in detail elsewhere [ 251.

Assessment marrow

of GM-CFC

in the blood and bone

Quantitative determinations of the GM-CFC in the blood and b.m. were performed by means of the standardized agar culture technique using serum from total body irradiated dogs as a source of CSA and in b.m. cell cultures additionally irradiated p.b. leukocytes ‘to achieve optimal growth conditions [ 2 1,22,24].

S-phase fraction of bone marrow GM-CFC

The S-phase fraction among the b.m. GM-CFC population was determined by exposing the cell suspensions prior to plating to the S-phase specific drug cytosine-arabinoside (Ara-C, Sigma, St.Louis, U.S.A.) [7], as described in the preceding paper [ 11. In short, b.m. cell suspensions of 2 x lo6 mononuclear cells per ml were incubated with and without Ara-C, 1.6 x 10mm4M. The S-phase fraction of b.m. GM-CFC was estimated from the difference between the numbers of colonies in the control cultures and those in the cultures with Ara-C treated cells. Cell size of GM-CFC marrow

from

regenerating

bone

Following perturbations of the b.m. after total body irradiation an overall increase in cell size has been found, which persisted in the regenerating GM-CFC compartment [ 13,19,34]. To establish whether such changes also occur in the regenerating b.m. after partial body irradiation the cell size distribution profiles were determined for the GM-CFC obtained from the partially repopulated b.m. of the humerus at day 36 after UBI. The cell size analysis of normal and regenerating GMCFC was performed by means of velocity sedimentation separation [ 121. Presentation of the data

If not stated otherwise, data are presented as mean values of:S.E.M. Smoothed curves were fitted to the data obtained from the velocity sedimentation separations for the single fractions of GM-CFC according to the method of Tukey [ 3 11.

250 Results

again between day 6 and 7, with the nadir at day 10, when the counts had decreased to 54% of the value before LBI. About day 21, the thrombocyte counts were back in the normal range and remained there despite some fluctuations up to day 380 after the second exposure.

Clinical observations

There was no sign of bleeding or hemorrhage at the time of maximum thrombocyte depression after either UBI or LBI. However, in contrast to the previous studies [25] there was an initial rise in the body temperature between day 7 and day 10, from the normal range of 38.3”C-38.8”C to 39.7”C in two dogs and 38.9”C in the third, and another rise between day 17 and day 27 to 39.1”C-39.9”C in all three dogs.

Lymphocytes

The maximum depression in the lymphocyte concentration occurred within the first 24 h after UBI when it dropped to 8 y0 of the average initial value (Fig. 3). Thereafter, it continuously increased, to a level of 50% of normal at day 55. LB1 again caused a rapid depression in the lymphocyte concentration within 24 h, to approximately 36% of the average value before LBI. The recovery within the following 15 days was faster than after the first exposure (UBI) and the counts were back in the range of those measured before LB1 at about day 35. However, the lymphocyte counts did not completely recover within the next 100 days or even up to day 360, i.e. they were approximately 20% lower than before treatment.

Thrombocytes

Following UBI, the thrombocytes showed a rapid decrease in the period between day 6 and day 13, when the counts reached their nadir (Fig. 2). Thereafter, a fast recovery occurred and the values were back in the normal range at day 28 at least. The thrombocyte concentration remained in the normal range up to day 55, i.e. the day before LB1 was performed. LB1 caused a rapid decrease

OJ

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Fig. 2. Changes in the thrombocyte

concentration

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20

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40

I11171

60

130 360

Irradiation

in the blood of dogs after sequential irradiation the lower body (LBI).

360

. of the upper body (UBI) and>

251 10

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, 20

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Days after

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380

Irradiation

Fig. 3. Changes in the lymphocyte concentration (Ly, 0 - - - - 0), and the granulocyte concentration (Gra, 0 -0) blood of dogs after sequential irradiation of the upper body (UBI) and the lower body (LBI).

Neutrophilic granulocytes After UBI, the neutrophilic granulocytes (segmented and band forms) first showed a slight drop within the first 3 days that was followed by a rapid decrease in the period up to day 7, when the counts reached the nadir at 29% of the pretreatment average values (Fig. 3). So far these changes were in accordance with those predicted on the basis of the results obtained after UBI in our previous studies [ 251. However, in all three dogs in the present experiment, the neutrophils showed a rapid but transient increase following the nadir in the period up to day 15, and a further rise above the initial values in the period up to day 31. In contrast, in the animals which in the previous experiments had received UBI [25], the neutrophils steadily rose between day 10 and day 22, at which point they had reattained slightly subnormal levels. However, the secondary decrease observed beyond day 30 in the present studies (Fig. 3) is quite similar to that found in the previous experi-

in the

ments after UBI [25]. Consequently, the neutrophils were subnormal (70% of the initial pretreatment values) when LB1 was performed. LB1 caused a moderate decrease in the granulocyte concentration, with the nadir at day 6, when it had dropped to 64% of the value measured the days before LBI. Thereafter a continuous increase took place to a slightly subnormal level between day 40 and day 60. At least 130 days after LB1 the granulocyte counts had returned to the normal pre-irradiation level. GM-CFC in the blood The GM-CFC per ml blood had decreased already at day 1 after UBI to extremely low numbers: 0.5 % of the average normal values (Fig. 4). In accordance with the results from the previous studies on UBI [25], there was a slight increase in the first 7 days. In general, the transient increase in the GM-CFC, commencing between day 7 to day 10, exhibiting a certain dip about day 20 and lasting up to day 34 resembled the pattern

252 LBI

300

J

if

200 z ,o m z ; P (0 100 7 0

0

-I

40

0 20 40 Days after Irradiation

60

130 360

380

Fig. 4. Changes in the concentration of granulocyte/macrophage progenitor cells (GM-CFC) in the blood of dogs after sequential irradiation of the upper body (UBI) and the lower body (LBI).

observed in the preceding UBI studies [ 251. However, the individual peak values in the GM-CFC numbers between day 10 and day 17 were much higher in the present experiments when compared to the former. The GM-CFC were clearly subnormal in the interval from day 36 to the day before LB1 was performed. LB1 caused an immediate decrease to about 10% of the average level before the exposure. Again a transient increase took place from day 10 to day 38 with an intermediate dip at day 21, followed by another depression after day 40. At about day 130 and one year after LBI, the GM-CFC had increased but were found clearly below the normal pre-treatment level. GM-CFC in aspirates from the bone marrow UBI caused a moderate decrease in the GM-CFC concentration in the protected b.m. of the iliac crest at day 1 that was followed by a transient return to the normal or slightly supranormal levels within the next 21 days (Fig. 5). A second decrease was found between day 36 and day 48.

In the irradiated b.m. sites, the GM-CFC had dropped to undetectable numbers on the first day after UBI. Already at day 7 small but significant numbers of GM-CFC were present in both the b.m. of the humerus and the scapula. The GM-CFC regeneration curve for the b.m. from the scapula showed a rapid increase in the period up to day 2 1 and a plateauing thereafter up to day 48 (Fig. 5). This pattern is identical to that established for UBI in previous studies [ 251, and is also found for the sternum at day 36 and day 48. For both sites the GM-CFC numbers were clearly subnormal at this latter period in time. The extremely fast increase in the GM-CFC numbers of the humerus in the interval from day 7 to day 21 to approximately 80 % of the preirradiation value is clearly out of line with the previously observed moderate rise to 43% [25]. Interestingly, the GM-CFC concentration then decreased progressively, and at day 48 had returned to the level predicted on the basis of previous results, i.e. 36% of the initial value. LB1 was followed by a similar GM-CFC re-

253

,f I I

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protected ??iliac crest

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protected o humerus 0 scapula v sternum

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20

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0 Days after

I1

20

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I-60

130 360

360

Irradiation

Fig. 5. Changes in the concentration of GM-CFC in four different bone marrow sites of dogs after sequential irradiation of the upper body (UBI) and the lower body (LBI). The bone marrow sites protected or irradiated during UBI or LB1 are indicated in the upper part of the figure.

sponse in the protected as well as in the irradiated b.m. sites as observed after UBI, except for certain quantitative differences. As seen in Fig. 5 none of the protected (but pre-irradiated) b.m. sites, i.e. the humerus, the scapula and the sternum, exhibited a drop in the GM-CFC numbers. In the period up to day 21, the GM-CFC concentration increased in all the three sites, just as observed in the protected b.m. of the iliac crest after UBI. Again, a second decrease occurred at day 36 and day 54, more pronounced in the b.m. of the scapula and sternum than of the humerus. The GM-CFC values in the humerus had reattained their normal levels by day 135, whereas the GM-CFC concentration in the scapula and the sternum was still subnormal at day 135, but clearly back in the normal range one year after LBI. The GM-CFC in the irradiated b.m. of the iliac crest were found decreased to undetectable numbers on the first day after LBI. The recovery curve was quite similar to that obtained for GM-CFC in

the irradiated b.m. of the scapula after UBI with significant GM-CFC numbers at day 7, an increase in the period up to day 21 and a subsequent plateauing at at least 30% of pre-treatment values until day 54. At day 135 the GM-CFC concentration had recovered to approximately 72% of the normal, and was found quite normal at day 380. Fraction of bone marrow GM-CFC

in S-phase

After each exposure, the S-phase fraction of the b.m. GM-CFC showed the same response pattern in protected and irradiated sites (Fig. 6): the values were at first strongly increased, alter which they continuously descended. After UBI, the S-phase fraction in the protected b.m. of the iliac crest was found increased from day 1 up to at least day 21, whereas in the irradiated regenerating b.m. of the humerus it remained increased up to day 48. LB1 again caused a strong but transient increase in the S-phase fraction in the protected (but

254 LBI

UBI o humerus

f I I

60 -

iliac crest

??

protected ??iliac crest

I I

f

protected o humerus

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0 Days after

,

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130

Irradiation

Fig. 6. Changes in the S-phase fraction of GM-CFC in the protected bone marrow and the irradiated bone marrow of dogs in the course of sequential irradiation of the upper body (UBI) and the lower body (LBI). The bone marrow sites protected or irradiated during UBI or LB1 are indicated at the top of the figure.

pre-irradiated and partially regenerated) b.m. of the humerus at day 7 and day 21, followed by slightly elevated levels up to day 54. In the irradiated b.m. of the iliac crest the S-phase fraction remained slightly elevated at least up to day 135 after LBI. Rib biopsy material Rib biopsies were performed at three different times after LBI. The results of the studies are presented in Table I. The absolute cell numbers (“cellularity”) per 1 cm rib specimen were found quite normal at day 110, day 191 and day 435 after UBI, i.e. the exposure of the respective sites. Cell size of regenerating GM-CFC after UBI The average velocity sedimentation profile derived from the pooled data for GM-CFC in the normal marrow of the humerus before exposure was uniform with a velocity sedimentation rate of 5.4 mm/h for the maximum of the GM-CFC (Fig. 7). The profile obtained from the pooled

data for regenerating GM-CFC in the irradiated humerus at day 36 after UBI showed a slight shift to the right at lower sedimentation rates, and some broadening of the peak region. This was associated with some extension of the profile into the direction of faster sedimentation rates up to a value of approximately 8.2 mm/h. However, overall the two profiles were not much different from each other.

TABLE I Total cell number ( x 10 - “) in the bone marrow of the rib as determined in 1 cm specimens”. Before irradiation

29.2 k 1.1

Days after UBI 110 (54)b

191 (135)

435 (379)

33.1 + 4.3

37.3 & 10.5

32.4 + 7.0

a Mean values f 1 S.E.M. b Numbers in parentheses = days after LBI.

255

Sedimentation

Velocity

(mm/hr)

Fig. 7. Velocity sedimentation profiles of GM-CFC in the bone marrow of the humerus under normal conditions and during regeneration at day 36 after irradiation ( -) of the upper body (- - - - ). Each profile was obtained from the pooled data from three dogs. The arrows on the abscissa indicate the sedimentation rate for the maximum of normal GM-CFC (N) and for the GM-CFC forming the extended maximum in the regenerating bone marrow (R).

Discussion UHBI in man involves the irradiation of some 57-64% of the total active bone marrow mass [ 8,25,29, data based on Ellis [ 911; quite normal blood cell counts are reported for the period between 6 and 8 weeks after UHBI [27]. The return of the granulocytes and platelets back into the normal range after either UHBI or LHBI is taken as an indication that the irradiated b.m. sites in total have recovered to such an extent that the second treatment given to the other half of the body will be tolerated again. However, it has to be considered that due to the specific functional organization of the hemopoietic system as a whole, it is the unexposed (protected) b.m. that compensates with immediate hyperactivity for the loss of large parts of the organ, as shown in experiments with mice and rabbits after partial-body or local irradiation [2,28,30]. Increased cell proliferation and even hyperplasia in the protected marrow, are to a great extent responsible for the recovery of the blood cell counts in the first few weeks after irradiation.

As a consequence, if the time interval between the first and the second treatment is too short, the marrow irradiated initially will not have recovered sufficiently to allow for an effective compensatory response and thus hematological complications will occur [lo]. The few b.m. examinations performed so far in man after HBI appear to show regeneration within 6 weeks and serial b.m. scans have failed to detect persistent damage 6-8 weeks after exposure [ 271. The results of the present studies and of the previous experiments [ 251 reveal some interesting aspects about how the b.m. irradiated during the first exposure, i.e. the UBI, regenerates and is able to compensate for the damage done to the bone marrow exposed at LBI. Bone marrow regeneration in sites exposed during UBI The results clearly show that for a dog, an UBI involving the irradiation of 72% of the total b.m. mass with a dose of 11.7 Gy allows for only a partial regeneration of the exposed marrow even within 40 and 80 days after treatment. The GM-CFC concentration in representative marrow sites (humerus, scapula, sternum, ribs) had returned to between 30 and 40% of the normal values, and the cellularity of the bone marrow of the rib was found to have recovered to 80 % [ 251. As also becomes evident from the present studies and the previous experiments [ 251 it needs approximately 3 weeks for the GM-CFC to achieve the above-mentioned degree of restoration. Thus it can be concluded that were the second treatment, i.e. LB1 given within 14 to 21 days after UBI, the compartment size of the GM-CFC, and probably also of the stem cells and other progenitor cells, would still be too small to compensate effectively for the damage caused by the second treatment to the b.m. in the other half of the body. According to clinical observation [ 10,15,27] a comparable situation can be assumed for sequential HBI of patients, though there may be certain differences depending on the irradiation conditions (dose and dose rate) and specific cyto-

256

kinetic parameters in man and the dog. UHBI in man involves sparing of some 40% of the b.m. in the lower body, in contrast to 27-28% in the dog. Thus there may be some differences in the seeding rate of stem cells from the protected marrow to the irradiated sites in favour of the human as discussed in the preceding paper [25]. Interestingly, in the present study, the GMCFC in the irradiated marrow of the humerus exhibited an enormous but transient increase between day 7 and day 21 after UBI, in contrast to previous experiments [25]. The reason for this difference observed only in the humeral marrow is not clear. It may be related to the events leading to the rise in the body temperature after irradiation, which was only observed in the present study, but not in the previous one [ 251. However, it is important to notice that after day 21, the GM-CFC in the humerus marrow declined again and stabilized at the same level as the GM-CFC in the scapula and the sternum, as had been predicted from previous experiments [25]. The prolonged recovery of the b.m. in the irradiated sites observed after day 21 of both UBI in the present and in previous studies [25] and LB1 [l] is obviously more or less independent of the respective volumes of protected and irradiated b.m. at the dose level of 11.7 Gy if the volumes exposed are not too different from each other (i.e. whether it is 28% protected and 72% irradiated or vice versa). This indicates that there may be some damage to the hemopoietic supportive stroma; this would be in agreement with findings obtained from rodents after irradiation of parts of their body with single doses of 10 Gy and up to 20 Gy [ 16,18,20,33]. In patients, the somewhat smaller radiation doses used for HBI may cause less damage to the stroma. On the other hand, the cell size distribution profile of the regenerating GM-CFC in the humerus marrow as determined at day 36 after UBI showed only small differences in comparison to the normal state. There was only a relative increase in the fraction of larger cells, which can mainly be attributed to the enhanced cycling at that time (cf. Fig. 6). However, the alterations in

the profile are quite small when compared to those observed in regenerating GM-CFC populations of mice and dogs after total body irradiation with doses in the range up to several Grays [ 13,191. Thus the regenerating GM-CFC in the partial body irradiated dogs, originating from a non-irradiated hemopoietic cell population, obviously shows no such pathophysiological alterations apart from an enhanced turnover. Compensatory response of bone marrow protected during LBI The GM-CFC compartment in the protected b.m.

sites of the upper body (humerus, scapula, sternum), though only partially regenerated from UBI, was able to respond immediately in a quite effective way to the damage caused by LB1 to the b.m. located in the lower half of the body: first, there was a strong increase in the proliferation of the GM-CFC that caused a remarkable rise in the GM-CFC numbers of the protected b.m. sites, i.e. the humerus, the scapula and the sternum. Second, there was no initial drop (“dip”) in the GMCFC numbers of the protected sites at day 1 after LB1 (as the second treatment) in contrast to the GM-CFC in the protected normal b.m. in the iliac crest after UBI [25] or in the humerus and scapula after LB1 [ 11. This indicates that in the partially regenerated b.m. sites there might have been an enhanced influx of pluripotent stem cells into the GM-CFC compartment as observed in rodents several weeks after partial body irradiation [4,30]. In general, the compensatory response pattern of the GM-CFC in the b.m. sites of the upper body after LB1 (given as the second treatment) is rather similar to that observed in the protected marrow of the iliac crest after UBI (cf. Figs. 5 and 6). This shows that the partially regenerated marrow in the 11.7 Gy irradiated stroma is able to compensate for the loss of large fractions of the total b.m. mass in a similar way as normal b.m. Blood granulocyte changes in relation to bone marrow function

At the time LB1 was performed in the present experiments, i.e. at day 56 after UBI the blood

257 granulocyte values were slightly subnormal in accordance to previous studies [25]. These findings are in accordance with the clearly subnormal GM-CFC numbers (less than 50%) found in the irradiated sites before LB1 was performed. On the other hand, the moderate decrease in the blood granulocyte concentration after LB1 clearly indicates that the partially regenerated b.m. in the upper body of the dogs (i.e. 72% of the total b.m. mass in normal animals) is able to effectively compensate for the abrogation of the 28 % of the b.m. in the lower body which comprises more than 30% of the total activity at the time of the irradiation. Consequently, the acute blood cell alterations (granulocytes, thrombocytes) do not become critical after the second treatment. However, in dogs that had already been treated with UBI, the LB1 caused a stronger depression of the granulocytes and the platelets when related to the values measured during the last days before LB1 (64 and 54x, respectively) than in dogs which had received LB1 as the only treatment (73 and 80%) respectively) [ 11. These findings among others indicate that at the time LB1 was performed, the marrow had not completely recovered from UBI. Long-term regeneration tected during LBI

of the bone marrow pro-

When compared to animals treated with UBI alone [25] the regeneration of the GM-CFC population (measured as GM-CFC/ 105, b.m. MNC) in the humerus, the scapula and the sternum in the animals receiving sequential HBI was not much different up to day 55 after LBI; i.e. it was delayed in the same way at least up to day 111 after UBI. As predicted from the previous experiments [25] the GM-CFC in the humerus showed the fastest regeneration. At least 420 days after treatment with UBI (360 days after LBI) the GM-CFC values were quite normal in all the sites. However, the findings obtained from the rib biopsies indicate that after UBI the b.m. cellularity may recover faster in dogs which received additional LB1 than in those treated with UBI alone [25]. However, it is an open question, whether

such findings can be generalized for all the other bones. The data obtained from murine experiments on sequential HBI do not contribute to the solution of this problem because the regeneration in the irradiated b.m. sites is quite fast after each exposure and because of the involvement of the spleen

[21* Bone marrow regeneration in sites exposed during LBI

Another important aspect that has to be considered in the context of sequential HBI is the repopulation and regeneration events in the infield irradiated b.m. sites that were irradiated during the second exposure, i.e. LB1 in the present studies. As has become evident from the discussion before, these processes in the irradiated marrow are of minor influence on the blood cell changes in the first few weeks and thus for the acute tolerance of the hemopoietic system to HBI. However, they will become quite important for hemopoietic tolerance at later times, if other hematotoxic treatments (i.e. local radiotherapy, chemotherapy) have to be performed after HBI. The dose of 11.7 Gy as applied in the present studies will reduce the resident hemopoietic progenitor and stem cells to about 10 - 8 if the survival D, = 0.6 Gy and n = 1 as curve parameters measured for the canine b.m. GM-CFC in vitro [23] are applied to the in vivo situation. Consequently, at these low probabilities, the reconstitution of the highly irradiated b.m. sites within the first weeks will depend mainly on the seeding of circulating hemopoietic cells from the protected marrow and their repopulating capacity (see [ 251 for a more detailed discussion), though their importance for later reconstitution is a matter of debate [28]. In context with sequential HBI, it has to be considered that the stem cells to be seeded to the irradiated marrow after the second exposure, i.e. LBI, originate from a partially regenerated but not normal marrow. The GM-CFC determined in the blood may allow some insight into the kinetics of the circulating hemopoietic cells after sequential HBI

258 and their relation to the regeneration events in the bm. AS discussed in previous papers where either UBI or LB1 was given as the only treatments [ 1,251 there was a clear relationship between the compensatory response of the GM-CFC compartment in the protected marrow and the GMCFC changes in the blood on the one hand and the repopulation and the regeneration kinetics in the irradiated b.m. on the other. The GM-CFC in the blood were clearly subnormal (at 30% of the normal value) 56 days after UBI when LB1 was performed. This depression is to be related to the fact that at that time after UBI, 72% of the total normal b.m. volume has not reattained its normal functional state. However, despite this depression in the concentration of circulating GM-CFC there must have been a significant seeding of stem cells and probably progenitor cells to the irradiated b.m. of the iliac crest within the first few days since already at day 7 the GM-CFC concentration had recovered to 7% of the normal value. Since this is the same level as that observed in animals which had received LB1 alone [l] the initial seeding of hemopoietic cells from the partially regenerated b.m. is obviously the same as from normal b.m. Data obtained from mice so far have shown that even if only 10% of the total bone marrow are protected against large doses of radiation, there is a significant seeding of circulating pluripotent stem cells (CFU,) from the protected marrow to the spleen already within the first hours after exposure [ 5,171. This occurs even though the number of CFU, per ml is dramatically depressed in the interval between 15 min and at least 24 h after the exposure [3]. The further restoration of the GM-CFC compartment in the irradiated b.m. beyond day 7 after LBI, as measured in the iliac crest, in principle showed the same pattern as observed in the same animals in the irradiated b.m. sites after UBI, i.e. in the humerus, scapula and sternum, and in the previous studies [ 251. The pattern of regeneration is at least similar to that observed for the GMCFC in the b.m. of the iliac crest in animals that received LB1 alone but no prior UBI [ 11. This general pattern is characterized by a relatively

rapid increase in the GM-CFC concentration in the interval up to day 21 followed by a plateau in the interval up to day 80 at least [25]. The restoration of the GM-CFC in the iliac crest up to day 56 after LB1 is obviously slightly delayed in the animals that had received UBI when compared to dogs given LB1 only; the plateau was at a level of about 30% of the normal pre-treatment value in the former in comparison to between 40% to 50% of the latter [ 11. However, at day 135 after LB1 at least the GM-CFC values in the irradiated b.m. of the iliac crest had recovered to the same level (70%) in both groups of dogs indicating that at that time UBI given as the first treatment no longer had an effect. After about 360 days the GM-CFC were found only slightly below the normal values. Acknowledgements The authors are indebted to Mrs. I. Konig, Mrs. G. Baur, Mrs. M. Buchenscheit and Mrs. R. NieD for their careful technical assistance. The work was supported by the Commission of the European Communities under Contract BI-60061-l (B) and the Bundesminister fur Umwelt, Naturschutz und Reaktorsicherheit, Contract St.Sch. 744 and St.Sch. 1.029.

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