International Journal of Radiation Biology, 2014; Early Online: 1–9 © 2014 Informa UK, Ltd. ISSN 0955-3002 print / ISSN 1362-3095 online DOI: 10.3109/09553002.2014.899443
Modelling hematological parameters after total body irradiation Christopher Oelkrug1, Nadja Hilger1, Uta Schönfelder2, Johannes Boltze1,3, Ulrich Sack1,3,4, Christian Fricke5, Guido Hildebrandt6, Thomas Keller7, Frank Emmrich1,3,4 & Stephan Fricke1,3,4
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1Fraunhofer Institute for Cell Therapy and Immunology (IZI), Leipzig, Germany, 2Evangelic Diaconic Hospital,
Div. Oncology and Hematology, Leipzig, Germany, 3Translational Centre for Regenerative Medicine, Universität Leipzig, Leipzig, Germany, 4Institute of Clinical Immunology, Universität Leipzig, Leipzig, Germany, 5Ambulante Dienste LUPS, Luzern, Switzerland, 6Department of Radiation Oncology, University Hospital of Rostock, Rostock, Germany, and 7Acomed Statistik, Leipzig, Germany
Introduction
Abstract Purpose: The time- and dose-dependent reconstitution of hematopoiesis after radiation exposure is strongly related to the stem cell population and can be used to predict hematological parameters. These parameters allow further insight into the hematopoietic system and might lead to the development of novel stem cell transplantation models. Materials and methods: CD4-/- C57Bl/6 mice, transgenic for human CD4 and HLA-DR3, were irradiated in a single (3, 6, 8 and 12 Gy) and fractionated (6 1 Gy, 6 1.5 Gy, 6 2 Gy; twice daily) dose regimen. Blood was analyzed weekly for red blood cells (RBC), hemoglobin concentration (Hb), hematocrit (HCT) and white blood cells (WBC). Organ and tissue damage after irradiation were examined by histopathology. Results: The recovery curves for RBC, Hb, HCT and WBC showed the same velocity ( 1 week) for all radiation doses (3–12 Gy) starting at different, dose-dependent times. The only dosedependent parameter was defined by the beginning of the recovery process (dose-dependent shift) and higher doses were related to a later recovery of the hematopoietic system. The RBC, Hb and HCT recovery was followed by a saturation curve reaching a final concentration independent of the radiation dose. Histological analysis of the bone marrow in the single dose cohort showed a dose-dependent reduction of the cellularity in the bone marrow cavities. The fractioned radiation dose cohort resulted in a regeneration of all bone marrow cavities. Conclusion: Specific functions were developed to describe the reconstitution of hematological parameters after total body irradiation.
The main function of the hematopoietic system is to maintain different blood cell types at a constant level, which show different turnover kinetics as 1.2 1011 day for granulocytes, 2.0 1011 day for erythrocytes or 1.5 1011 day for platelets and are dependent on an unlimited replicative and pluripotent potential of the stem cells in the bone marrow (Fliedner et al. 2002a, 2002b). The hematopoietic system is sensitive to radiation damage, which often leads to radiation induced lethality (Zhou and Mi 2005). To further understand the radiation-induced pathophysiology of the hematopoietic system, it is necessary to investigate this system in special developed models. The time- and dose-dependent reconstitution of the hematopoietic system after radiation exposure plays a key role in the development of a stem cell transplantation model. The hematological parameters are able to further define the stem cell population and the resulting white blood cell population. Modelling these parameters allows further insight into the development of novel stem cell transplantation models and the prediction of reconstitution in irradiated subjects. The hematopoietic cell subsets show different degrees of radiation sensitivity and some sub-fractions of the stem cells seem to be radioresistant. In human stem cell transplantation after lethal irradiation, the participation of host derived hematopoietic cells is still a major problem which often causes graft rejection. Especially long-term reconstituting (LTR) stem cells play a key role in the generation of a functional stem cell transplantation model (Trevisan and Iscove 1995, Trevisan et al. 1996, Zhao et al. 2005). These LTR cells are estimated with 0.001–0.01% in the bone marrow
Keywords: Hematopoiesis, total body irradiation (TBI), transplantation, radiation
Correspondence: Christopher Oelkrug, Fraunhofer Institute for Cell Therapy and Immunology, Perlickstraße 01, 04103 Leipzig, Germany. Tel: 49 341 355 363121. Fax: 49 341 35536 9921. E-mail: [email protected] (Received 29 June 2013; revised 17 October 2013; accepted 21 February 2014)
1
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2 C. Oelkrug et al. and are normally at rest and radioresistant. Therefore, the reconstitution of hematopoiesis in lethally irradiated CD4-/- C57Bl/6 mice transgenic for human CD4 and HLA-DR3 (triple transgenic mice [TTG]) was investigated (Laub et al. 2000, 2001, 2002, Fricke et al. 2009, 2010, 2012, Fricke 2011). The TTG mice have a complete functional murine immune system which is modified with regard to CD4 and HLA-DR. In this setting the presence of human or murine CD4 after transplantation can be used as a marker for chimerism even in a syngeneic setting. This model was developed to observe the engraftment of allogeneic and syngeneic stem cells to induce murine CD4 expression after transplantation. In contrast to this, an endogenous recovery in hematopoiesis will lead to human CD4 and HLA-DR3 expression. To model the hematopoietic recovery, TTG mice were irradiated in a single (3, 6, 8 and 12 Gy) and fractionated (6 1 Gy, 6 1.5 Gy, 6 2 Gy; twice daily) dose regimen. Blood was analyzed weekly for red blood cells (RBC), hemoglobin concentration (Hb), hematocrit (HCT) and white blood cells (WBC). Organ or tissue damage due to the irradiation of the TTG mice was determined through histopathological examination. The analysis of the bone marrow in the single-dose cohort showed a dose-dependent reduction of the cellularity in the bone marrow cavities. The fractioned radiation dose cohort resulted in a regeneration of all bone marrow cavities. In addition, the described irradiation model could also be used to model radioprotective effects of substances on victims from radiation accidents.
Materials and methods Ethics statement All mice were housed, treated and handled in accordance with the guidelines of the University of Leipzig Animal Care Committee and the Regional Board of Animal Care for Leipzig (animal experiment registration number 24/06, 28/08, and 55/11).
Animals TTG are murine CD4 k/o mice expressing the human CD4 and HLA-DR3 molecules on a stable C57Bl/6 background as described previously (Fricke et al. 2009, 2010, 2012, Fricke 2011). The mice were fed ad libitum. All animals were maintained under standardized conditions in the Medical Experimental Centre of the University of Leipzig.
Table II. Fractionated-dose total body irradiation (TBI) experiment. Single dose [Gy] Time after Time [days] 0 6 8 13 20 27 34 41 48 55 Total
radiation
0.00
1.00
1.5
2
Total
0 2 7 14 21 28 35 42 49
4 4 4 4 4 4 4 4 4 4 40
4 4 4 4 4 4 4 4 4 4 40
4 4 4 4 4 4 4 4 4 1 37
4 4 4 4 3 3 3 3 3 3 34
16 16 16 16 15 15 15 15 15 12 151
Irradiation protocol For the total body irradiation (TBI) of the TTG mice, an X-ray apparatus (D3225, Orthovoltage, Gulmay Medical, Camberley, UK) was adjusted for radiation exposure in parallel for four animals in a Plexiglas container (divided in five spaces of 0.5 4.0 cm each). Initially a radiation doseresponse curve was developed. For determination of the lethal radiation dose, 16 mice underwent single TBI with radiation doses from 3–12 Gy (200 kV, dose rate 1.14 Gy/min). Four mice were used as sham-irradiated controls. To model the hematological parameters after TBI, mice were irradiated in a single (3, 6, 8 and 12 Gy) and fractionated (6 fractions; total dose 6, 9 and 12 Gy) dose regimen. The animals irradiated using a fractionated regimen were treated twice a day for three consecutive days with single doses of 1 Gy, 1.5 Gy, or 2 Gy up to total doses of 6 Gy, 9 Gy or 12 Gy, respectively. As shown in Tables I and II, four animals were treated per dose group (tables display time-point of blood collection [days], time after irradiation, dosage [Gy], [total number of animals in the experiment on the horizontal axis for each experimental day, the total number of animals tested during the experiment concerning each radiation dose on the vertical axis]). After day 2 (post irradiation), weekly measurements were performed (number of animals and valid measurements for single and fractionated dose experiments). Blood was analyzed weekly for red blood
Table I. Single-dose total body irradiation (TBI) experiment. Single dose [Gy] Time after Time [days] 0 6 8 13 20 27 34 41 48 55 Total
radiation 0 2 7 14 21 28 35 42 49
0.00 3.00 4 4 4 4 4 4 4 4 4 4 40
4 4 4 4 4 4 4 4 4 4 40
6.00
8.00
12.00
Total
4 4 4 4 4 4 4 4 4 4 40
4 4 4 4 1 0 0 0 0 0 17
4 4 4 2 0 0 0 0 0 0 14
20 20 20 18 13 12 12 12 12 12 151
Figure 1. Determination of the delivered radiation dose. To analyze the administered dose rate for following irradiation experi ments the absorbed dose was calculated in J/kg (Gy).
Figure 2. Non-linear regression models of function parameters function (dose, time) for Hb, HCT, RBC and WBC. Time was modelled using exponential decay and sigmoidal increase or decay. These functions are correlated to damage processes and growth processes. Models for time dependent cell recurrence in single-dose radiation Hb (A), HCT (B) and RBC (C); Fractioned dose radiation Hb (D), HCT (E), WBC (F) and RBC (G). Survival plot showing animal survival in the fractioned dose regimen. (G and H; supplementary data from Fricke et al. 2009). This Figure is reproduced in color in the online version of International Journal of Radiation Biology.
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Hematological parameters after TBI 3
4 C. Oelkrug et al. Table III. Red blood cells (RBC) (single dose). 95% Confidence Interval Parameter C1 T1 C2 t2 m2 k2 T2
Estimate
Std. Error
Lower bound
Upper bound
10.837 6.660 8.228 6.089 0.126 2.248 1.976
0.223 0.447 0.135 0.527 0.126 0.539 0.351
10.395 5.775 7.961 5.045 0.124 1.182 1.281
11.279 7.546 8.495 7.132 0.376 3.314 2.670
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cells (RBC), hemoglobin concentration (Hb), hematocrit (HCT) and white blood cells (WBC).
Hematological parameters of TTG
Blood (140 ml) was collected through heparinized capillaries (Greiner Biochemica, Flacht, Germany) by retroorbitally bleeding, followed by the administration of an antibiotic ophthalmic ointment. Hemoglobin concentration was determined using an Animal Blood Counter (SCIL, Viernheim, Germany), which had been calibrated for mouse blood, within 2 h after blood collection. To model the hematological parameters, murine blood was analyzed for RBC (red blood cells), Hb (hemoglobin concentration), HCT (hematocrit) and WBC (white blood cell count).
Histology Bone marrow, gut, heart and liver of all transgenic mice were analyzed histologically after the end of the irradiation experiments (day 55). Organs were prepared as described according to the protocol established by us previously (Fricke et al. 2010).
Statistical analysis All data are presented as means SD. Non-linear regression models for describing the time course of hematological parameters as a function of dose and time were developed by ACOMED statistics, Leipzig, Germany by using the SPSS 15.0 software package (SPSS Science, Erkrath, Germany). Statistical analysis and graphic presentation were made using Sigma Plot 10.0/ Sigma Stat 3.5 (Systat, Erkrath, Germany).
Results Determination of lethal radiation toxicity in TTG mice Initial to the total body irradiation (TBI) experiments, the administered dose rate was analyzed and the absorbed dose was calculated in J/kg (Gy), (Figure 1).
Comment Initial values time constant of decay Final values shift of recovery t2 m2.dosek2 time constant of recovery
To determine the lethal radiation dose in triple transgeneic mice for ongoing experiments, groups of four TTG mice underwent TBI with single doses of X-rays ranging from 0–12 Gy followed by analysis of survival, and recovery of peripheral red blood cells (RBC), hemoglobin concentration (Hb), hematocrit (HCT) and white blood cells (WBC). Single dose TBI of mice with doses 8 Gy led to death of all mice within 19 days, while mice irradiated with single doses 8 Gy survived (Figure 2A, 2B). The Kaplan-Meier plot for the survival of single dose irradiated mice is shown in Figure 2H. For the fractioned dose experiments with exception of one animal all survived, a survival analysis was not meaningful.
Non-linear regression model Non-linear regression models of function parameter function (dose, time) were developed. Time was modelled using two types of functions including the exponential decay: C.exp(-t/T), with C amplitude, T time constant describing velocity of change and the sigmoidal increase (or decay): C/(1 exp(-t/T)). Both functions are related to typical damage- and growth-processes, respectively. Additionally, time shifts were introduced by using t ti instead of t. This refers to a shift along the x-axis (time). Dose dependency was introduced as m.dosek-factors. If amplitude was dose-dependent, it was multiplied by this factor. If time-shift depended on dose, this factor was added to time-shift. Non-linear regression estimated parameters Ci, ti, Ti, m and k of these functions. A further proof of validity was to include dose 0 into the models. The resulting models should have correctly reflected behavior with dose 0. The investigated time-points were related to t 0 and t 6 days (initial radiation).
Single-dose exposure To establish a model which describes Hb, HCT and RBC (Figure 2A–C) reconstitution after single-dose irradiation,
Table IV. Hematocrit (HCT) (single dose). 95% Confidence Interval Parameter C1 T1 C2 t2 m2 k2 T2
Estimate
Std. Error
Lower bound
Upper bound
57.052 6.243 43.249 5.781 0.091 2.397 1.646
1.043 0.377 0.621 0.449 0.086 0.510 0.289
54.989 5.496 42.020 4.893 0.080 1.387 1.074
59.116 6.989 44.478 6.670 0.262 3.406 2.217
Comment Initial values time constant of decay Final values shift of recovery t2 m2.dosek2 time constant of recovery
Hematological parameters after TBI 5 Table V. Hemoglobin concentration (Hb) (single dose). 95% Confidence Interval Parameter
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C1 T1 C2 t2 m2 k2 T2
Estimate
Std. Error
17.527 7.385 13.799 6.030 0.170 2 2.203
0.304 0.444 0.191 0.396 0.014 0.255
Lower bound
Upper bound
Comment
18.129 8.264 14.177 6.813 0.199
Initial values time constant of decay Final values
16.924 6.506 13.422 5.247 0.142 not estimated 1.698
several assumptions were made. First, time constants are not dose dependent. The only dose-dependency parameters can be observed at the beginning of the RBC recovery (dose-dependent shift, function 1). The parameters to develop the model functions were determined as initial values (C1), time constant of decay (T1), final values (C2), shift of the recovery (t2, m2, k2) and the time constant of the recovery (T2). The parameters are given in Table III for RBC, Table IV for HCT and Table V for Hb for the singledose exposure. It was not possible to model a function for the WBC, because principle behavior is different followed by a dose-dependent decrease which is then followed by an increase in the WBC. The model function describing Hb developed for fractioned doses is suitable for single doses, too. However, it was not possible to find an appropriate model, when k is included as an independent parameter. Therefore, k was set 2. Same model and same assumptions as for RBC (function 1) are used for modeling HCT.
Fractionated dose exposure For the development of a model for Hb, HCT, WBC and RBC (Figure 2D–G) reconstitution the absolute cell concentration was determined during the observation period. Since irradiation was administered at day 6, the time considered for this model ranges from day 6 to day 55. Therefore, all time-points within these models refer to the original time values minus 6 days. For the recovery, the same velocity ( 1 week) was observed for all radiation doses, but the recovery started at different, dose-dependent time-points. Hereby, larger doses led to later recovery of hematopoiesis. The recovery was also followed by a saturation curve reaching a final concentration that was independent from the radiation dose itself. The time constant of the recovery process was independent on radiation and dose itself, and a
2.707
shift of recovery t2 m2.dosek2 time constant of recovery
secondary constant was applied to model a time dependent shift within each model. The complete developed model was described by the sum of both processes. Parameters were determined by non-linear regression. Graphical analysis demonstrated validity of the model. For the model, several assumptions were made. First, time constants were not dose-dependent. The only dose-dependent parameter was seen at the beginning of the recovery process that represented a dose dependent shift in Hb, HCT and WBC (Figure 2D–F). The concentration of Hb, HCT and WBC showed a decrease comparing the initial data to the end point at day 49. With exception of m1 and t2, all parameters in the WBC (function 3) model were found to have significant influence. Because m1 was not found to be significant different from 0, dose-dependency as postulated above is not shown. Therefore a second 8-parametric model was fitted. It is interesting to note that k was similar to strength of dosedependency as found for RBC. The parameters to develop the model functions were determined as initial values (C1), time constant of decay (T1), final values (C2), shift of the recovery (t2, m2, k2) and the time constant of the recovery (T2). The parameters are given in Table VI for HCT, Table VII for Hb, Table VIII for WBC and Table IX for RBC (function 2) in the fractioned dose exposure.
Histology The organ and tissue damage was analyzed in mice after single-dose TBI with doses from 0–12 Gy or a fractionated TBI with doses from 0–12 Gy using single doses of 0–2 Gy (6 1 Gy, 6 1.5 Gy, 6 2 Gy; twice daily). Figures 3 and 4 show the histology of the bone marrow cavities, the gut system, the hearts and the livers of irradiated mice. The application of the single-dose TBI showed a dosedependent reduction of the cellularity in the bone marrow
Table VI. Hematocrit (HCT) (fractioned dose). 95% Confidence Interval Parameter C1 T1 C2 t2 m2 k2 T2
Estimate
Std. Error
Lower bound
Upper bound
Comment
56.298 7.734 43.093 6.478 4.073 2.034 2.097
1.149 0.522 0.678 0.520 0.600 0.211 0.252
54.024 6.701 41.751 5.448 2.887 1.617 1.598
58.572 8.767 44.435 7.508 5.260 2.450 2.597
Initial values time constant of decay Final values shift of recovery t2 m2.dosek2 time constant of recovery
6 C. Oelkrug et al. Table VII. Hemoglobin concentration (Hb) (fractioned dose). 95% Confidence Interval Parameter
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C1 T1 C2 t2 m2 k2 T2
Estimate
Std. Error
Lower bound
Upper bound
17.837 8.923 13.659 7.123 3.766 2.049 2.364
0.332 0.572 0.213 0.530 0.580 0.216 0.256
17.180 7.792 13.236 6.074 2.619 1.622 1.858
18.495 10.055 14.081 8.172 4.914 2.477 2.871
cavities and an increased replacement of bone marrow cells by marrow adipose cells (Figure 3A, 3E, 3I, 3N, 3R). With higher radiation doses, the gut system demonstrated more lesions of the mucosa, an increasing inflammable cellular infiltration and ulcera (Figure 3B, 3F, 3K, 3O, 3S). Liver tissue was less sensitive to X-ray irradiation. Heart tissue showed no damage caused by irradiation (Figure 3C, 3G, 3L, 3P, 3T). With higher radiation doses, a fatty degeneration of the liver tissue and a reduced cell density were found (Figure 3D, 3H, 3M, 3Q, 3U). After application of the fractioned TBI, all bone marrow cavities showed regeneration with a normal distribution of blood cells leading to a survival of all mice (Figure 4A, 4E, 4I, 4N). There was a recurrence of islets with a prevalent form of erythropoiesis, endothelial cells and reticulum cells. The gut system showed an intact barrier and no signs of tissue destruction but signs of activation of lymphoid follicles were visible (Figure 4B, 4F, 4K, 4O). Also, a regular structure of the liver could be observed in all mice receiving fractioned doses (Figure 4D, 4H, 4M, 4Q). The heart tissue showed no sign of irradiation caused damage (Figure 4C, 4G, 4L, 4P).
Discussion One of the major effects in the hematopoietic system due to total body irradiation (TBI) is bone marrow aplasia, which impairs the hematopoietic function and leads to leucopenia, erythropenia and thrombocytopenia (Zhou and Mi 2005). Radiation caused cell damage by genomic DNA disruption, changes in membrane composition or indirect cell damages from toxic molecules is observed in bone marrow cavities (Brocklehurst 2001, Mothersill and Seymour 2002, 2003, Zhao et al. 2005) and leads to apoptosis. These damages are normally monitored 4 h after the radiation exposure (Paris et al. 2001). For the determination and model generation we used the peripheral blood cell
Comment Initial values time constant of decay Final values shift of recovery t2 m2.dosek2 time constant of recovery
count as possible indicators of bone marrow functionality and hematopoietic recovery after TBI. The recovery curves for RBC, Hb, HCT and WBC showed the same velocity ( 1 week) for all doses but starting at different, dose-dependent times. The only dosedependent parameter was defined by the beginning of the recovery process (dose-dependent shift). Furthermore, higher doses were related to a later recovery of the hemato poietic system. The RBC, Hb and HCT recovery was then followed by a saturation curve reaching a final concentration independent of the radiation dose. The contribution of LTR cells might be of relevance in the explanation of the dose-dependent hematopoietic recovery, since this subset is radioresistant and the initial LTR concentration in the bone marrow might be of relevance (Trevisan and Iscove 1995, Trevisan et al. 1996, Zhao et al. 2005). The determination of radiation-induced organ and tissue damage due to the total body exposure of the TTG mice was also investigated through histopathology. Here, the analysis of the bone marrow in the single-dose TBI cohort showed a dose-dependent reduction of the cellularity in the bone marrow cavities (Cao et al. 2011). The fractionated-dose TBI cohort resulted in a regeneration of all bone marrow cavities. Since the bone marrow microenvironment is pivotal for the hematopoietic recovery process these results are of major interest in TBI (Trevisan and Iscove 1995, Trevisan et al. 1996, Zhao et al. 2005). Fliedner et al. (2002a, 2002b) state that irradiated bone marrow result in bone marrow hemorrhage and cell loss due to a dysfunction between the cellular growth pressure and blood flow dynamics. Several studies concerning modelling hematological parameters include canines and human data, especially data collected after radiation accidents and chronic irradiation exposure (space travel experiments) (Vacha and Znojil 1975, Hu and Cucinotta 2011, Hu et al. 2012, Smirnova 2012).
Table VIII. White blood cells (WBC) (fractioned dose). 95% Confidence Interval Parameter
Estimate Std. Error Lower bound
C0 C1 t1 m1 T1 t2 m2 k T2
0.895 1.423 2.247 0.396 2.686 1.334 3.814 2.104 2.142
0.022 0.162 0.936 0.324 0.542 0.824 0.621 0.227 0.390
0.852 1.104 0.395 1.037 1.614 0.297 2.585 1.654 1.371
Upper bound
Comment
0.938 1.743 4.099 0.245 3.758 2.965 5.044 2.554 2.914
refers to final values (antilog: 7.8 103/mm3) refers to amplitude of increase refers to shift of initial decay. Radiation factor describing dose dependency time constant of decay refers to shift of increase time shift of decrease shift2 m2 dosek time constant of increase
Hematological parameters after TBI 7 Table IX. Red blood cells (RBC) (fractioned dose). 95% Confidence Interval Parameter C1 T1 C2 t2 m2 k2 T2
Estimate
Std. Error
Lower bound
Upper bound
10.551 8.335 8.097 6.909 4.852 1.818 2.490
0.217 0.572 0.136 0.600 0.694 0.202 0.272
10.122 7.204 7.829 5.722 3.479 1.418 1.951
10.979 9.466 8.366 8.096 6.225 2.217 3.028
Comment Initial values time constant of decay Final values shift of recovery t2 m2.dosek2 time constant of recovery
Functions: Function 1: RBC (single dose)
RBC (t , d ) C1 ⋅ exp(t / T1 )
C2 1 exp((t t 2 m2 ⋅ dose k2 )/ T2 )
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Function 2: RBC (fractioned dose)
RBC (t , d ) C1 ⋅ exp(t / T1 )
C2 1 exp((t t 2 m2 ⋅ dose k2 )/ T2 )
Function 3: WBC (fractioned dose)
WBC (t , d ) C 0 C1 /(1exp((t t1 )/ T1 )) C1 /(1 exp((t t 2 m2 ⋅ dose k )/ T2 ))
Due to the data collected, in these chronic irradiation exposure was investigated. Interestingly, all mathematical models show an initial dose-dependent decline which was followed by a stabilization, maintenance, and partial recovery of the analyzed cell subsets and is similar to our model established.
Dainiak (2002) has analyzed data published by Cronkite and Fliedner concerning the decline in the lymphocyte, granulocyte and erythrocyte counts after irradiation. This decline is often preceded by an initial phase of granulocytosis. The granulocyte count also normalizes within 1–3 months after irradiation.
Figure 3. Histological analysis by kaolin-aniline-orange G (KAO) staining of bone marrow and hematoxylin eosin (HE) staining for gut, heart and liver from triple transgenic mice (TTG) irradiated with single-dose total body irradiation (TBI) from 0–12 Gy. Sham-irradiated control (A–D), 3 Gy (E–H), 6 Gy (I–M), 8 Gy (N–Q), 12 Gy (R–U). Shown at 20 original magnification. This Figure is reproduced in color in the online version of International Journal of Radiation Biology.
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8 C. Oelkrug et al.
Figure 4. Histological analysis by kaolin-aniline-orange G (KAO) staining of bone marrow and hematoxylin eosin (HE) staining for gut, heart and liver from triple transgenic mice (TTG) irradiated with fractionated dose total body irradiation (TBI) of 6 1 Gy, 6 1.5 Gy, and 6 2 Gy (twice daily). Sham-irradiated control mice (A–D), 6 1 Gy (E–H), 6 1.5 Gy (I–M), 6 2 Gy (N–Q). Shown at 20 original magnification. This Figure is reproduced in color in the online version of International Journal of Radiation Biology.
The use of the TTG mice analyzes different radiation doses and regimens (single versus fractioned) in order to generate model functions to describe the hematopoietic regeneration. These model functions can be further used for transplantation experiments of immune and stem cells in order to investigate their supportive effects and compare them with the established functions. Furthermore, it was shown how radiation influenced the organs of TTG mice and if we were able to establish a radiation configuration which leads to an ablation of the hematopoietic system without the damage of the organs. These findings might be of relevance in the refinement of strategies in the treatment of hematological malignancies concerning irradiation protocols or even helps to further understand possible effects on the hematopoietic system and its recovery after nuclear accidents. The developed model could also be of relevance in the investigation on radio-protective effects of substances on victims from radiation accidents.
Acknowledgements We thank the colleagues from the Translational Centre for Regenerative Medicine, University of Leipzig for providing and breeding the triple transgenic (TTG) mice, Ms Stephanie Tuche and Mrs Ramona Blaschke for the preparation of the histological slides, Mrs Jutta Jahns for preparing the irradiation for the recipient mice. The work presented in this paper was funded by the German Federal Ministry of Education and Research (BMBF 0313452, PtJ-Bio, 0313909).
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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Hematological parameters after TBI 9 bone marrow grafts with high levels of CD4() CD25() FoxP3() T cells can lead to engraftment failure. Cytometry A 81:476–488. Hu S, Cucinotta FA. 2011. Characterization of the radiationdamaged precursor cells in bone marrow based on modeling of the peripheral blood granulocytes response. Health Phys 101:67–78. Hu S, Smirnova OA, Cucinotta FA. 2012. A biomathematical model of lymphopoiesis following severe radiation accidents – potential use for dose assessment. Health Phys 102:425–436. Laub R, Brecht R, Dorsch M, Valey U, Wenk K, Emmrich F. 2002. Anti-human CD4 induces peripheral tolerance in a human CD4, murine CD4-, HLA-DR advanced transgenic mouse model. J Immunol 169:2947–2955. Laub R, Dorsch M, Meyer D, Ermann J, Hedrich HJ, Emmrich F. 2000. A multiple transgenic mouse model with a partially humanized activation pathway for helper T cell responses. J Immunol Meth 246:37–50. Laub R, Dorsch M, Wenk K, Emmrich F. 2001. Induction of immunologic tolerance to tetanus toxoid by anti-human CD4 in HLA-DR3()/human CD4()/murine CD4(-) multiple transgenic mice. Transplant Proc 33:2182–2183. Mothersill C, Seymour C. 2003. Low-dose radiation effects: Experimental hematology and the changing paradigm. Exp Hematol 31:437–445. Mothersill C, Seymour CB. 2002. Bystander and delayed effects after fractionated radiation exposure. Radiat Res 158:626–633.
Paris F, Fuks Z, Kang A, Capodieci P, Juan G, Ehleiter D, Haimovitz-Friedman A, Cordon-Cardo C, Kolesnick R. 2001. Endothelial apoptosis as the primary lesion initiating intestinal radiation damage in mice. Science 293:293–297. Smirnova OA. 2012. Comparative analysis of the dynamics of thrombocytopoietic, granulocytopoietic, and erythropoietic systems in irradiated humans: A modeling approach. Health Phys 103:787–801. Trevisan M, Iscove NN. 1995. Phenotypic analysis of murine long-term hemopoietic reconstituting cells quantitated competitively in vivo and comparison with more advanced colony-forming progeny. J Exp Med 181:93–103. Trevisan M, Yan XQ, Iscove NN. 1996. Cycle initiation and colony formation in culture by murine marrow cells with longterm reconstituting potential in vivo. Blood 88:4149–4158. Vacha J, Znojil V. 1975. [Use of a mathematical model of erythropoiesis to study the recovery process in mice exposed to acute x-ray irradiation]. Biofizika 20:872–879. Zhao Y, Zhan Y, Burke KA, Anderson WF. 2005. Soluble factor(s) from bone marrow cells can rescue lethally irradiated mice by protecting endogenous hematopoietic stem cells. Exp Hematol 33:428–434. Zhou Y, Mi MT. 2005. Genistein stimulates hematopoiesis and increases survival in irradiated mice. J Radiat Res 46: 425–433.
Modelling hematological parameters after total body irradiation Christopher Oelkrug1, Nadja Hilger1, Uta Schönfelder2, Johannes Boltze1,3, Ulrich Sack1,3,4, Christian Fricke5, Guido Hildebrandt6, Thomas Keller7, Frank Emmrich1,3,4 & Stephan Fricke1,3,4
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1Fraunhofer Institute for Cell Therapy and Immunology (IZI), Leipzig, Germany, 2Evangelic Diaconic Hospital,
Div. Oncology and Hematology, Leipzig, Germany, 3Translational Centre for Regenerative Medicine, Universität Leipzig, Leipzig, Germany, 4Institute of Clinical Immunology, Universität Leipzig, Leipzig, Germany, 5Ambulante Dienste LUPS, Luzern, Switzerland, 6Department of Radiation Oncology, University Hospital of Rostock, Rostock, Germany, and 7Acomed Statistik, Leipzig, Germany
Introduction
Abstract Purpose: The time- and dose-dependent reconstitution of hematopoiesis after radiation exposure is strongly related to the stem cell population and can be used to predict hematological parameters. These parameters allow further insight into the hematopoietic system and might lead to the development of novel stem cell transplantation models. Materials and methods: CD4-/- C57Bl/6 mice, transgenic for human CD4 and HLA-DR3, were irradiated in a single (3, 6, 8 and 12 Gy) and fractionated (6 1 Gy, 6 1.5 Gy, 6 2 Gy; twice daily) dose regimen. Blood was analyzed weekly for red blood cells (RBC), hemoglobin concentration (Hb), hematocrit (HCT) and white blood cells (WBC). Organ and tissue damage after irradiation were examined by histopathology. Results: The recovery curves for RBC, Hb, HCT and WBC showed the same velocity ( 1 week) for all radiation doses (3–12 Gy) starting at different, dose-dependent times. The only dosedependent parameter was defined by the beginning of the recovery process (dose-dependent shift) and higher doses were related to a later recovery of the hematopoietic system. The RBC, Hb and HCT recovery was followed by a saturation curve reaching a final concentration independent of the radiation dose. Histological analysis of the bone marrow in the single dose cohort showed a dose-dependent reduction of the cellularity in the bone marrow cavities. The fractioned radiation dose cohort resulted in a regeneration of all bone marrow cavities. Conclusion: Specific functions were developed to describe the reconstitution of hematological parameters after total body irradiation.
The main function of the hematopoietic system is to maintain different blood cell types at a constant level, which show different turnover kinetics as 1.2 1011 day for granulocytes, 2.0 1011 day for erythrocytes or 1.5 1011 day for platelets and are dependent on an unlimited replicative and pluripotent potential of the stem cells in the bone marrow (Fliedner et al. 2002a, 2002b). The hematopoietic system is sensitive to radiation damage, which often leads to radiation induced lethality (Zhou and Mi 2005). To further understand the radiation-induced pathophysiology of the hematopoietic system, it is necessary to investigate this system in special developed models. The time- and dose-dependent reconstitution of the hematopoietic system after radiation exposure plays a key role in the development of a stem cell transplantation model. The hematological parameters are able to further define the stem cell population and the resulting white blood cell population. Modelling these parameters allows further insight into the development of novel stem cell transplantation models and the prediction of reconstitution in irradiated subjects. The hematopoietic cell subsets show different degrees of radiation sensitivity and some sub-fractions of the stem cells seem to be radioresistant. In human stem cell transplantation after lethal irradiation, the participation of host derived hematopoietic cells is still a major problem which often causes graft rejection. Especially long-term reconstituting (LTR) stem cells play a key role in the generation of a functional stem cell transplantation model (Trevisan and Iscove 1995, Trevisan et al. 1996, Zhao et al. 2005). These LTR cells are estimated with 0.001–0.01% in the bone marrow
Keywords: Hematopoiesis, total body irradiation (TBI), transplantation, radiation
Correspondence: Christopher Oelkrug, Fraunhofer Institute for Cell Therapy and Immunology, Perlickstraße 01, 04103 Leipzig, Germany. Tel: 49 341 355 363121. Fax: 49 341 35536 9921. E-mail: [email protected] (Received 29 June 2013; revised 17 October 2013; accepted 21 February 2014)
1
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2 C. Oelkrug et al. and are normally at rest and radioresistant. Therefore, the reconstitution of hematopoiesis in lethally irradiated CD4-/- C57Bl/6 mice transgenic for human CD4 and HLA-DR3 (triple transgenic mice [TTG]) was investigated (Laub et al. 2000, 2001, 2002, Fricke et al. 2009, 2010, 2012, Fricke 2011). The TTG mice have a complete functional murine immune system which is modified with regard to CD4 and HLA-DR. In this setting the presence of human or murine CD4 after transplantation can be used as a marker for chimerism even in a syngeneic setting. This model was developed to observe the engraftment of allogeneic and syngeneic stem cells to induce murine CD4 expression after transplantation. In contrast to this, an endogenous recovery in hematopoiesis will lead to human CD4 and HLA-DR3 expression. To model the hematopoietic recovery, TTG mice were irradiated in a single (3, 6, 8 and 12 Gy) and fractionated (6 1 Gy, 6 1.5 Gy, 6 2 Gy; twice daily) dose regimen. Blood was analyzed weekly for red blood cells (RBC), hemoglobin concentration (Hb), hematocrit (HCT) and white blood cells (WBC). Organ or tissue damage due to the irradiation of the TTG mice was determined through histopathological examination. The analysis of the bone marrow in the single-dose cohort showed a dose-dependent reduction of the cellularity in the bone marrow cavities. The fractioned radiation dose cohort resulted in a regeneration of all bone marrow cavities. In addition, the described irradiation model could also be used to model radioprotective effects of substances on victims from radiation accidents.
Materials and methods Ethics statement All mice were housed, treated and handled in accordance with the guidelines of the University of Leipzig Animal Care Committee and the Regional Board of Animal Care for Leipzig (animal experiment registration number 24/06, 28/08, and 55/11).
Animals TTG are murine CD4 k/o mice expressing the human CD4 and HLA-DR3 molecules on a stable C57Bl/6 background as described previously (Fricke et al. 2009, 2010, 2012, Fricke 2011). The mice were fed ad libitum. All animals were maintained under standardized conditions in the Medical Experimental Centre of the University of Leipzig.
Table II. Fractionated-dose total body irradiation (TBI) experiment. Single dose [Gy] Time after Time [days] 0 6 8 13 20 27 34 41 48 55 Total
radiation
0.00
1.00
1.5
2
Total
0 2 7 14 21 28 35 42 49
4 4 4 4 4 4 4 4 4 4 40
4 4 4 4 4 4 4 4 4 4 40
4 4 4 4 4 4 4 4 4 1 37
4 4 4 4 3 3 3 3 3 3 34
16 16 16 16 15 15 15 15 15 12 151
Irradiation protocol For the total body irradiation (TBI) of the TTG mice, an X-ray apparatus (D3225, Orthovoltage, Gulmay Medical, Camberley, UK) was adjusted for radiation exposure in parallel for four animals in a Plexiglas container (divided in five spaces of 0.5 4.0 cm each). Initially a radiation doseresponse curve was developed. For determination of the lethal radiation dose, 16 mice underwent single TBI with radiation doses from 3–12 Gy (200 kV, dose rate 1.14 Gy/min). Four mice were used as sham-irradiated controls. To model the hematological parameters after TBI, mice were irradiated in a single (3, 6, 8 and 12 Gy) and fractionated (6 fractions; total dose 6, 9 and 12 Gy) dose regimen. The animals irradiated using a fractionated regimen were treated twice a day for three consecutive days with single doses of 1 Gy, 1.5 Gy, or 2 Gy up to total doses of 6 Gy, 9 Gy or 12 Gy, respectively. As shown in Tables I and II, four animals were treated per dose group (tables display time-point of blood collection [days], time after irradiation, dosage [Gy], [total number of animals in the experiment on the horizontal axis for each experimental day, the total number of animals tested during the experiment concerning each radiation dose on the vertical axis]). After day 2 (post irradiation), weekly measurements were performed (number of animals and valid measurements for single and fractionated dose experiments). Blood was analyzed weekly for red blood
Table I. Single-dose total body irradiation (TBI) experiment. Single dose [Gy] Time after Time [days] 0 6 8 13 20 27 34 41 48 55 Total
radiation 0 2 7 14 21 28 35 42 49
0.00 3.00 4 4 4 4 4 4 4 4 4 4 40
4 4 4 4 4 4 4 4 4 4 40
6.00
8.00
12.00
Total
4 4 4 4 4 4 4 4 4 4 40
4 4 4 4 1 0 0 0 0 0 17
4 4 4 2 0 0 0 0 0 0 14
20 20 20 18 13 12 12 12 12 12 151
Figure 1. Determination of the delivered radiation dose. To analyze the administered dose rate for following irradiation experi ments the absorbed dose was calculated in J/kg (Gy).
Figure 2. Non-linear regression models of function parameters function (dose, time) for Hb, HCT, RBC and WBC. Time was modelled using exponential decay and sigmoidal increase or decay. These functions are correlated to damage processes and growth processes. Models for time dependent cell recurrence in single-dose radiation Hb (A), HCT (B) and RBC (C); Fractioned dose radiation Hb (D), HCT (E), WBC (F) and RBC (G). Survival plot showing animal survival in the fractioned dose regimen. (G and H; supplementary data from Fricke et al. 2009). This Figure is reproduced in color in the online version of International Journal of Radiation Biology.
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Hematological parameters after TBI 3
4 C. Oelkrug et al. Table III. Red blood cells (RBC) (single dose). 95% Confidence Interval Parameter C1 T1 C2 t2 m2 k2 T2
Estimate
Std. Error
Lower bound
Upper bound
10.837 6.660 8.228 6.089 0.126 2.248 1.976
0.223 0.447 0.135 0.527 0.126 0.539 0.351
10.395 5.775 7.961 5.045 0.124 1.182 1.281
11.279 7.546 8.495 7.132 0.376 3.314 2.670
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cells (RBC), hemoglobin concentration (Hb), hematocrit (HCT) and white blood cells (WBC).
Hematological parameters of TTG
Blood (140 ml) was collected through heparinized capillaries (Greiner Biochemica, Flacht, Germany) by retroorbitally bleeding, followed by the administration of an antibiotic ophthalmic ointment. Hemoglobin concentration was determined using an Animal Blood Counter (SCIL, Viernheim, Germany), which had been calibrated for mouse blood, within 2 h after blood collection. To model the hematological parameters, murine blood was analyzed for RBC (red blood cells), Hb (hemoglobin concentration), HCT (hematocrit) and WBC (white blood cell count).
Histology Bone marrow, gut, heart and liver of all transgenic mice were analyzed histologically after the end of the irradiation experiments (day 55). Organs were prepared as described according to the protocol established by us previously (Fricke et al. 2010).
Statistical analysis All data are presented as means SD. Non-linear regression models for describing the time course of hematological parameters as a function of dose and time were developed by ACOMED statistics, Leipzig, Germany by using the SPSS 15.0 software package (SPSS Science, Erkrath, Germany). Statistical analysis and graphic presentation were made using Sigma Plot 10.0/ Sigma Stat 3.5 (Systat, Erkrath, Germany).
Results Determination of lethal radiation toxicity in TTG mice Initial to the total body irradiation (TBI) experiments, the administered dose rate was analyzed and the absorbed dose was calculated in J/kg (Gy), (Figure 1).
Comment Initial values time constant of decay Final values shift of recovery t2 m2.dosek2 time constant of recovery
To determine the lethal radiation dose in triple transgeneic mice for ongoing experiments, groups of four TTG mice underwent TBI with single doses of X-rays ranging from 0–12 Gy followed by analysis of survival, and recovery of peripheral red blood cells (RBC), hemoglobin concentration (Hb), hematocrit (HCT) and white blood cells (WBC). Single dose TBI of mice with doses 8 Gy led to death of all mice within 19 days, while mice irradiated with single doses 8 Gy survived (Figure 2A, 2B). The Kaplan-Meier plot for the survival of single dose irradiated mice is shown in Figure 2H. For the fractioned dose experiments with exception of one animal all survived, a survival analysis was not meaningful.
Non-linear regression model Non-linear regression models of function parameter function (dose, time) were developed. Time was modelled using two types of functions including the exponential decay: C.exp(-t/T), with C amplitude, T time constant describing velocity of change and the sigmoidal increase (or decay): C/(1 exp(-t/T)). Both functions are related to typical damage- and growth-processes, respectively. Additionally, time shifts were introduced by using t ti instead of t. This refers to a shift along the x-axis (time). Dose dependency was introduced as m.dosek-factors. If amplitude was dose-dependent, it was multiplied by this factor. If time-shift depended on dose, this factor was added to time-shift. Non-linear regression estimated parameters Ci, ti, Ti, m and k of these functions. A further proof of validity was to include dose 0 into the models. The resulting models should have correctly reflected behavior with dose 0. The investigated time-points were related to t 0 and t 6 days (initial radiation).
Single-dose exposure To establish a model which describes Hb, HCT and RBC (Figure 2A–C) reconstitution after single-dose irradiation,
Table IV. Hematocrit (HCT) (single dose). 95% Confidence Interval Parameter C1 T1 C2 t2 m2 k2 T2
Estimate
Std. Error
Lower bound
Upper bound
57.052 6.243 43.249 5.781 0.091 2.397 1.646
1.043 0.377 0.621 0.449 0.086 0.510 0.289
54.989 5.496 42.020 4.893 0.080 1.387 1.074
59.116 6.989 44.478 6.670 0.262 3.406 2.217
Comment Initial values time constant of decay Final values shift of recovery t2 m2.dosek2 time constant of recovery
Hematological parameters after TBI 5 Table V. Hemoglobin concentration (Hb) (single dose). 95% Confidence Interval Parameter
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C1 T1 C2 t2 m2 k2 T2
Estimate
Std. Error
17.527 7.385 13.799 6.030 0.170 2 2.203
0.304 0.444 0.191 0.396 0.014 0.255
Lower bound
Upper bound
Comment
18.129 8.264 14.177 6.813 0.199
Initial values time constant of decay Final values
16.924 6.506 13.422 5.247 0.142 not estimated 1.698
several assumptions were made. First, time constants are not dose dependent. The only dose-dependency parameters can be observed at the beginning of the RBC recovery (dose-dependent shift, function 1). The parameters to develop the model functions were determined as initial values (C1), time constant of decay (T1), final values (C2), shift of the recovery (t2, m2, k2) and the time constant of the recovery (T2). The parameters are given in Table III for RBC, Table IV for HCT and Table V for Hb for the singledose exposure. It was not possible to model a function for the WBC, because principle behavior is different followed by a dose-dependent decrease which is then followed by an increase in the WBC. The model function describing Hb developed for fractioned doses is suitable for single doses, too. However, it was not possible to find an appropriate model, when k is included as an independent parameter. Therefore, k was set 2. Same model and same assumptions as for RBC (function 1) are used for modeling HCT.
Fractionated dose exposure For the development of a model for Hb, HCT, WBC and RBC (Figure 2D–G) reconstitution the absolute cell concentration was determined during the observation period. Since irradiation was administered at day 6, the time considered for this model ranges from day 6 to day 55. Therefore, all time-points within these models refer to the original time values minus 6 days. For the recovery, the same velocity ( 1 week) was observed for all radiation doses, but the recovery started at different, dose-dependent time-points. Hereby, larger doses led to later recovery of hematopoiesis. The recovery was also followed by a saturation curve reaching a final concentration that was independent from the radiation dose itself. The time constant of the recovery process was independent on radiation and dose itself, and a
2.707
shift of recovery t2 m2.dosek2 time constant of recovery
secondary constant was applied to model a time dependent shift within each model. The complete developed model was described by the sum of both processes. Parameters were determined by non-linear regression. Graphical analysis demonstrated validity of the model. For the model, several assumptions were made. First, time constants were not dose-dependent. The only dose-dependent parameter was seen at the beginning of the recovery process that represented a dose dependent shift in Hb, HCT and WBC (Figure 2D–F). The concentration of Hb, HCT and WBC showed a decrease comparing the initial data to the end point at day 49. With exception of m1 and t2, all parameters in the WBC (function 3) model were found to have significant influence. Because m1 was not found to be significant different from 0, dose-dependency as postulated above is not shown. Therefore a second 8-parametric model was fitted. It is interesting to note that k was similar to strength of dosedependency as found for RBC. The parameters to develop the model functions were determined as initial values (C1), time constant of decay (T1), final values (C2), shift of the recovery (t2, m2, k2) and the time constant of the recovery (T2). The parameters are given in Table VI for HCT, Table VII for Hb, Table VIII for WBC and Table IX for RBC (function 2) in the fractioned dose exposure.
Histology The organ and tissue damage was analyzed in mice after single-dose TBI with doses from 0–12 Gy or a fractionated TBI with doses from 0–12 Gy using single doses of 0–2 Gy (6 1 Gy, 6 1.5 Gy, 6 2 Gy; twice daily). Figures 3 and 4 show the histology of the bone marrow cavities, the gut system, the hearts and the livers of irradiated mice. The application of the single-dose TBI showed a dosedependent reduction of the cellularity in the bone marrow
Table VI. Hematocrit (HCT) (fractioned dose). 95% Confidence Interval Parameter C1 T1 C2 t2 m2 k2 T2
Estimate
Std. Error
Lower bound
Upper bound
Comment
56.298 7.734 43.093 6.478 4.073 2.034 2.097
1.149 0.522 0.678 0.520 0.600 0.211 0.252
54.024 6.701 41.751 5.448 2.887 1.617 1.598
58.572 8.767 44.435 7.508 5.260 2.450 2.597
Initial values time constant of decay Final values shift of recovery t2 m2.dosek2 time constant of recovery
6 C. Oelkrug et al. Table VII. Hemoglobin concentration (Hb) (fractioned dose). 95% Confidence Interval Parameter
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C1 T1 C2 t2 m2 k2 T2
Estimate
Std. Error
Lower bound
Upper bound
17.837 8.923 13.659 7.123 3.766 2.049 2.364
0.332 0.572 0.213 0.530 0.580 0.216 0.256
17.180 7.792 13.236 6.074 2.619 1.622 1.858
18.495 10.055 14.081 8.172 4.914 2.477 2.871
cavities and an increased replacement of bone marrow cells by marrow adipose cells (Figure 3A, 3E, 3I, 3N, 3R). With higher radiation doses, the gut system demonstrated more lesions of the mucosa, an increasing inflammable cellular infiltration and ulcera (Figure 3B, 3F, 3K, 3O, 3S). Liver tissue was less sensitive to X-ray irradiation. Heart tissue showed no damage caused by irradiation (Figure 3C, 3G, 3L, 3P, 3T). With higher radiation doses, a fatty degeneration of the liver tissue and a reduced cell density were found (Figure 3D, 3H, 3M, 3Q, 3U). After application of the fractioned TBI, all bone marrow cavities showed regeneration with a normal distribution of blood cells leading to a survival of all mice (Figure 4A, 4E, 4I, 4N). There was a recurrence of islets with a prevalent form of erythropoiesis, endothelial cells and reticulum cells. The gut system showed an intact barrier and no signs of tissue destruction but signs of activation of lymphoid follicles were visible (Figure 4B, 4F, 4K, 4O). Also, a regular structure of the liver could be observed in all mice receiving fractioned doses (Figure 4D, 4H, 4M, 4Q). The heart tissue showed no sign of irradiation caused damage (Figure 4C, 4G, 4L, 4P).
Discussion One of the major effects in the hematopoietic system due to total body irradiation (TBI) is bone marrow aplasia, which impairs the hematopoietic function and leads to leucopenia, erythropenia and thrombocytopenia (Zhou and Mi 2005). Radiation caused cell damage by genomic DNA disruption, changes in membrane composition or indirect cell damages from toxic molecules is observed in bone marrow cavities (Brocklehurst 2001, Mothersill and Seymour 2002, 2003, Zhao et al. 2005) and leads to apoptosis. These damages are normally monitored 4 h after the radiation exposure (Paris et al. 2001). For the determination and model generation we used the peripheral blood cell
Comment Initial values time constant of decay Final values shift of recovery t2 m2.dosek2 time constant of recovery
count as possible indicators of bone marrow functionality and hematopoietic recovery after TBI. The recovery curves for RBC, Hb, HCT and WBC showed the same velocity ( 1 week) for all doses but starting at different, dose-dependent times. The only dosedependent parameter was defined by the beginning of the recovery process (dose-dependent shift). Furthermore, higher doses were related to a later recovery of the hemato poietic system. The RBC, Hb and HCT recovery was then followed by a saturation curve reaching a final concentration independent of the radiation dose. The contribution of LTR cells might be of relevance in the explanation of the dose-dependent hematopoietic recovery, since this subset is radioresistant and the initial LTR concentration in the bone marrow might be of relevance (Trevisan and Iscove 1995, Trevisan et al. 1996, Zhao et al. 2005). The determination of radiation-induced organ and tissue damage due to the total body exposure of the TTG mice was also investigated through histopathology. Here, the analysis of the bone marrow in the single-dose TBI cohort showed a dose-dependent reduction of the cellularity in the bone marrow cavities (Cao et al. 2011). The fractionated-dose TBI cohort resulted in a regeneration of all bone marrow cavities. Since the bone marrow microenvironment is pivotal for the hematopoietic recovery process these results are of major interest in TBI (Trevisan and Iscove 1995, Trevisan et al. 1996, Zhao et al. 2005). Fliedner et al. (2002a, 2002b) state that irradiated bone marrow result in bone marrow hemorrhage and cell loss due to a dysfunction between the cellular growth pressure and blood flow dynamics. Several studies concerning modelling hematological parameters include canines and human data, especially data collected after radiation accidents and chronic irradiation exposure (space travel experiments) (Vacha and Znojil 1975, Hu and Cucinotta 2011, Hu et al. 2012, Smirnova 2012).
Table VIII. White blood cells (WBC) (fractioned dose). 95% Confidence Interval Parameter
Estimate Std. Error Lower bound
C0 C1 t1 m1 T1 t2 m2 k T2
0.895 1.423 2.247 0.396 2.686 1.334 3.814 2.104 2.142
0.022 0.162 0.936 0.324 0.542 0.824 0.621 0.227 0.390
0.852 1.104 0.395 1.037 1.614 0.297 2.585 1.654 1.371
Upper bound
Comment
0.938 1.743 4.099 0.245 3.758 2.965 5.044 2.554 2.914
refers to final values (antilog: 7.8 103/mm3) refers to amplitude of increase refers to shift of initial decay. Radiation factor describing dose dependency time constant of decay refers to shift of increase time shift of decrease shift2 m2 dosek time constant of increase
Hematological parameters after TBI 7 Table IX. Red blood cells (RBC) (fractioned dose). 95% Confidence Interval Parameter C1 T1 C2 t2 m2 k2 T2
Estimate
Std. Error
Lower bound
Upper bound
10.551 8.335 8.097 6.909 4.852 1.818 2.490
0.217 0.572 0.136 0.600 0.694 0.202 0.272
10.122 7.204 7.829 5.722 3.479 1.418 1.951
10.979 9.466 8.366 8.096 6.225 2.217 3.028
Comment Initial values time constant of decay Final values shift of recovery t2 m2.dosek2 time constant of recovery
Functions: Function 1: RBC (single dose)
RBC (t , d ) C1 ⋅ exp(t / T1 )
C2 1 exp((t t 2 m2 ⋅ dose k2 )/ T2 )
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Function 2: RBC (fractioned dose)
RBC (t , d ) C1 ⋅ exp(t / T1 )
C2 1 exp((t t 2 m2 ⋅ dose k2 )/ T2 )
Function 3: WBC (fractioned dose)
WBC (t , d ) C 0 C1 /(1exp((t t1 )/ T1 )) C1 /(1 exp((t t 2 m2 ⋅ dose k )/ T2 ))
Due to the data collected, in these chronic irradiation exposure was investigated. Interestingly, all mathematical models show an initial dose-dependent decline which was followed by a stabilization, maintenance, and partial recovery of the analyzed cell subsets and is similar to our model established.
Dainiak (2002) has analyzed data published by Cronkite and Fliedner concerning the decline in the lymphocyte, granulocyte and erythrocyte counts after irradiation. This decline is often preceded by an initial phase of granulocytosis. The granulocyte count also normalizes within 1–3 months after irradiation.
Figure 3. Histological analysis by kaolin-aniline-orange G (KAO) staining of bone marrow and hematoxylin eosin (HE) staining for gut, heart and liver from triple transgenic mice (TTG) irradiated with single-dose total body irradiation (TBI) from 0–12 Gy. Sham-irradiated control (A–D), 3 Gy (E–H), 6 Gy (I–M), 8 Gy (N–Q), 12 Gy (R–U). Shown at 20 original magnification. This Figure is reproduced in color in the online version of International Journal of Radiation Biology.
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8 C. Oelkrug et al.
Figure 4. Histological analysis by kaolin-aniline-orange G (KAO) staining of bone marrow and hematoxylin eosin (HE) staining for gut, heart and liver from triple transgenic mice (TTG) irradiated with fractionated dose total body irradiation (TBI) of 6 1 Gy, 6 1.5 Gy, and 6 2 Gy (twice daily). Sham-irradiated control mice (A–D), 6 1 Gy (E–H), 6 1.5 Gy (I–M), 6 2 Gy (N–Q). Shown at 20 original magnification. This Figure is reproduced in color in the online version of International Journal of Radiation Biology.
The use of the TTG mice analyzes different radiation doses and regimens (single versus fractioned) in order to generate model functions to describe the hematopoietic regeneration. These model functions can be further used for transplantation experiments of immune and stem cells in order to investigate their supportive effects and compare them with the established functions. Furthermore, it was shown how radiation influenced the organs of TTG mice and if we were able to establish a radiation configuration which leads to an ablation of the hematopoietic system without the damage of the organs. These findings might be of relevance in the refinement of strategies in the treatment of hematological malignancies concerning irradiation protocols or even helps to further understand possible effects on the hematopoietic system and its recovery after nuclear accidents. The developed model could also be of relevance in the investigation on radio-protective effects of substances on victims from radiation accidents.
Acknowledgements We thank the colleagues from the Translational Centre for Regenerative Medicine, University of Leipzig for providing and breeding the triple transgenic (TTG) mice, Ms Stephanie Tuche and Mrs Ramona Blaschke for the preparation of the histological slides, Mrs Jutta Jahns for preparing the irradiation for the recipient mice. The work presented in this paper was funded by the German Federal Ministry of Education and Research (BMBF 0313452, PtJ-Bio, 0313909).
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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