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Determination of boron distribution in rat's brain, kidney and liver
ARTICLE IN PRESS Applied Radiation and Isotopes 67 (2009) S369–S373
Contents lists available at ScienceDirect
Applied Radiation and Isotopes journal homepage: www.elsevier.com/locate/apradiso
Determination of boron distribution in rat’s brain, kidney and liver Ali Pazirandeh a,c,, Behnam Jameie b, Maysam Zargar a a b c
Nuclear Engineering Department, Science and Research Branch, Islamic Azad University, Tehran, Iran Neuroscience Lab, Cellular & Molecular Research Center, Iran University of Medical Sciences, Tehran, Iran Science and Technology Research Center, AEOI, Tehran, Iran
a r t i c l e in fo
Keywords: Boron-10 Borax Cr-39 plastic Alpha track counting Liver Brain Kidney
abstract To determine relative boron distribution in rat’s brain, liver and kidney, a mixture of boric acid and borax, was used. After transcardial injection of the solution, the animals were sacrificed and the brain, kidney and liver were removed. The coronal sections of certain areas of the brain were prepared by freezing microtome. The slices were sandwiched within two pieces of CR-39. The samples were bombarded in a thermal neutron field of the TRR pneumatic facility. The alpha tracks are registered on CR-39 after being etched in NaOH. The boron distribution was determined by counting these alpha tracks CR-39 plastics. The distribution showed non-uniformity in brain, liver and kidney. & 2009 Elsevier Ltd. All rights reserved.
1. Introduction The cytotoxic effect of boron neutron capture therapy is due to the nuclear reaction of neutrons with 10B leading to splitting of 11B into two highly energetic ions: alpha particle and 7Li with a maximum Q-value of 2.79 MeV (in 93.7% of these reactions also a 0.478 MeV gamma is produced). The resulting ions are high linear energy transfer (LET) particles, which give a high biological effect. The short range of the ions in tissue (5–9 mm) restricts radiation damage to the cells in which boron absorption takes place. In BNCT, it is important that the boron compound selectively concentrate in tumor cells to destroy tumor cells without significantly damaging the surrounding normal tissues. Practically, the boron carrier is concentrated mainly in the tumor, but, nevertheless, some remain in blood and surrounding tissues. As a result, it may lead to 10B(n,a)7Li reactions in healthy tissues. The compound uptake as well as the boron distribution in the targeted tissue are important factors in the success of the treatment (Suzuki et al., 2004). Due to heterogeneity of the brain and tumor structures, and liquid between brain gaps, the boron compound is distributed also rather heterogeneously. This effect makes the bio-distribution of boron in the brain, especially in the tumor, un-desirable for successful treatment by BNCT (Awad et al., 2005). One may think that a high boron concentration is advantageous because it is more effective in the destruction of malignant
Corresponding author at: Nuclear Engineering Department, Science and Research Branch, Islamic Azad University, Tehran, Iran. Tel./fax: +98 2144869656. E-mail addresses: [email protected], [email protected] (A. Pazirandeh).
0969-8043/$ - see front matter & 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.apradiso.2009.03.057
cells. On the other hand, because of the high absorption probability of 10B for thermal neutrons, high boron concentration decreases the thermal neutron flux in the tumor. This property decreases the thermal flux and as a result also the absorption rate. The most desirable neutron beam for deep seated tumor exists in epithermal neutrons (1–10 keV). Epithermal neutrons passing through skin, cranium and healthy brain towards the tumor would slow down. However, the distribution of thermal neutrons is not a pure Maxwellian with kT ¼ 0.025 eV. Because of 1/v absorption cross section of 10B, as the boron concentration increases in the tumor and its surroundings, more thermal neutrons are absorbed and the neutron spectrum becomes harder. This effect causes the Maxwellian distribution peak shift to the higher energies, which leads to less reaction rates. On the other hand, the human brain consists of 78% water and 22% heavy molecules (Barth et al., 2003), which influences slower neutron moderation, especially in the low energy range. Therefore, the boron concentration in the tumor should be adjusted as such to optimize the 10B(n,a)7Li reaction. In this study, we have measured the boron distribution in rat’s brain, kidney and liver to find out the real distribution of 10 B in the rats’ organs.
2. Material and methods Animal models for human brain tumors have played a significant role in experimental neuro-oncology for almost four decades (Barth et al., 2003; Weisacker et al., 1981). Although no currently available animal brain tumor model exactly simulates human high grade gliomas; rat’s brain tumor models have provided much useful information that has helped us to better
ARTICLE IN PRESS S370
A. Pazirandeh et al. / Applied Radiation and Isotopes 67 (2009) S369–S373
Table 1 Some information about rats used in the experiment. Item
Quantity
No. of rats Sex Weight (g) Age (d) Slice thickness brain Slide dimension (mm) Reactor power Neutron flux Etching agent Etching period
3 male 240, 280, 300 60, 70, 75 40 mm 1 1 40 kW E106 n/cm2 s 6.25 M NaOH 30 min
Table 2 Composition of two solutions used in the experiment. Solution Compositions no.
Mass
1
1 g+250 mg 70 mg
7.57 0.324E20
0.65E20
2 g+500 mg 50 mg
6.88 0.910E20
3.64E20
pH
B10 content/ ml
Injected B10
Fig. 3. Ion tracks of rat’s forebrain on CR-39.
Fig. 1. I Total rat’s brain.
Fig. 4. Alpha tracks in CR-39 using SRIM.
700 Number of alpha-Tracks/slide
2
Boric acid+borax, H3BO3+Na2B4O7 Boric acid+borax, H3BO3+Na2B4O7
Water (ml)
600 500
Forebrain_Horizontal_Top
FBH_Background
FB_Vertical_Top
FBH_Bottom
400 300 200 100 0 -100 0
Fig. 2. Rat forebrain lobe cuts.
2
4
6 8 10 Slide Number
12
14
Fig. 5. Alpha track counts in rat’s brain along horizontal and vertical lines in rat’s forebrain.
ARTICLE IN PRESS A. Pazirandeh et al. / Applied Radiation and Isotopes 67 (2009) S369–S373
understand human brain tumors and potentially may lead to innovative clinical strategies for their treatment. In human tumor treatment, normally, two boron compounds have been used clinically: sodium borocaptate or BSH (Na2B12H11SH) and boronophenylalanine BPA. In this paper, the results of our measurements to determine the distribution of boron in rat’s brain, kidney and liver, which performed on three rats using a solution, i.e., a mixture of boric acid and borax, with a pH of nearly 7 are reported.
3. Experimentation Adult male Sprague-Dawely rats were used in this study, to measure the boron distribution in brain, kidney and liver, (see Table 1). Ten minutes after induction of anesthesia by Ketamine (0.7 ml/kg), 1 ml of boron solution was injected transcardially via the left ventricle, the animal left untouched while the heart was
S371
still beating. The second injection also carried out in left ventricle in the same dose. Table 2 provides the composition of the boron solutions. After 15 min, the animal was scarified and the brain, kidneys and the liver were removed and frozen. By using freezing microtome, coronal sections of 40 mm were prepared. To keep the slices intact, they were treated with H–CHO—formaldehyde. Certain sections from different parts of the organs were sandwiched with two pieces of CR-39 (allyl diglycol carbonate) plastic to record 1.48 MeV a and 0.83 MeV 7Li particles generated by 10B(n,a) 7Li reaction, as well as 0.59 MeV protons originated from the 14N(n,p)14C reaction 14N is the biological abundant nuclide (Ogura et al., 2004). Recorded recoil protons are due to 1 H(n,n0 )1H reactions in the tissue and in the plastic itself because of epithermal neutron scattering from the hydrogen nucleus. The sandwiched brain slices were bombarded in the thermal neutron pneumatic facility of the Tehran Research Reactor (TRR) in a neutron flux of 106 n/cm2 s for 15 min. Thermal neutrons captured by 10B may lead to the production of the high energetic alpha
Fig. 6. A few CR-39 alpha tracks slides of rat’s liver slices.
Fig. 7. A few CR-39 alpha tracks slides of rat’s brain slices.
ARTICLE IN PRESS S372
A. Pazirandeh et al. / Applied Radiation and Isotopes 67 (2009) S369–S373
particle. Some alpha particles produced in the slices may escape and penetrate into the plastic CR-39. After the irradiation, the plastic pieces were etched in 6.25 mole NaOH at 65 1C for 1 h. The etched CR-39 pieces were viewed on a PC-based optical microscopy equipped with a digital camera that revealed the ion tracks of alphas, lithium and protons. The boron distribution was determined by counting the alpha tracks.
4. Results Fig. 1 shows the photo of the removed rat’s brain. The slices of two brain lobe cuts are shown in Fig. 2. The small slices of brain were cut in two parallel horizontal lines and one vertical line, see
Fig. 2. These slices were chosen for boron distribution determination. Fig. 3 shows the typical ion tracks on the plastic of a forebrain slice. The larger tracks on the plastic are due to alphas and lithium ions and small ones are identified as recoiled protons. Fig. 4 shows the range of helium and lithium ions in plastic using the Monte Carlo code SRIM2003. In Fig. 5, the relative track density along top and bottom horizontal lines and along a vertical line, in two brain lobes, are plotted. The results of these measurements revealed that due to the brain structure, the relative boron concentration varies as shown in Fig. 5. The accompanying error bars apply to the other curves as well. As indicated in Section 3, relative boron density in rat’s brain tended to be as close as possible to the density for a possible brain cancer therapy. Therefore, relative boron density in rat’s
Fig. 8. A few CR-39 alpha tracks slides of rat’s kidney slices.
ARTICLE IN PRESS A. Pazirandeh et al. / Applied Radiation and Isotopes 67 (2009) S369–S373
brain slices was close to the in vivo density. Figs. 6–8 show the spread of 10B in different part of the liver, brain and kidney.
5. Conclusions We found by counting alpha tracks due to 10B(n,a)7Li reactions on CR-39 plastic as shown in Fig. 7, that the boron distribution in brain due to its complex structure is highly space dependent. The pattern of this distribution of 10B could be very important from the therapeutic point of view. Accurate measurements of the 10B distribution in the biological models with a sensitivity of a few ppm is essential for evaluating the potential usefulness of various 10 B-delivery compounds. For this purpose, we can easily recognize 10 B-rich regions in the samples by a cursory glance area in the images of the digital camera induced by alpha tracks. It should be borne in mind that human brain is much different from rat’s brain with respect to the structure and composition. In BNCT treatment, especially in human brain, the shape of epithermal neutron beams and the degree of moderation of neutrons in brain play an important role in destruction of malignant tissues. Liquids around tumor influence great deal in moderation of epithermal and scattering of neutrons. These affect the success of treatment.
S373
Acknowledgments The authors would like to thank the Tehran Research Reactor Operating Staff for irradiating samples and the Cellular and Molecular Research Center of Iran University of Medical Sciences for their technical support. References Awad, D.I., Lutz, T., Silke, L., Wessolowski, H., Gabel, D., 2005. Interaction of Na2B12H11SH with liposomes: influence on zeta potential and particle size. Journal of Organomettalic Chemistry 690, 2732–2735. Barth, R.F., Yang, W., Coderre, J.A., 2003. Rat brain tumor models to assess the efficacy of boron neutron capture therapy: a critical evaluation. Journal of Neuro-Oncology, 6261–6274. Ogura, K., Yanagie, H., Eriguchi, M., Lehmann, E.H., Kuelne, G., Bayon, G., Kobayashi, H., 2004. Neutron capture autoradiographic study of the biodistribution of 10B in tumor-bearing mice. Applied Radiation and Isotopes 61, 585–590. Suzuki, M., Nagata, K., Masunaga, S., Kinashi, Y., Sakurai, Y., Maruhashi, A., Ono, K., 2004. Biodistribution of 10B in a rat liver tumor model following intra-arterial administration of BSH/ degradable starch microspheres (DSM) emulsion. Applied Radiation and Isotopes 61, 933–937. Weisacker, M., Deen, D.F., Rosenblum, H.T., Gutin, P.H., Barker, M., 1981. The 9L rat brain tumor: description and application of an animal model. Journal of Neurology 224, 183–192.
Contents lists available at ScienceDirect
Applied Radiation and Isotopes journal homepage: www.elsevier.com/locate/apradiso
Determination of boron distribution in rat’s brain, kidney and liver Ali Pazirandeh a,c,, Behnam Jameie b, Maysam Zargar a a b c
Nuclear Engineering Department, Science and Research Branch, Islamic Azad University, Tehran, Iran Neuroscience Lab, Cellular & Molecular Research Center, Iran University of Medical Sciences, Tehran, Iran Science and Technology Research Center, AEOI, Tehran, Iran
a r t i c l e in fo
Keywords: Boron-10 Borax Cr-39 plastic Alpha track counting Liver Brain Kidney
abstract To determine relative boron distribution in rat’s brain, liver and kidney, a mixture of boric acid and borax, was used. After transcardial injection of the solution, the animals were sacrificed and the brain, kidney and liver were removed. The coronal sections of certain areas of the brain were prepared by freezing microtome. The slices were sandwiched within two pieces of CR-39. The samples were bombarded in a thermal neutron field of the TRR pneumatic facility. The alpha tracks are registered on CR-39 after being etched in NaOH. The boron distribution was determined by counting these alpha tracks CR-39 plastics. The distribution showed non-uniformity in brain, liver and kidney. & 2009 Elsevier Ltd. All rights reserved.
1. Introduction The cytotoxic effect of boron neutron capture therapy is due to the nuclear reaction of neutrons with 10B leading to splitting of 11B into two highly energetic ions: alpha particle and 7Li with a maximum Q-value of 2.79 MeV (in 93.7% of these reactions also a 0.478 MeV gamma is produced). The resulting ions are high linear energy transfer (LET) particles, which give a high biological effect. The short range of the ions in tissue (5–9 mm) restricts radiation damage to the cells in which boron absorption takes place. In BNCT, it is important that the boron compound selectively concentrate in tumor cells to destroy tumor cells without significantly damaging the surrounding normal tissues. Practically, the boron carrier is concentrated mainly in the tumor, but, nevertheless, some remain in blood and surrounding tissues. As a result, it may lead to 10B(n,a)7Li reactions in healthy tissues. The compound uptake as well as the boron distribution in the targeted tissue are important factors in the success of the treatment (Suzuki et al., 2004). Due to heterogeneity of the brain and tumor structures, and liquid between brain gaps, the boron compound is distributed also rather heterogeneously. This effect makes the bio-distribution of boron in the brain, especially in the tumor, un-desirable for successful treatment by BNCT (Awad et al., 2005). One may think that a high boron concentration is advantageous because it is more effective in the destruction of malignant
Corresponding author at: Nuclear Engineering Department, Science and Research Branch, Islamic Azad University, Tehran, Iran. Tel./fax: +98 2144869656. E-mail addresses: [email protected], [email protected] (A. Pazirandeh).
0969-8043/$ - see front matter & 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.apradiso.2009.03.057
cells. On the other hand, because of the high absorption probability of 10B for thermal neutrons, high boron concentration decreases the thermal neutron flux in the tumor. This property decreases the thermal flux and as a result also the absorption rate. The most desirable neutron beam for deep seated tumor exists in epithermal neutrons (1–10 keV). Epithermal neutrons passing through skin, cranium and healthy brain towards the tumor would slow down. However, the distribution of thermal neutrons is not a pure Maxwellian with kT ¼ 0.025 eV. Because of 1/v absorption cross section of 10B, as the boron concentration increases in the tumor and its surroundings, more thermal neutrons are absorbed and the neutron spectrum becomes harder. This effect causes the Maxwellian distribution peak shift to the higher energies, which leads to less reaction rates. On the other hand, the human brain consists of 78% water and 22% heavy molecules (Barth et al., 2003), which influences slower neutron moderation, especially in the low energy range. Therefore, the boron concentration in the tumor should be adjusted as such to optimize the 10B(n,a)7Li reaction. In this study, we have measured the boron distribution in rat’s brain, kidney and liver to find out the real distribution of 10 B in the rats’ organs.
2. Material and methods Animal models for human brain tumors have played a significant role in experimental neuro-oncology for almost four decades (Barth et al., 2003; Weisacker et al., 1981). Although no currently available animal brain tumor model exactly simulates human high grade gliomas; rat’s brain tumor models have provided much useful information that has helped us to better
ARTICLE IN PRESS S370
A. Pazirandeh et al. / Applied Radiation and Isotopes 67 (2009) S369–S373
Table 1 Some information about rats used in the experiment. Item
Quantity
No. of rats Sex Weight (g) Age (d) Slice thickness brain Slide dimension (mm) Reactor power Neutron flux Etching agent Etching period
3 male 240, 280, 300 60, 70, 75 40 mm 1 1 40 kW E106 n/cm2 s 6.25 M NaOH 30 min
Table 2 Composition of two solutions used in the experiment. Solution Compositions no.
Mass
1
1 g+250 mg 70 mg
7.57 0.324E20
0.65E20
2 g+500 mg 50 mg
6.88 0.910E20
3.64E20
pH
B10 content/ ml
Injected B10
Fig. 3. Ion tracks of rat’s forebrain on CR-39.
Fig. 1. I Total rat’s brain.
Fig. 4. Alpha tracks in CR-39 using SRIM.
700 Number of alpha-Tracks/slide
2
Boric acid+borax, H3BO3+Na2B4O7 Boric acid+borax, H3BO3+Na2B4O7
Water (ml)
600 500
Forebrain_Horizontal_Top
FBH_Background
FB_Vertical_Top
FBH_Bottom
400 300 200 100 0 -100 0
Fig. 2. Rat forebrain lobe cuts.
2
4
6 8 10 Slide Number
12
14
Fig. 5. Alpha track counts in rat’s brain along horizontal and vertical lines in rat’s forebrain.
ARTICLE IN PRESS A. Pazirandeh et al. / Applied Radiation and Isotopes 67 (2009) S369–S373
understand human brain tumors and potentially may lead to innovative clinical strategies for their treatment. In human tumor treatment, normally, two boron compounds have been used clinically: sodium borocaptate or BSH (Na2B12H11SH) and boronophenylalanine BPA. In this paper, the results of our measurements to determine the distribution of boron in rat’s brain, kidney and liver, which performed on three rats using a solution, i.e., a mixture of boric acid and borax, with a pH of nearly 7 are reported.
3. Experimentation Adult male Sprague-Dawely rats were used in this study, to measure the boron distribution in brain, kidney and liver, (see Table 1). Ten minutes after induction of anesthesia by Ketamine (0.7 ml/kg), 1 ml of boron solution was injected transcardially via the left ventricle, the animal left untouched while the heart was
S371
still beating. The second injection also carried out in left ventricle in the same dose. Table 2 provides the composition of the boron solutions. After 15 min, the animal was scarified and the brain, kidneys and the liver were removed and frozen. By using freezing microtome, coronal sections of 40 mm were prepared. To keep the slices intact, they were treated with H–CHO—formaldehyde. Certain sections from different parts of the organs were sandwiched with two pieces of CR-39 (allyl diglycol carbonate) plastic to record 1.48 MeV a and 0.83 MeV 7Li particles generated by 10B(n,a) 7Li reaction, as well as 0.59 MeV protons originated from the 14N(n,p)14C reaction 14N is the biological abundant nuclide (Ogura et al., 2004). Recorded recoil protons are due to 1 H(n,n0 )1H reactions in the tissue and in the plastic itself because of epithermal neutron scattering from the hydrogen nucleus. The sandwiched brain slices were bombarded in the thermal neutron pneumatic facility of the Tehran Research Reactor (TRR) in a neutron flux of 106 n/cm2 s for 15 min. Thermal neutrons captured by 10B may lead to the production of the high energetic alpha
Fig. 6. A few CR-39 alpha tracks slides of rat’s liver slices.
Fig. 7. A few CR-39 alpha tracks slides of rat’s brain slices.
ARTICLE IN PRESS S372
A. Pazirandeh et al. / Applied Radiation and Isotopes 67 (2009) S369–S373
particle. Some alpha particles produced in the slices may escape and penetrate into the plastic CR-39. After the irradiation, the plastic pieces were etched in 6.25 mole NaOH at 65 1C for 1 h. The etched CR-39 pieces were viewed on a PC-based optical microscopy equipped with a digital camera that revealed the ion tracks of alphas, lithium and protons. The boron distribution was determined by counting the alpha tracks.
4. Results Fig. 1 shows the photo of the removed rat’s brain. The slices of two brain lobe cuts are shown in Fig. 2. The small slices of brain were cut in two parallel horizontal lines and one vertical line, see
Fig. 2. These slices were chosen for boron distribution determination. Fig. 3 shows the typical ion tracks on the plastic of a forebrain slice. The larger tracks on the plastic are due to alphas and lithium ions and small ones are identified as recoiled protons. Fig. 4 shows the range of helium and lithium ions in plastic using the Monte Carlo code SRIM2003. In Fig. 5, the relative track density along top and bottom horizontal lines and along a vertical line, in two brain lobes, are plotted. The results of these measurements revealed that due to the brain structure, the relative boron concentration varies as shown in Fig. 5. The accompanying error bars apply to the other curves as well. As indicated in Section 3, relative boron density in rat’s brain tended to be as close as possible to the density for a possible brain cancer therapy. Therefore, relative boron density in rat’s
Fig. 8. A few CR-39 alpha tracks slides of rat’s kidney slices.
ARTICLE IN PRESS A. Pazirandeh et al. / Applied Radiation and Isotopes 67 (2009) S369–S373
brain slices was close to the in vivo density. Figs. 6–8 show the spread of 10B in different part of the liver, brain and kidney.
5. Conclusions We found by counting alpha tracks due to 10B(n,a)7Li reactions on CR-39 plastic as shown in Fig. 7, that the boron distribution in brain due to its complex structure is highly space dependent. The pattern of this distribution of 10B could be very important from the therapeutic point of view. Accurate measurements of the 10B distribution in the biological models with a sensitivity of a few ppm is essential for evaluating the potential usefulness of various 10 B-delivery compounds. For this purpose, we can easily recognize 10 B-rich regions in the samples by a cursory glance area in the images of the digital camera induced by alpha tracks. It should be borne in mind that human brain is much different from rat’s brain with respect to the structure and composition. In BNCT treatment, especially in human brain, the shape of epithermal neutron beams and the degree of moderation of neutrons in brain play an important role in destruction of malignant tissues. Liquids around tumor influence great deal in moderation of epithermal and scattering of neutrons. These affect the success of treatment.
S373
Acknowledgments The authors would like to thank the Tehran Research Reactor Operating Staff for irradiating samples and the Cellular and Molecular Research Center of Iran University of Medical Sciences for their technical support. References Awad, D.I., Lutz, T., Silke, L., Wessolowski, H., Gabel, D., 2005. Interaction of Na2B12H11SH with liposomes: influence on zeta potential and particle size. Journal of Organomettalic Chemistry 690, 2732–2735. Barth, R.F., Yang, W., Coderre, J.A., 2003. Rat brain tumor models to assess the efficacy of boron neutron capture therapy: a critical evaluation. Journal of Neuro-Oncology, 6261–6274. Ogura, K., Yanagie, H., Eriguchi, M., Lehmann, E.H., Kuelne, G., Bayon, G., Kobayashi, H., 2004. Neutron capture autoradiographic study of the biodistribution of 10B in tumor-bearing mice. Applied Radiation and Isotopes 61, 585–590. Suzuki, M., Nagata, K., Masunaga, S., Kinashi, Y., Sakurai, Y., Maruhashi, A., Ono, K., 2004. Biodistribution of 10B in a rat liver tumor model following intra-arterial administration of BSH/ degradable starch microspheres (DSM) emulsion. Applied Radiation and Isotopes 61, 933–937. Weisacker, M., Deen, D.F., Rosenblum, H.T., Gutin, P.H., Barker, M., 1981. The 9L rat brain tumor: description and application of an animal model. Journal of Neurology 224, 183–192.