Mouse cardiac tissue received greater protection from and had better tolerance to the maximum dose of FLASH versus conventional radiation

doi:10.21203/rs.3.rs-3118807/v1

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Mouse cardiac tissue received greater protection from and had better tolerance to the maximum dose of FLASH versus conventional radiation

https://doi.org/10.21203/rs.3.rs-3118807/v1

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Radiation therapy is used to treat patients with various types of cancer by killing tumor cells or inhibiting their growth. However, conventional radiation (CONV) not only shrinks the tumor, but also irradiates the surrounding normal tissue, causing normal tissue toxicity. Here, we investigated the potential benefits of high-dose FLASH irradiation therapy applied over a short period and compared the effects of FLASH irradiation and CONV irradiation on mouse cardiac tissue. Our results showed that compared to CONV irradiation, FLASH irradiation resulted in lower mortality and prevented radiation-induced cardiac fibrosis, according to in the survival curves of irradiated mice. FLASH irradiation was more effective than CONV irradiation at higher doses and showed a protective effect and greater cardiac tissue tolerance. These findings can be used to evaluate the efficacy, safety, and potential side effects of FLASH radiation therapy in animal models before application to human patients. Thus, our results on cardiac tissue changes in mice after FLASH irradiation in this study will be an important reference for predicting and measuring cardiotoxicity in preclinical and clinical applications of FLASH treatment in the future.

Health sciences/Cardiology

Health sciences/Medical research

Ionizing radiation has been used for decades to treat cancer in > 50% of patients, and technical advancements in radiotherapy (RT) have improved treatment efficiency significantly [1, 2]. While the conventional (CONV) dose-rate RT has been reported to reduce tumor size, it has shown radiation-induced toxicity effects in the surrounding normal tissue [3, 4, 5]. Moreover, although thoracic RT for breast, esophageal, and lung cancer is used as tumor therapy, the cardiac tissue, a highly oxygen-dependent tissue, is affected negatively because of the influence of the reactive oxygen species released during RT, making it difficult to treat tumors [6, 7].

Radiation-induced cardiac diseases (RICDs), such as coronary artery disease, cardiomyopathy, valve disease, arrhythmia, and pericardial disease, activate an acute inflammatory pathway, leading to a chronic pathogenic cascade and cardiac fibrosis [8, 9]. Since these RICDs develop progressively over 10–20 years, early detection using irradiation is difficult [6]. Therefore, it is necessary to develop treatments for RICDs.

FLASH radiation (average dose rate: > 40 Gy/s) was first introduced in 2014. It delivers ultrahigh doses of radiation, which are several times higher than the clinically used CONV radiation (average dose rate: > 5 Gy/min) [10, 11]. FLASH radiation has been reported to reduce the tumor size and to increase normal tissue sparing. To reduce radiation-induced toxicity commonly associated with CONV radiation, FLASH radiation has been studied in preclinical models, such as of radiation-induced lung fibrosis, hematopoiesis damage, and neurocognitive deficits, but the mechanism remains unclear [12–14]. In particular, long-term cardiac studies using FLASH radiation in the preclinical stage are rare compared to studies using CONV radiation.

Although radiographic research using mouse models has been conducted in the past 10 years [15], the biophysical correlation with the murine model remains unclear. Moreover, the findings have varied across studies [15, 16].

This study investigated the effects of CONV and FLASH radiation on cardiac tissue from mice by assessing animal survival and measuring fibrosis in the cardiac tissue. Through this study, the effects and potential benefits of FLASH radiation therapy will be investigated. Based on the study findings, guidelines for cardiac tissue radiation experiments on mice at the preclinical stage can be developed and used as an important reference for clinical treatment.

Irradiation devices and dosimetry

FLASH irradiation was performed using the Dongnam Institute of Radiological and Medical Sciences (DIRAMS) linear accelerator (LINAC). The DIRAMS LINAC operates at a radio frequency (RF) of 5.712 GHz and produces 6 MeV electron beams at 2.5 MW RF power. To irradiate the mouse cardiac tissue with FLASH electron beams, a copper shielding block with a thickness of 5 mm and a hole diameter of 10 mm was used. The mean energy of the electron beam used in this study was approximately 5.7 MeV, and the repetition rate of the beams was 150 Hz. The distance between the primary foil and mouse surface was 30 cm.

A film dosimetry method using a radiochromic film (GAFchromic™ MD-V3, Ashland, NJ, USA) supporting doses of approximately 100 Gy was used for the FLASH beam-dose measurement. The average dose per pulse, dose rate, and instantaneous dose rate of electron beams were 0.77 Gy/pulse, 115.5 Gy/s, and 3.08 X 10⁵ Gy/s, respectively [17, 18]. CONV radiation was shielded using a block that allowed only the cardiac tissue to be irradiated. The irradiation conditions and experiments that were performed are outlined in Supplementary Table S1.

Mice and animal care

C57BL/6N male mice aged 7 weeks, each with a body weight of 22 ± 2 g, were purchased from Doo Yeol Biotech (Seoul, Korea). All experimental procedures were performed in accordance with the guidelines of the DIRAMS Animal Care. This study was approved by the Institutional Animal Experimentation Ethics Committee of the Dongnam Institute of Radiological and Medical Sciences (Dirams AEC-2021-007). Our study was designed according to the ARRIVE guidelines.

Irradiation

The 7-week-old C57BL/6N male mice were divided randomly into three groups, and each mouse received a single fraction (10 and 20 Gy) of irradiation: control (0 Gy, n = 12), FLASH (40 Gy/s, n = 12 animals per group and experiment), and CONV (0.067 Gy/s, n = 8–9 animals per group and experiment) groups. All mice were anesthetized intraperitoneally with a mixture of alfaxalone (60 mg/kg, Careside, Gyeonggi-do, Korea) and xylazine (5 mg/kg, Bayer, Seoul, Korea) before irradiation. The survival of the mice was checked daily, and the mice were euthanized at 6, 12, and 16 months. The control mice were anesthetized, but not irradiated. Individual identification of mice was performed by piercing their ears.

Processing and histochemical examination

The mice from all three groups (n = 1–4 animals per group and experiment) were euthanized using CO₂ asphyxiation, and the cardiac tissues were collected 6, 12, and 16 months post irradiation. The tissues were fixed with 4%-phosphate buffered formalin, embedded in paraffin, and sectioned. The paraffin sections of 5 µm thickness were stained with hematoxylin-eosin to determine the histomorphometry of the irradiated cardiac tissue.

Masson and trichrome staining

The tissue sections were hydrated using graded ethanol solutions (100%, 90%, 80%, and 70%), incubated in Bouin’s solution for 60 min at 60°C, and washed with running water to remove the yellow color. After staining with Weigert’s iron hematoxylin, the slides were washed with running water for 10 min at room temperature. Then, the slides were washed in distilled water and stained in Biebrich scarlet-acid fuchsin solution for 15 min.

After further washing, the slides were stained with phosphomolybdic acid/phosphotungstic acid for 10 min and aniline blue for 10 min. The slides were then rinsed in 1% acetic acid solution, dehydrated, and mounted using a permanent mounting solution.

Immunohistochemistry

The paraffin sections of the control and the irradiated cardiac tissues were baked, deparaffinized in xylene for 8 min, and hydrated using graded ethanol solutions (100%, 90%, 80%, and 70%). Antigen retrieval was performed using a citrate buffer (Sigma-Aldrich, St. Louis, MO, USA) at a pH of 6. The slides were incubated in 0.3% H₂O₂ for 20 min, blocked with normal horse serum in phosphate-buffered saline (PBS), and incubated with mouse anti-alpha-smooth muscle actin (SMA; Santa Cruz Biotechnology, CA, USA) and mouse anti-myosin heavy chain (MHC; R&D system, MAB4470, 1:300) at 4°C overnight. After washing with PBS, the slides were incubated with a secondary antibody (biotinylated anti-mouse IgG; Vector Laboratories Inc., Burlingame, CA, USA) at room temperature for 30 min. Thereafter, the slides were washed with PBS for 5 min and then incubated with prepared VECTASTAIN Elite ABC Reagent as the vector for 30 min. The slides were then incubated with 3–3'-diaminobenzidine (vector) for 5–8 min, counterstained with Mayer’s hematoxylin (Abcam), dehydrated and occluded using a consul-mount solution (Fisher Scientific, Hampton, NH, USA), and covered with a coverslip.

Tissue analysis

The images of the stained tissue were observed using pathology slide scanners and the Aperio image scope software (Leica, Wetzlar, Germany) to determine the structural changes in the irradiated cardiac tissue by comparing the irradiated tissue with tissue from the control group.

The fibrotic area of the cardiac tissue was observed under a light microscope (Nikon Eclipse 80i; Nikon Corporation; Tokyo, Japan) with separate areas for the left ventricle (LV), septum, and right ventricle (RV). The images were quantified using Image J software (NIH, Bethesda, MD, USA).

Statistical analysis

All experimental data are expressed as the means ± standard errors of the mean. The comparisons between the values were performed using one-way analysis of variance with Scheffe's post-hoc tests. All statistical analyses were performed using PASW (SPSS Inc., Chicago, IL, USA), and the statistical significance was set at P < 0.05.

FLASH-irradiated mice showed a higher survival rate than CONV-irradiated mice

Figure 1 shows the overall flow of FLASH- and CONV-irradiation. The examined mice were monitored daily, and the survival curve in Fig. 2 shows the animals that died during the experiment. Figure 2 shows that upon administering an irradiation dose of 20 Gy over 350 days, 2 and 1 mice died in the CONV and FLASH groups, respectively. By the 465th day of irradiation, 10 Gy CONV and 20 Gy FLASH resulted in a higher survival rate. These results show that the survival rate with FLASH is high under high-dose irradiation.

No differences in the morphological changes of the cardiac tissue were observed between CONV- and FLASH-irradiated mice at approximately 12 months

The area of fibrosis in the cardiac tissue of the irradiated mice was evaluated using Masson’s trichrome staining (MT) images at 6 and 12 months post irradiation and measured using histomorphometry (Figs. 3 and 4). As shown in Fig. 3, the collagen deposition in the cardiac tissue of CONV- and FLASH-irradiated mice showed no changes compared with that in the controls. As shown in Fig. 4, some collagen accumulation was observed in the LV, septum, and RV of CONV- and FLASH-irradiated mice 12 months post irradiation compared with the controls; however, there were no differences in the MT staining (Fig. 4a) and quantified data (Fig. 4b–d) between the CONV and FLASH groups.

Next, we determined the fibroblast activation of the endothelial cells and cardiomyocytes within the cardiac tissue post irradiation. Immunohistochemical staining of the sections with anti-alpha SMA was performed to assess radiation-induced cardiac fibrosis between the CONV and FLASH groups. The SMA + area of the cardiac tissue increased in the septum and RV of the CONV- and FLASH-irradiated mice compared with the controls; however, the CONV and FLASH groups showed similar tendencies, and there were no differences 12 months post irradiation (Fig. 5).

Radiation-induced cardiac fibrosis was less observed in FLASH-irradiated mice than in CONV-irradiated mice

We compared cardiac tissue toxicity between the C57BL/6 mice irradiated with CONV and FLASH at doses of 10 and 20 Gy each. Sixteen months post irradiation with CONV and FLASH, the mice were sacrificed, and the collagen deposition in the fibrous cardiac tissue was assessed using MT staining.

The cardiac tissue in the LV, septum, and RV irradiated with CONV and FLASH was partly replaced by fibrous scar tissue and tended to show collagen deposition as the radiation dose increased (Fig. 6a). Figure 6b shows the results of epicardial thickness quantification at 16 months post irradiation. Furthermore, the 20 Gy CONV (2.0 ± 0.2 µm) group showed a significantly higher epicardial thickness of irradiated cardiac tissue (P < 0.05) than the control (0.4 ± 0.1 µm), 10 Gy FLASH (1.1 ± 0.1 µm), and 20 Gy FLASH (1.3 ± 0.2 µm) groups. The fibrotic area in the LV and septum increased in the 20 Gy CONV group compared with the other groups; however, the difference was not statistically significant (Fig. 6c, 6d).

As shown in Fig. 6e, the 20 Gy CONV (7.7 ± 0.9%) group showed excessive formation of fibrous scar tissue and a significantly higher fibrotic area in the RV than the control (0.4 ± 0.1%), 10 Gy FLASH (3.2 ± 0.1%), and 20 Gy FLASH (4.2 ± 0.4%) groups (P < 0.05). These results indicated that the fibrotic area and collagen deposition in the RV increased the most post irradiation with 20 Gy CONV.

CONV-irradiated mice showed greater radiation-induced cellular response than FLASH-irradiated mice

The cardiac tissue sections were obtained from mice irradiated with 10 and 20 Gy CONV and FLASH at 16 months. These sections were stained with SMA. CONV radiation led to the fibroblast activation of cardiomyocytes and endothelial-to-mesenchymal transition within the cardiac tissue of the LV, septum, and RV compared with FLASH and control radiation at 16 months post irradiation (Fig. 7a). The quantified data of the SMA + area in the septum of the CONV-irradiated mice (10 Gy: 1.8 ± 0.5%, 20 Gy: 3.2 ± 1.1%; n = 6) and FLASH-irradiated mice (10 Gy: 2.8 ± 0.4%, 20 Gy: 2.6 ± 0.6%; n = 6) showed a gradual dose-dependent increase compared with the controls (0.3 ± 0.2%); however, this was not significant (Fig. 7b). Compared with the controls (2.5 ± 0.74%), the SMA + area in the RV of the CONV-irradiated mice (10 Gy: 4.0 ± 0.6%, 20 Gy: 14.9 ± 1.5%; n = 6) and FLASH-irradiated mice (10 Gy: 5.1 ± 0.9%, 20 Gy: 6.2 ± 0.4%; n = 6) showed a similar tendency to that observed in the fibrosis area of the LV (Fig. 7c). Notably, the SMA + area in the RV increased slightly in the 20 Gy CONV group (P < 0.05). These findings suggested that radiation-induced cardiac fibrosis can be observed more frequently in the RV than in the septum and it may also occur more frequently with CONV radiation than with FLASH radiation.

The protective effect of FLASH was better distinguished from that of CONV in the muscle structure than in the vascular structure

After confirming the radiation-induced fibrosis of the cardiac tissue at 16 months of irradiation (Figs. 6 and 7), the thickness and microvascular structure of the blood vessel wall were visualized. As shown in Fig. 8, vessel wall thickness and collagen deposition in the arteries increased in the CONV and FLASH groups compared with the control group; however, the difference was insignificant. The cardiac tissue irradiated with FLASH showed increased volume and enlargement of the muscle fibers and radiation-induced hypertrophy of the myocardium. In contrast, CONV irradiation decreased the volume and size of the muscle fibers, and radiation-induced atrophy was observed (Fig. 8). Therefore, the radiation-induced cellular responses occurred chronically, and the myocardial changes, more frequently observed in the CONV group, were irreversible.

In this study, FLASH radiation [17, 18] was compared with CONV radiation to examine the effect of reducing cardiac toxicity, and the degree of cardiac toxicity caused by radiation was measured by evaluating parameters such as cardiac fibrosis. FLASH radiation appears to have a protective effect on normal tissues, potentially reducing the toxicity of radiation therapy [19, 20]. The initial results of a study on FLASH radiotherapy were shown to be promising, but further studies are needed to fully evaluate its effectiveness [21].

Cardiac fibrosis is a potential side effect of radiation exposure, which can lead to excessive buildup of collagen in the cardiac tissue [22, 23]. Radiation exposure can also cause inflammation of the cardiac tissue, which can activate immune cells such as fibroblasts, producing more collagen and contributing to the fibrotic process [24, 25].

Our results suggested that the effect of radiation on mouse survival rates not only varied in a dose-dependent manner but possibly also according to the type of radiation exposure. At the higher dose of 20 Gy, the survival rate of the FLASH-irradiated group was higher than that of the CONV- irradiated group (Fig. 2).

In addition, the difference in cardiac fibrosis according to FLASH and CONV irradiation may be seen clearly when observed for a long period of time at the high dose of 20 Gy. Our results showed that there was difference in the damage depending on the area of the cardiac tissue that was irradiated, and in addition, that radiation mostly affected the RV. Several researchers have reported that post cardiac irradiation, the RV was more greatly affected by radiation than the LV [24, 26, ]. Other sequelae of cardiac fibrosis that have been observed are vascular damage and structural changes in the muscles due to blood flow damage [27, 28].

In addition, radiation-induced vascular damage in the cardiac tissue was supported by the results of this study. There was a tendency for vascular damage 16 months post CONV or FLASH irradiation (Fig. 8). However, further clarification of vascular changes at the molecular level is needed. Moreover, the size of cardiomyocytes, which comprise the most abundant cell type in the myocardium, determines hypertrophy and atrophy depending on the signaling pathway, and this phenomenon is determined by protein synthesis and degradation of cardiac muscle [29, 30]. After irradiation, the muscle structure of cardiac tissue changed, and atrophy was observed in CONV-irradiated mice and hypertrophy in FLASH-irradiated mice. Whether this is caused by other mechanisms or muscle damage should be studied further.

These results suggested the development of RT through the biological evaluation of mouse cardiac tissue using FLASH irradiation. The reduction in normal tissue side effects using FLASH irradiation may play an important role in starting a new paradigm for RT.

This study had some limitations. First, the investigation method of this study was limited to the murine model. Second, this study used a much higher dose than that frequently used in clinical practice to confirm the protective effect of the FLASH irradiation method on tissue. These experimental methods may also pose difficulties with the application of the results to clinical practice. Compared with the tissue response to CONV irradiation, the tissue response to FLASH irradiation has not yet been clarified at the molecular level; therefore, future studies need to further investigate its relevance.

In conclusion, FLASH irradiation was more effective than CONV at higher doses in terms of protective effect and cardiac tissue tolerance. The purpose of this study was to evaluate the efficacy, safety, and potential side effects of FLASH radiation therapy in animal models prior to application in human patients. Therefore, the data on cardiac tissue changes in mice after FLASH irradiation in this study can be used as a reference for predicting and measuring cardiotoxicity in future preclinical and clinical applications of FLASH treatment.

Acknowledgments

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (No.2020R1F1A1071857, No. 2020R1F1A1070824, No.2021R1F1A1057443, No.2022R1F1A1076225) and the Dongnam Institute of Radiological and Medical Sciences (DIRAMS) grant funded by the Korean government (MSIT) (50604–2023).

Funding: This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (No.2020R1F1A1071857, No. 2020R1F1A1070824, No.2021R1F1A1057443, No.2022R1F1A1076225) and the Dongnam Institute of Radiological and Medical Sciences (DIRAMS) grant funded by the Korean government (MSIT) (50604–2023).

Competing interests: The author(s) declare no competing interests. Data availability: All data generated or analysed during this study are included in this published article and its supplementary information files.

Authors' contributions: W.J. conceived and planned the experiments. M.-Y.C., H.K., K.W.J., J.Y.K, and W.J. carried out the experiments. S.K.K., S.J.L., H.L., M.L., D.H.J., T.Y., C.W.C, and Y.M.M. planned and carried out the stimulation. W.J. and M.-Y.C. contributed to sample preparation. K.W.J. and J.Y.K. contributed to the interpretation of the results. M.-Y.C. contributed most to the writing of the manuscript. All authors provided critical feedback and helped shape the research, analysis, and writing of the manuscript. All authors read and approved the final manuscript.

Consent to participate: Not applicable.

Consent for publication: Not applicable.

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No competing interests reported.

SupplementaryMaterial1.docx
Supplementary Information Supplementary Table S1.

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Mouse cardiac tissue received greater protection from and had better tolerance to the maximum dose of FLASH versus conventional radiation

Status:

Version 1

Abstract

Figures

Introduction

Methods

Irradiation devices and dosimetry

Mice and animal care

Irradiation

Processing and histochemical examination

Masson and trichrome staining

Immunohistochemistry

Tissue analysis

Statistical analysis

Results

FLASH-irradiated mice showed a higher survival rate than CONV-irradiated mice

Radiation-induced cardiac fibrosis was less observed in FLASH-irradiated mice than in CONV-irradiated mice

CONV-irradiated mice showed greater radiation-induced cellular response than FLASH-irradiated mice

Discussion

Declarations

References

Additional Declarations

Supplementary Files

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