Tolerance of mouse cardiac tissue to the maximum dose of FLASH versus conventional radiation

doi:10.21203/rs.3.rs-3346651/v1

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Tolerance of mouse cardiac tissue to the maximum dose of FLASH versus conventional radiation

https://doi.org/10.21203/rs.3.rs-3346651/v1

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Background: FLASH radiation (average dose rate: >40 Gy/s) delivers ultrahigh doses of radiation, which are several times higher than the clinically used conventional (CONV) radiation doses (average dose rate: >5 Gy/min). This study aimed to investigate the potential benefits of FLASH radiation therapy and compare the effects of FLASH radiation at the maximum dose and CONV radiation on mouse cardiac tissue.

Methods: C57BL/6 mice were divided into three groups: control (0 Gy), FLASH (40 Gy/s), and CONV (0.067 Gy/s). The mice treated with 10 and 20 Gy of CONV or FLASH radiation were euthanized at 6, 12, and 16 months. The effect of FLASH radiation was evaluated by plotting the survival curve of irradiated mice. Radiated cardiac tissues were examined using Masson’s trichrome staining (MT) and immunohistochemistry. Fibrous scar formation and alpha-smooth muscle actin-positive areas were quantitatively evaluated separately in the left ventricle, septum, and right ventricle (RV).

Results: Shorter survival was observed in the 20 Gy CONV irradiation group than in the 20 Gy FLASH irradiation group. Histological evaluation revealed higher radiation-induced cardiac fibrosis in the RV of the 20 Gy CONV group than in the control, 10 Gy CONV and FLASH, and 20 Gy FLASH groups (P<0.05).

Conclusions: FLASH radiation was more effective than CONV radiation at higher doses and displayed a protective effect and greater cardiac tissue tolerance. The results of this study will help improve cancer treatment outcomes, reduce radiation side effects, shorten treatment duration, and add to radiation biology literature.

animals

actins

immunohistochemistry

radiobiology

Ionizing radiation has been used for decades to treat cancer in more than 50% of all patients, and technical advancements in radiotherapy (RT) have improved treatment efficiency significantly [1, 2]. While conventional (CONV) dose rate RT reduces the tumor size, it also results in radiation-induced toxicity in the surrounding normal tissue [3–5]. Moreover, although thoracic RT is used to treat breast, esophageal, and lung cancer, the cardiac tissue, which is highly oxygen-dependent, is adversely affected by 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]. As 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 those delivered in CONV radiation (average dose rate: > 5 Gy/min) [10, 11]. Compared to CONV radiation, FLASH radiation has been reported to reduce the tumor size while sparing the surrounding normal tissues. Thus, although FLASH radiation has been studied as a potential RT alternative in preclinical models of radiation-induced lung fibrosis, suppression of hematopoiesis, and neurocognitive deficits, among others, the mechanism underlying the radiotherapeutic effects of FLASH radiation 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 several radiographic studies involving mouse models have been conducted in the past 10 years [15, 16], further studies on the biophysical correlation of clinical disease with mouse models are needed.

This study aimed to investigate 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 could 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 linear accelerator (LINAC) at the Dongnam Institute of Radiological and Medical Sciences (DIRAMS). 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 involving a radiochromic film (GAFchromic™ MD-V3, Ashland, NJ, USA) that supports 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 3.08×10⁵ Gy/s, respectively [17, 18]. CONV radiation was performed using a shielding block that allowed only the cardiac tissue to be irradiated. The irradiation conditions and experiments that were performed are outlined in Additional file 1.

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 and reported according to the ARRIVE guidelines.

Irradiation

The 7-week-old C57BL/6N male mice were randomly divided into three groups: 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). Other than those in the control group, each mouse received a single fraction dose (10 and 20 Gy) of irradiation. All mice were anesthetized intraperitoneally with a mixture of alfaxalone (60 mg/kg, Careside, Gyeonggi-do, Korea) and xylazine (5 mg/kg, Bayer, Leverkusen, Germany) before irradiation. The control mice were anesthetized but not irradiated. Individual identification of mice was performed by piercing their ears.

Survival analysis

The survival of the mice was observed for up to 16 months after irradiation, and euthanasia was performed at 6, 12, and 16 months (n = 2–4 animals per group) after irradiation for analysis. The survival rates were determined by calculating the percentage of dead mice per surviving mouse. All mice were euthanized via CO₂ asphyxiation for tissue harvesting.

Processing and histochemical examination

The cardiac tissues from mice in all three groups were collected at 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. Then, the slides were 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 pH 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, Santa Cruz, CA, USA) and mouse anti-myosin heavy chain (MHC; MAB4470, 1:300, R&D System, Minneapolis, MN, USA) 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 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, Cambridge, UK), 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 (IBM Corp., Armonk, NY, 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 percentage of animals that died during the experiment. Figure 2 shows that upon administering an irradiation dose of 20 Gy, two and one mice died in the CONV and FLASH groups, respectively, within 350 days. By the 465th day after irradiation with 10 Gy CONV or 20 Gy FLASH, a higher survival rate was observed among the irradiated mice. 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 fibrotic area in the cardiac tissue of the irradiated mice was evaluated using images of the MT-stained tissues 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 significant changes compared with that in the control mice. As shown in Fig. 4, some collagen accumulation was observed in the LV, septum, and RV of CONV- and FLASH-irradiated mice at 12 months post irradiation compared with the control mice; however, there were no significant 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-positive 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 between the groups at 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 radiation at doses of 10 and 20 Gy each. At 16 months after CONV and FLASH irradiation, the mice were sacrificed, and 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 radiation was partly replaced by fibrous scar tissue and tended to show increased collagen deposition with increasing radiation dose (Fig. 6a). Figure 6b depicts the epicardial thickness 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, d).

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 did (P < 0.05). These results indicated that the fibrotic area and collagen deposition in the RV increased the most after 20 Gy CONV radiation.

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 radiation and control radiation at 16 months post irradiation (Fig. 7a). The quantified data of the SMA-positive 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 control mice (0.3 ± 0.2%); however, this was not significant (Fig. 7b). Compared with the controls (2.5 ± 0.74%), the SMA-positive 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) were similar in size compared to that observed in the fibrotic area of the LV (Fig. 7c). Notably, the SMA-positive area in the RV slightly increased 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 radiation 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, which were observed more frequently in the CONV group, were irreversible.

In this study, the effect of FLASH radiation [17, 18] was compared with that of CONV radiation in terms of the degree of cardiac toxicity caused by radiation 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, thereby 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 also varied 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 caused by FLASH and CONV irradiation may be observed clearly when cardiac tissues exposed to the high dose of 20 Gy are observed over a long period. Our results showed that there was difference in the damage depending on the area of the cardiac tissue that was irradiated and 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 was [24, 26]. Other sequelae of cardiac fibrosis that have been observed are vascular damage and structural changes in the muscles owing to blood flow damage [27, 28].

In addition, radiation-induced vascular damage in the cardiac tissue was supported by the results of this study. Vascular damage was observed at 16 months after 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 whereas hypertrophy was observed in FLASH-irradiated mice. Whether this event is caused by other mechanisms or muscle damage should be investigated further.

The finding that high-dose FLASH-irradiated mouse heart tissue is less prone to radiation-induced toxicity compared to CONV irradiation confirms the benefits of advancing RT technology. The reduction in side effects impacting the normal tissue exposed to FLASH radiation may play an important role in developing a new strategy for RT.

This study had some limitations. First, the investigative 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 the 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 should further investigate the tissue response to FLASH irradiation and its relevance.

FLASH radiation was more effective than CONV radiation 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 RT 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.

DIRAMS Dongnam Institute of Radiological and Medical Sciences

LINAC Linear accelerator

LV Left ventricle

MHC Myosin heavy chain

MT Masson’s trichrome

PBS Phosphate-buffered saline

RF Radio frequency

RV Right ventricle

SMA Smooth muscle actin

Ethics approval and consent to participate: 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). Consent to participate is not applicable, as the study did not involve any human participants.

Consent for publication: Not applicable.

Availability of data and materials: All data generated or analyzed during this study are included in this published article and its supplementary information files.

Competing interests: The authors declare no competing interests.

Funding

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (Grant 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).

Authors’ contributions: TY and WJ conceived and planned the experiments. MYC., HK, KWJ, JYK, and WJ carried out the experiments. SKK, SJL, HL, ML, DHJ, CWC, and YMM planned and carried out the stimulation. WJ and MYC contributed to sample preparation. KWJ and JYK contributed to the interpretation of the results. MYC 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.

Acknowledgements

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No competing interests reported.

SupplementaryMaterial1.docx
Additional File File name: Additional file 1 File format: .docx Title of data: Comparison of the irradiation conditions between FLASH and conventional radiation Description of data: The table presents a summary of irradiation conditions and experiments for FLASH and conventional radiation.

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Tolerance of mouse cardiac tissue to the maximum dose of FLASH versus conventional radiation

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Version 1

Abstract

Figures

Background

Methods

Irradiation devices and dosimetry

Mice and animal care

Irradiation

Survival analysis

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

Conclusions

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

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