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Increasing evidence suggests that ultra-high-dose-rate (UHDR) radiation could result in similar tumor control as conventional (CONV) radiation therapy (RT) while reducing toxicity to surrounding healthy tissues. Considering that radiation toxicity to gonadal tissues can cause hormone disturbances and infertility in young patients with cancer, the purpose of this study was to assess the possible role of UHDR-RT in reducing toxicity to healthy gonads in mice compared with CONV-RT.
Methods and Materials
Radiation was delivered to the abdomen or pelvis of female (8 or 16 Gy) and male (5 Gy) C57BL/6J mice, respectively, at conventional (∼0.4 Gy/s) or ultrahigh (>100 Gy/s) dose rates using an IntraOp Mobetron linear accelerator. Organ weights along with histopathology and immunostaining of irradiated gonads were used to compare toxicity between radiation modalities.
Results
CONV-RT and UHDR-RT induced a similar decrease in uterine weights at both studied doses (∼50% of controls), which indicated similarly reduced ovarian follicular activity. Histologically, ovaries of CONV- and UHDR-irradiated mice exhibited a comparable lack of follicles. Weights of CONV- and UHDR-irradiated testes were reduced to ∼30% of controls, and the percentage of degenerate seminiferous tubules was also similar between radiation modalities (∼80% above controls). Pairwise comparisons of all quantitative data indicated statistical significance between irradiated (CONV or UHDR) and control groups (from P ≤ .01 to P ≤ .0001) but not between radiation modalities.
Conclusions
The data presented here suggest that the short-term effects of UHDR-RT on the mouse gonads are comparable to those of CONV-RT.
Introduction
Ionizing radiation is an effective treatment for cancer, but its curative potential is limited by a variety of acute and late toxic effects on normal tissues.
FLASH radiation therapy (RT), which is defined as ultra-high dose rate radiation (UHDR-RT) (>40 Gy/s), has been recently shown to result in reduced toxicity to healthy tissues while maintaining similar tumor control as conventional (CONV) RT (∼0.1 Gy/s) in preclinical models.
Thus, the use of FLASH-RT could lead to an increase in therapeutic ratio with greater potential for tumor control. The in vivo biological response of normal tissue sparing is referred to as “the FLASH effect” and has been reported mostly using electron and proton beams.
The molecular mechanisms underlying this observed protective effect are largely unknown, but proposed mechanisms include differential regulation of pro- and anti-inflammatory molecules, DNA damage and repair, apoptosis, and senescence to list a few.
The existing preclinical data uniformly support the notion that FLASH-RT provides comparable tumor control as CONV-RT, although the evidence concerning the proposed reduced toxicity of FLASH on normal tissues is less compelling. Most observations to date are derived from mouse studies using moderately radiosensitive tissues, namely the brain, the lung, and the skin.
Reduced cognitive deficits after FLASH irradiation of whole mouse brain are associated with less hippocampal dendritic spine loss and neuroinflammation.
Despite a growing body of evidence pointing to the existence of a FLASH effect, conflicting data continue to emerge as a number of studies have failed to demonstrate sparing across different systems, tissues, and species.
Ultra high dose rate (35 Gy/sec) radiation does not spare the normal tissue in cardiac and splenic models of lymphopenia and gastrointestinal syndrome.
Perstin A, Poirier Y, Sawant A, Tambasco M. Physics contribution quantifying the DNA-damaging effects of FLASH irradiation with plasmid DNA. 2022;113:437-447.
Discrepancies in study results have been attributed to a lack of homogeneity on the irradiation beam parameters used across experiments, and recommendations on standards for uniformly reporting physical aspects of FLASH have been made to contribute to alleviate this issue.
In addition to male and female infertility and the detrimental consequences of premature ovarian failure in women, endocrine dysfunction can impair proper sexual development in prepubertal children undergoing treatment for hematologic malignancies.
Given the general lack of preclinical studies assessing a possible protective role of FLASH-RT on healthy gonads, the present study was conducted to evaluate the effects of a single fraction of UHDR-RT compared with CONV-RT on the mouse female and male reproductive organs as models of acute radiation injury. Intriguingly, analysis of organ weights along with thorough histopathologic and immunohistochemical evaluation of irradiated tissues suggested that the short-term effects of UHDR-RT on the mouse gonads are comparable to those of CONV-RT.
Methods and Materials
Mouse models and care
Mouse usage was approved by the Institutional Animal Care and Use Committee at the Ohio State University. C57BL/6J mice were purchased from The Jackson Laboratory at 6 weeks of age and allowed to acclimate for a minimum of 2 weeks before irradiation. Mice were housed under normal husbandry conditions (≤5 animals per cage) in a vivarium with a 12-hour light/dark cycle. For high-dose abdominal irradiation, body weight and overall health condition were assessed by applying the Mouse Intervention Scoring System 3 following recommended removal criteria.
Adult (2-4 months old) C57BL/6J mice were irradiated at both UHDR and CONV dose rates using a Mobetron (IntraOp, Sunnyvale, CA) linear accelerator. Detailed information on irradiation beam parameters is presented in Table E1. Mice were anesthetized with isoflurane in air and positioned supine in a custom 3-dimensionally printed polylactic acid plastic immobilization device (Fig. 1A, 1B). A 6-mm thick, 3.5- × 4-cm copper collimator was used to target radiation to the abdominal (female) and pelvic (male) regions (Fig. 1A, 1C). For abdominal irradiation in female mice, the xiphoid process of the sternum was used to mark the cranial end of the irradiation field (Fig. 1B). For pelvic irradiation in male mice, the scrotal area was placed in the center of the irradiation field (Fig. 1B). GafChromic EBTXD (Ashland Advanced Materials, Bridgewater, NJ) films were placed beneath the irradiated region for dosimetry and the exit dose was recorded for each mouse (Fig. 1D, Table E1). Percent depth dose (PDD) as well as lateral and longitudinal dose profiles at depth of maximum dose (dmax) were acquired using GafChromic film in Solid Water phantom slabs (Fig. 1E).
Figure 1Mouse irradiation setup. (A) Plastic immobilization device comprising a base (right) and a top piece secured to a copper collimator (left). A 3.5- × 4-cm
opening within the top piece ensures reproducible placement of the collimator. (B) Plastic immobilization device holding an anesthetized mouse (left) before securing the copper collimator. The blue outline on the left panel indicates the placement of the 3.5- × 4-cm2 abdominal irradiation field (female). The red laser pointer on the right panel indicates the center of the 3.5- × 4-cm2 pelvic irradiation field (male). GafChromic film (marked by asterisk in panel A) was placed underneath the mouse for exit dosimetry. (C) Fully assembled immobilization device and setup configurations (Conf) for Conf35 (left) and Conf18 (right). (D) Example of an exit dosimetry film (top) and corresponding isodose map (bottom) on the pelvic region of a male mouse. (E) Percent depth dose (PDD) and longitudinal and lateral dose profiles acquired at maximum dose using GafChromic film in solid water phantom slabs.
The Mobetron is a mobile electron linear accelerator capable of delivering 9 MeV electrons at conventional dose rates of the order of ∼10 Gy/min as well as UHDRs >40 Gy/s. Two different source-to-surface distance (SSD) configurations were used in the irradiations, with collimator placement at the exit window of the linear accelerator (SSD = 18.3 cm, Conf18) or with an applicator (SSD = 35 cm, Conf35), as shown in Fig. 1C. The Mobetron delivers UHDR-RT at discrete combinations of pulse width, pulse repetition frequency (PRF), and number of pulses. The dose and dose rate in the UHDR realm are not independent parameters, and only a discrete set of doses and dose rates can be achieved using a particular collimator and SSD configuration. PDDs, dose profiles, and dose at dmax were obtained through film dosimetry in solid water for both energies at the 2 SSD configurations. The UDHR data are measured at 60 Hz PRF and 4 μs pulse width, and correction factors are applied to the dose per pulse when changing PRF and pulse width to obtain the intended dose and dose rate. The setup parameters used for the 3 sets of experiments included in this study are outlined in Table E1. Representative PDDs and profile data are shown in Fig. 1E.
Approach to the analysis of ovarian toxicity
Given that counting ovarian follicle numbers accurately can be challenging, the quantitative assessment of radiation-induced ovarian follicle loss in this study was performed indirectly by measuring changes in uterine weight.
The effect of raloxifene on the uterine weight response in immature mice exposed to 17beta-estradiol, 1,1,1-trichloro-2, 2-bis(p-chlorophenyl)ethane, and methoxychlor.
In mice undergoing normal estrus cycles, late tertiary ovarian follicles secrete estradiol which causes cyclical increases in uterine weight. Radiation induced loss of ovarian follicles is expected to lead to reduced estradiol levels and subsequent decreases in uterine weight that manifest with variable latency depending on the radiation dose delivered. Qualitative assessment of ovarian follicle damage was performed histologically as well as by immunohistochemistry (see Histopathologic Analyses for details on classification of follicles). Ovarian follicle sensitivity to radiation varies depending on the follicle's maturation stage.
In this study, a dose ≥16 Gy induced immediate (within days) degeneration and loss of all follicle stages including existing late tertiary follicles. Thus, changes in uterine weight at this dose were noticeable within a few days after irradiation. Day 6 postirradiation was defined as the endpoint for this dose as mice receiving 16 Gy experience gastrointestinal syndrome and meet early removal criteria around this time. In contrast, a dose ≤8 Gy caused immediate loss of small follicles while preserving existing (secondary and tertiary) follicles with no immediate changes in uterine weights. Thus, this dose was used to study radiation induced loss of small follicles. Uterine weights were evaluated 2 months after irradiation at this dose based on follicular maturation time (from primordial to late tertiary), which is estimated to be about 1.5 months in mice. Under this premise, a latency period of at least 2 months would be sufficient for small follicles surviving 8 Gy to grow into tertiary follicles and exert estrogenic influence on the uterus.
For all experiments, fresh uterine weights were recorded at necropsy and are presented as percentage of body weight except for 16 Gy. Weight loss occurs acutely at the latter dose, making comparison to unirradiated controls less reliable; thus, uterine weight data are presented in milligrams instead.
Approach to the analysis of testicular toxicity
Testicular weights were used as a readout of radiation-induced toxicity. Due to the exquisite sensitivity of germ cells to radiation, a dose of 5 Gy was chosen for these experiments to allow for survival of at least a subset of spermatogonia and subsequent evaluation of tubular repopulation.
The endpoint for assessing radiation toxicity on testicular germ cells was set at 35 days postirradiation as it takes mouse spermatogonia ∼35 days to mature into spermatozoa and achieve some level of restitution of the seminiferous epithelium.
For all experiments, fresh testis weights were recorded at necropsy and are presented as percentage of body weight. The testis and epididymis were subjected to histopathologic evaluation. The percentage of seminiferous tubules exhibiting degeneration was used as additional quantitative evidence of radiation-induced testicular toxicity (see Histopathologic Analyses).
Tissue preparation and histology
Tissue used for histology was fixed with 10% pH-buffered formalin (23-245-685; Fisher Scientific) for 48 to 72 hours at room temperature, embedded in paraffin, and cut into 4-μm sections for staining with hematoxylin and eosin (H&E).
Histopathologic analyses
Ovarian toxicity
Histopathologic evaluation of ovaries and uteri was performed by a board-certified veterinary pathologist (M.C.C.) following recommended nomenclature.
Ovarian follicle damage was evaluated qualitatively on representative middle sections of ovaries stained with H&E, which was complemented by immunohistochemical staining with an oocyte marker (mouse vasa homologue [MVH]) to highlight the presence or absence of oocytes within follicles. Follicle stages were identified according to morphologic criteria as primordial (oocyte surrounded by a single layer of flattened pregranulosa cells), primary (oocyte surrounded by a single layer of cuboidal to columnar granulosa cells), secondary (≥2 layers of granulosa cells and a zona pellucida between oocyte and granulosa cells), tertiary (multiple layers of granulosa cells and small antrum), and late tertiary follicles (large antrum, oocyte and corona radiata are no longer attached to the wall of the follicle).
In this study, primordial and primary follicles are also collectively referred to as small follicles whereas secondary and tertiary follicles are referred to as medium/large follicles. The histomorphology of the uterus is dependent on hormonal changes and can be correlated with specific phases of the estrus cycle and cycle abnormalities in rodents.
These histologic features were used in this study as additional evidence of radiation-induced ovarian toxicity to facilitate comparisons between CONV- and UHDR-irradiated mice.
Testicular toxicity
The testis and epididymis were subjected to histopathologic evaluation by a board-certified veterinary pathologist (M.C.C.) following standard nomenclature.
Quantification of the percentage of degenerate seminiferous tubules was done using micrographs of H&E-stained sections. Tubules exhibiting an intact epithelium containing spermatogonia, spermatocytes, and spermatids were counted as nondegenerate. Sertoli cell-only tubules (tubules completely lacking germ cells) as well as tubules exhibiting partial depletion of germ cells, degenerating or apoptotic germ cells, or Sertoli cell cytoplasmic vacuolation were classified as degenerate.
The percentage of degenerate tubules was estimated from observing at least 120 to 150 tubules per mouse.
Immunostaining
Immunostaining was performed using a Lab Vision Autostainer 360 (Thermo Scientific) as per manufacturer's instructions. Briefly, a citrate-based solution (S1699; Dako) was used for epitope retrieval at ∼95°C in a steamer for 20 minutes. A 3% H2O2/PBS solution was used for endogenous enzyme block. Once in the autostainer, slides were incubated with 2.5% normal horse serum for 20 minutes then incubated with rabbit anti-MVH (Ab13840; Abcam) diluted to 1 µg/mL in Dako Antibody Diluent (S3022) for 30 minutes, rinsed, and then incubated with ImmPRESS Horse Anti-Rabbit HRP (MP-7401; Vector Laboratories) for 30 minutes. DAB (K3468; Dako) was applied for 2.5 minutes.
Statistical analysis
The Student t test was used for pairwise comparisons of mean organ weights and percent testicular degeneration. GraphPad Prism, version 9.2.0 (332) for Windows (GraphPad Software), was used for analysis. A P value <.05 was considered statistically significant.
Results
8-Gy UHDR-RT delivered in a single pulse induces similar acute ovarian toxicity as CONV-RT
To evaluate the level of toxicity of UHDR-RT on the ovary compared with CONV-RT, C57BL/6J mice were anesthetized and positioned supine in a custom 3-dimensionally printed polylactic acid plastic immobilization device. The abdomen was placed in the center of a 3.5- × 4-cm
irradiation field using a copper collimator (Fig. 1A-C). An IntraOp Mobetron linear accelerator was used to deliver radiation at both UHDR and CONV dose rates (Fig. 1C-E). We initially evaluated the effect of a dose of 8 Gy on small ovarian follicles when delivered in a single pulse at UHDR (2.31E+06 Gy/s) or CONV (0.436 Gy/s) dose rates. Detailed information on irradiation beam parameters is presented in Table E1. Radiation to the ovary is expected to cause loss of ovarian follicles. The quantitative assessment of radiation-induced ovarian follicle loss in this study was performed indirectly by measuring changes in uterine weight as the uterus responds to estradiol secreted by active ovarian follicles. A detailed description of the experimental approach to the analysis of ovarian toxicity, including the rationale for determining endpoints to measure uterine weights according to radiation dose, is provided in methods. Mice were euthanized 2 months postirradiation, fresh uterine weights were recorded, and the reproductive tract was subjected to histopathologic evaluation. On gross examination of the uterus, both UHDR- and CONV-irradiated mice exhibited slightly elongated uterine horns, which appeared smaller in diameter than control horns (Fig. 2A). Uterine weights of both UHDR- and CONV-irradiated mice were decreased to ∼50% of control mice, which suggested comparably reduced ovarian follicle activity (Fig. 2B). UHDR- and CONV-irradiated uteri exhibited histologic features of reduced ovarian follicular activity, namely a narrow lumen and an endometrium lined by low columnar epithelium (Fig. 2C). Features of estrogenic stimulation such as luminal dilatation were only observed in control mice (Fig. 2C). In agreement with these findings, the ovaries of both UHDR- and CONV-irradiated mice lacked follicles of all types (see Methods and Materials for classification of ovarian follicles) and were primarily composed of sheets of round cells reminiscent of ovarian interstitial cells (Fig. 2D). Immunohistochemistry for the MVH protein, a germ cell marker expressed in the cytoplasm of oocytes, highlighted the lack of oocytes within follicles in middle sections of ovaries (Fig. 2E). The absence of tertiary ovarian follicles 2 months after a dose of 8 Gy was interpreted to be the result of acute radiation toxicity to small follicles (see Methods and Materials for details on timing of follicular maturation). Late tertiary follicles are the source of estrogen and their absence in irradiated ovaries explains the reduced uterine weights and accompanying histologic features of reduced estrogenic stimulation. Taken together, these findings suggest that the acute toxic effects of single pulse UHDR-RT on the adult mouse ovaries are similar to those of CONV-RT.
Figure 2Effect of conventional (CONV) and ultra-high dose rate (UHDR) radiation therapy on the reproductive tract of female mice 2 months after receiving 8 Gy to the abdomen. (A) Macroscopic appearance of uteri and ovaries (indicated within dashed lines on control). Scale bar: 5 mm. (B) Uterine weights. Data are presented as mean ± SD; n = 5 to 6 mice per group; ns, P > .05; **, P ≤ .01. (C) Hematoxylin and eosin–stained sections of uteri. Lumen (asterisks) was narrow in CONV- and UHDR-irradiated uteri. Insets: higher magnification of surface epithelium. Tall columnar epithelium with necrotic debris (hormonally induced) is shown in control. (D) Hematoxylin and eosin–stained sections of ovaries. Normal follicles of different sizes composed of an oocyte (O) and surrounding granulosa cells (G) are apparent in control ovaries (smaller follicles [arrows] are magnified in inset). Follicles of all types are lacking in CONV- and UHDR-irradiated ovaries, which are composed of sheets of round cells (magnified in insets). (E) Mouse vasa homologue immunostaining of ovaries. Brown cytoplasmic staining highlights oocytes within follicles. Insets: higher magnification of boxed areas showing normal follicles of different types in control ovaries (arrows and arrowheads) and the lack of follicles of all types in CONV- and UHDR-irradiated ovaries. Note that the more mature oocytes in tertiary follicles stain faintly (arrowhead). Scale bars: 100 µm.
16-Gy UHDR-RT delivered in multiple pulses induces similar acute ovarian toxicity as CONV-RT
To evaluate the toxic effect of UHDR-RT on ovarian follicles when delivered in multiple pulses, 16 Gy was given to the abdomen of C57BL/6J female mice in UHDR (234 Gy/s) or CONV (0.436 Gy/s) dose-rate modes as previously described. Detailed information on irradiation beam parameters is presented in Table E1. All mice were euthanized 6 days postirradiation (see Methods and Materials for description of the experimental approach and determination of endpoints for evaluation of the reproductive organs). No difference in the percentage of body weight loss was observed between irradiated groups (∼22-25% of baseline at day 6). Fresh uterine weights were recorded, and ovaries and uteri were subjected to histopathologic evaluation. Upon gross examination, all irradiated uteri, regardless of the modality, were reduced in size and appeared thinner than control uteri (Fig. 3A). Uterine weights of both UHDR- and CONV-irradiated mice were decreased to ∼50% of control mice, suggesting similarly reduced ovarian activity compared with unirradiated uteri (Fig. 3B). As expected from these findings, all irradiated uteri exhibited histologic features of reduced ovarian follicular activity, such as a narrow lumen lined by cuboidal epithelial cells (Fig. 3C). In agreement with these findings, histologic analysis of ovaries of UHDR- and CONV-irradiated mice demonstrated a lack of intact follicles (including tertiary follicles) and corpora lutea (Fig. 3D). In contrast to control ovaries, only remnants of degenerate medium to large follicles were recognized in UHDR- and CONV-irradiated ovaries. Follicle remnants were composed of viable or degenerate oocytes lacking surrounding granulosa cells (Fig. 3D). Signs of degeneration observed in oocytes were nuclear pyknosis or karyorrhexis, hypereosinophilic cytoplasm, and/or cytoplasmic fragmentation (Fig. 3D). Immunohistochemistry for MVH highlighted oocytes within degenerate medium/large follicle remnants as well as the absence of small follicles (Fig. 3E). Small degenerate corpora lutea composed of vacuolated cells were also observed (Fig. 3D). Overall, these acute ovarian changes were indistinguishable between UHDR- and CONV-irradiated mice. In addition to depleting the more radiation-sensitive small follicles, this dose acutely damaged mature follicles which resulted in immediate reduction of uterine weights and accompanying histologic features of reduced estrogenic stimulation in all irradiated mice. Taken together, these results suggest that UHDR-RT delivered in multiple pulses induces similar acute ovarian toxicity as CONV-RT.
Figure 3Effect of conventional (CONV) and ultra-high dose rate (UHDR) radiation therapy on the reproductive tract of female mice 6 days after receiving a dose of 16 Gy to the abdomen. (A) Macroscopic appearance of uteri and ovaries (indicated within dashed lines on control). Scale bar: 5 mm. (B) Uterine weights. Data are presented as mean ± SD; n = 4 to 5 mice per group; ns, P > .05; **, P ≤ .01; ***, P ≤ .001. (C) Hematoxylin and eosin–stained sections of uteri. The lumen (asterisks) was narrow in CONV- and UHDR-irradiated uteri. Insets: higher magnification of surface epithelium. Tall columnar epithelium with mitotic figures (hormonally induced) is shown in control. (D) Hematoxylin and eosin–stained sections of ovaries. Intact follicles (boxes in control) or remnants of follicles (boxes in CONV- and UHDR-irradiated ovaries) are shown at higher magnification on bottom panels. Intact follicles are composed of oocytes (O) and granulosa cells (G). Remnants of follicles contain oocytes (presumably nondegenerate [left] and degenerate [right]) and lack granulosa cell layers. Small follicles (small oocyte and single layer of granulosa cells [arrows]) are only found in control ovaries. Only remnants of corpora lutea (CL) are visible in CONV- and UHDR-irradiated ovaries. (E) Mouse vasa homologue immunostaining of ovaries. Brown cytoplasmic staining highlights oocytes within intact follicles (boxes in control) or follicle remnants (boxes in CONV and UHDR). Mature oocytes in tertiary follicles stain faintly (arrowhead in control ovary). Oocytes within small follicles (arrows in control) are absent in CONV and UHDR ovaries. Scale bars: 100 µm.
5-Gy UHDR-RT delivered in a single pulse induces similar acute testicular toxicity as CONV-RT
To evaluate the effect of UHDR-RT on testicular germ cells when delivered in a single pulse, 5 Gy were delivered to the pelvis of adult mice in UHDR (2.35E+06 Gy/s) or CONV (0.436 Gy/s) dose rate modes (Table E1). To this end, the scrotal region of mice was placed in the center of a 3.5- × 4-cm irradiation field using a copper collimator as described for female mice (Fig. 1B). Mice were euthanized 35 days postirradiation, testicular weights were recorded, and the testes and epididymides were subjected to histopathologic evaluation (see Methods and Materials for description of the experimental approach and determination of endpoints). On gross examination, the testes of CONV- and UHDR-irradiated mice were significantly smaller than those of control mice, and testis weight was reduced to ∼30% of control mice for both modalities (Fig. 4A, 4B). Histologically, testis of CONV- and UHDR-irradiated mice exhibited widespread germ cell degeneration and loss (Fig. 4C). No sperm was observed in the epididymis of mice in either of the irradiated groups (Fig. 4C), indicating that spermatogenesis was similarly reduced. Immunostaining of testis sections for the MVH protein highlighted the marked, general reduction in the number of germ cells in all irradiated testes (Fig. 4D). In agreement with organ weight, the percentage of degenerate tubules was strikingly similar (∼80%) between UHDR- and CONV-irradiated mice (Fig. 4E), indicating that the acute toxic effects of UHDR-RT on spermatogenic cells at the studied dose and dose rate are comparable to those induced by CONV-RT.
Figure 4Effect of conventional (CONV) and ultra-high dose rate (UHDR) radiation therapy on the testis of mice 35 days after receiving a dose of 5 Gy to the pelvis. (A) Macroscopic appearance of testis. Scale bar: 5 mm. (B) Testis weight. (C) Hematoxylin and eosin–stained sections of testis and epididymis. Asterisks indicate examples of seminiferous tubules that are completely devoid of germ cells (magnified in insets on CONV and UHDR testes) as well as the lack of spermatozoa within the epididymis in CONV and UHDR testes. Spermatozoa are present in magnified inset on control. (D) Mouse vasa homologue immunostaining of testis highlights reduction in germ cell numbers in irradiated testes. (E) Percentage of seminiferous tubules exhibiting degenerative changes. Data in panels B and E are presented as mean ± SD; n = 3 to 4 mice per group; ns, P > .05; ****, P ≤ .0001. Scale bars: 100 µm.
The proposed normal tissue sparing effect of FLASH-RT has generated great interest among radiation scientists as the number of preclinical studies assessing this intriguing phenomenon continues to increase. Radiation injury to gonadal tissues can result in significant morbidities in young patients with cancer and, therefore, a possible role of UHDR-RT in protecting healthy gonads carries great translational impact.
To assess this possibility and to extend the existing data on FLASH-RT on acutely responding tissues, the present study evaluated the effects of a single fraction of electron radiation at UHDRs compared with conventional dose rates on the mouse female and male gonads. Analysis of organ weights along with histopathologic and immunohistochemical evaluation of irradiated tissues demonstrated that the short-term effects of UHDR-RT on the mouse gonads are comparable to those of CONV-RT.
Several levels of biological evidence are provided in this study that demonstrate similar gonadal toxicity between UHDR-RT and CONV-RT. Different parts of the female reproductive tract were assessed to comprehensively evaluate the magnitude of damage to the ovaries and facilitate comparison between radiation modalities. The uterus responds to changes in hormones secreted by the ovary and, as such, was used as a measure of ovarian follicular activity within appropriate time frames. We tested total doses >6 to 7 Gy because the sparing effect of FLASH-RT on normal tissues has been predominantly described above this range.
Considering radiation sensitivity by follicular type as well as follicular maturation time, a dose of 8 Gy was used to evaluate the effect of UHDR-RT on early follicular stages with protracted evaluation of uterine changes, whereas a dose of 16 Gy was used to assess toxicity of FLASH on mature follicles and its immediate effect on uterine weight and histopathology. Taken together, the data presented here demonstrated that UHDR- and CONV-RT damaged ovarian follicles similarly.
The applicability of FLASH-RT in clinical settings could be limited if only doses >7 Gy were able to confer normal tissue sparing. Few studies have evaluated the protective effect of UHDR-RT at doses relevant to fractionated or hypofractionated regimens currently used clinically.
Ultra high dose rate (35 Gy/sec) radiation does not spare the normal tissue in cardiac and splenic models of lymphopenia and gastrointestinal syndrome.
To this end, we evaluated the biological effect of a dose of 5 Gy on the mouse testis. Radiation-induced killing of germ cells translates into decreased testicular weights which were used in this study as a quantitative readout of radiation-induced toxicity. Due to the exquisite radiation sensitivity of spermatogenic germ cells, this dose was selected to allow survival of some spermatogonia and subsequent evaluation of tubular repopulation. Our data from organ weights along with the percentage of degenerate seminiferous tubules demonstrated that UHDR- and CONV-RT caused comparable damage to germ cells and the seminiferous epithelium overall.
Male and female germ cells are among the most radiation sensitive cell types in the body along with progenitor cells of the intestinal epithelium, hematopoietic progenitors, and lymphocytes.
The extreme radiosensitivity of some of these cell types, such as the intestinal epithelial, spermatogenic, and marrow cell lines, is linked to their high proliferation rates whereas in other cases, namely oocytes and lymphocytes, the reason for this extreme sensitivity is not fully understood.
Our results indicating a lack of sparing of UHDR-RT on the gonads of mice are not entirely unexpected as previous studies focusing on other highly radiation sensitive tissues have either failed to demonstrate an effect or have shown effects of a smaller magnitude than those observed in less radiosensitive tissues. For instance, Venkatesulu et al investigated the effect of single and fractionated UHDR-RT in mouse models of radiation-induced lymphopenia and reported equal depletion of lymphocytes between UHDR- and CONV-RT.
Ultra high dose rate (35 Gy/sec) radiation does not spare the normal tissue in cardiac and splenic models of lymphopenia and gastrointestinal syndrome.
Chabi et al analyzed the effect of FLASH-RT on human hematopoietic progenitor cells in mice with and without leukemia. Depending on the model, a spectrum of outcomes ranging from milder to more severe toxicity of FLASH-RT was observed following total body irradiation.
Similarly, the reported effects of FLASH-RT on mouse models of intestinal toxicity range from preventing death after a traditionally lethal dose of radiation to increasing severity of gastrointestinal syndrome and decreasing survival.
Ultra high dose rate (35 Gy/sec) radiation does not spare the normal tissue in cardiac and splenic models of lymphopenia and gastrointestinal syndrome.
Intriguingly, a number of studies have shown modest increases in intestinal crypt survival acutely, but whether these changes had an effect on the clinical severity of gastrointestinal syndrome was not reported (eg, body weight changes and overall survival).
Differences in irradiation beam parameters used across different institutions are thought to be a major contributor to the conflicting experimental results of FLASH regarding normal tissues effects. In an effort to mitigate these issues, recommendations on critical physical parameters as well as standards for uniformly reporting physical aspects of FLASH have been made.
One limitation of the present study is the inability to assess the effect of dose rate as an independent variable as the irradiation device used herein operates at discrete pulse repetition frequencies and pulse widths, and dose and dose rate are interdependent. Nonetheless, it should be emphasized that doses, dose rates (mean and instantaneous), and overall treatment times used in our study were kept within recommended ranges in all experiments. In addition, we thoroughly report beam parameter details and provide information on prescribed dose and corresponding dosimetry, thus demonstrating that the suggested optimal beam delivery conditions to trigger the FLASH effect were met. Another potential limitation of this study is the relatively low number of mice used. However, the low intragroup variability in organ weights and histologic findings as well as a high level of consistency of results between replicated experiments support the significance of the findings.
Conclusion
To the best of our knowledge, this is the first study evaluating the effect of UHDR-RT on healthy gonadal tissues of mice. Our data suggest that the short-term effects of UHDR-RT on the mouse gonads are comparable to those of CONV-RT. This study extends and complements the current knowledge on the effects of FLASH-RT on acutely responding tissues and reinforces the notion that not all UHDR radiation will universally decrease toxicity to healthy tissues. In addition, our inability to trigger normal tissue protection using FLASH-RT despite adhering to recommendations and best practices concerning its main physical aspects attests to the complexity of this phenomenon and the need for further investigation.
Acknowledgments
We thank Johanna Rawlings and Kevin Thorburn for assistance with histology and immunohistochemistry.
Reduced cognitive deficits after FLASH irradiation of whole mouse brain are associated with less hippocampal dendritic spine loss and neuroinflammation.
Ultra high dose rate (35 Gy/sec) radiation does not spare the normal tissue in cardiac and splenic models of lymphopenia and gastrointestinal syndrome.
Perstin A, Poirier Y, Sawant A, Tambasco M. Physics contribution quantifying the DNA-damaging effects of FLASH irradiation with plasmid DNA. 2022;113:437-447.
The effect of raloxifene on the uterine weight response in immature mice exposed to 17beta-estradiol, 1,1,1-trichloro-2, 2-bis(p-chlorophenyl)ethane, and methoxychlor.
Sources of support: This work was supported by grants R01CA108633, R01CA169368, RC2CA148190, and U10CA180850-01 (National Cancer Institute) and the Ohio State University Comprehensive Cancer Center (all to Dr Chakravarti). The histology and immunohistochemistry work was supported by the Comparative Pathology and Digital Imaging Shared Resource at the Ohio State University.
Disclosures: Dr Chakravarti reports a relationship with Varian Medical Systems that includes funding grants. Dr Blakaj reports a relationship with IntraOp Medical that includes funding grants. Dr Cetnar reports a relationship with the American Association of Physicists in Medicine that includes travel reimbursement and a board membership and serves as Associate Section Editor for Advances in Radiation Oncology. Dr McElroy reports a relationship with the Ohio State University Department of Radiation Oncology that includes employment and nonfinancial support. Dr Fleming serves as Associate Senior Editor for Advances in Radiation Oncology. Dr Ayan serves as Associate Section Editor for Advances in Radiation Oncology. No other disclosures were reported.
Research data are stored in an institutional repository and will be shared upon request to the corresponding author.
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