It is known that in humans after the radiotherapy ends, several late effects appear in the tissues of organs irradiated which in turn may change their functionality. These late effects, well described for the skin 16 weeks after irradiation in humans and after 4 weeks after irradiation in animal models, consist of an initial inflammatory process followed by an intense production of collagen fibers characterizing the fibrosis process [18, 19]. However, until this manuscript has been produced, similar effects in the calvaria skin were not described.
Therefore, in the present study, the calvaria skin was studied after a 15-Gy x-ray irradiation according to previous results , which reported this dose as appropriate for obtaining a chronic effect of radiation, and after this dose has been confirmed by the nuclear physicist from the Instituto Sul Paranaense de Oncologia (ISPON) placed in Ponta Grossa City, Paraná, Brazil.
In general, for organs and for the skin of other body parts than calvaria, fibrosis is reported after inflammation. In turn, inflammation is described as a consequence of the action of ionizing radiation which changes the macromolecules building organs and tissues, especially those from the cell membranes which are broken to generate molecules known as reactive oxygen species (ROS). ROS produce high oxidative stress that is responsible for the sequential damage of cells of the tissues and organs. For example, ROS break the endothelium of the small blood vessels to start the inflammatory cell process. The increase in oxidative stress also induces an increase in the enzymes that have the function to kidnap the ROS reducing the damages [21, 22]. Although the detection of ROS and ROS-associated enzymes can be performed by specific methods, the presence of inflammatory cells and the changes in morphology of the connective tissues (i.e., of collagen fibers or vessels) already indicates that an oxidative stress is happening. Thus, after an initial response to radiation damages, the connective cells start the regeneration of the injured structures. The more visible and detectable aspect of this regeneration process is the production of new collagen fibers, i.e., a fibrosis. However, after irradiation, this process occurs without appropriate control, which means that it may affect the functionality of different organs of the body .
On the other hand, it is known that MPPs are responsible for remodeling extracellular matrix components and are regulated by endogenous tissue inhibitors of metalloproteinases (TIMPs) . The relationship between the MMPs, TIMPs, and TGF-β1 has been explored in the heart, lung, and enteritis fibrosis [24,25,26]. However, for the skin, this relationship has been poorly explored . Thus, we evaluated the expression of MMP-9 in the calvaria skin day 4 to day 25 after the irradiation. The results showed that the number of MMP-9-positive cells in the dermis decreases from day 4 but significantly on days 9, 14, and 25 compared to controls. Although without a visible inflammatory cell influx in the dermis of the calvaria skin during this period, the MMP-9 results indicate a reduction in the remodeling of the extracellular matrix components of the dermis which may confirm the absence of a fibrosis process during these times evaluated.
In spite of this, results corroborate what has been described for the skin that covers the most regions of the body [28, 29], related with drastic but temporary atrophy of sebaceous glands and hair follicles. We found that between day 4 and day 14, these epidermal components disappeared. The reduced number of BrdU-positive cells in the basal layer indicates a precocious effect of radiation in the cell proliferative process. However, on day 25, the presence of BrdU-positive cells in the epidermis, close to sebaceous glands as well as in the hair follicle, indicates that the proliferative process was reestablished. Of note, the use of stem cells obtained from different tissues has been investigated in vitro and in vivo to induce the regenerative damage produced by radiotherapy [13, 30, 31]. Therefore, the reestablishment of the cell proliferation observed in our results suggests the presence of the stem cells in these epidermal structures or its migration from another region to assume this proliferative status. However, the dynamics of stem cells in skin submitted to radiation needs to be better investigated in our model study: the calvaria skin.
Regardless, the literature shows that a myofibroblast differentiation occurs and this causes the fibrosis process in the dermis of calvaria skin, and we did not observe cell proliferation after irradiation, playing in favor of an interruption of this process. Although it had been reported in previous studies [11, 20] that in the skin of other parts of the body, the fibrosis may occurs at least from week 4 after a single dose of 15 Gy and that it is preceded by an inflammation, we did not observe this process in the calvaria skin until the day 25. This finding was also confirmed by the reduction of the number of MMP-9-expressing cells in the dermis, MMP-9 being considered an active molecule involved in the inflammatory process. This may be partly explained because in the calvaria skin, and some other regions of the head, the low density and thickness of the subcutaneous layers containing blood vessels may reduce the generation of inflammation. However, we do not exclude the possibility of a subsequent fibrosis process starting after the time period here considered, opening the way for future studies prolonging the observation after day 25.
Concerning the extracellular matrix in the dermis, it is known that in a normal condition, the ratio of types I and III collagen fibers in covered skin remained constant throughout childhood and young adult life . However, it is interesting that collagen fibrils in the dermis are hybrid molecules formed by type I and type III collagen, in which type III collagen is located at the periphery of the collagen fibrils [33, 34]. In this context, the general descriptions for the radiation effects in the dermis of skin covering most body regions are an increase in its thickness as a result of the collagen fiber deposition, an increase in the fiber diameter, and the replacement of the subcutaneous fat and skeletal muscle by a fibrotic tissue .
In our investigation, the Masson’s staining allowed us to detect the two types of collagen fibers in the control group (type I collagen fibers in blue; type III collagen fibers in red [35, 36]). However, in the irradiated calvaria skin, it was possible to determine a significant change in the pattern of the color of collagen fibers by Masson staining not previously well explored. Irradiation produced alterations of the collagen morphology with a deep change, going from a fibrous and waive arrangement to a complete fiber degradation in some regions, with amorphous aspect. These changes were detected at all time points, becoming more evident at day 9, suggesting that the amorphous aspect of the collagen fibers resulted from the dissociation of type III collagen, which increased the collagen maturation index for type III collagen, without any increase in the collagen synthesis. Type III collagen fibers were predominant on day 4 and day 25, and type I collagen fibers on day 9 and day 14. The predominance of both collagen fibers may be the result of their disorganization produced by the radiation and not caused by new collagen synthesis, considering that proliferation of dermal cells was never detected.
In conclusion, our findings showed that a 15-Gy x-ray irradiation to the calvaria skin evaluated until day 25 (1) changed the morphology of collagen fibers in the dermis to an amorphous aspect in few days after irradiation, (2) produced a temporary morphological damage of the sebaceous gland and hair follicles, and (3) did not determine a visible inflammatory process, cell proliferation, or fibrosis process in the dermis, differing from the results already described in the literature for the skin of other body parts.