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Single dose gamma irradiation induced angiogenesis in rat skin hair follicles

Background: Hair follicle cycling usually associated with prominent changes in skin vascularization; through follicular dermal papilla production of angiogenic factors. The early response of hair follicles to ionizing irradiation (IR) is induction of early anagen hair and appearance of new hair formation. Material and Method: Fifty rats were equally divided into 2 groups; control and γ-rays (10Gy) as a single dose, skin biopsy was taken from dorsum of the rat 72 hours after irradiation. Skin biopsy was examined histopathological and with immunohistochemical staining CD31. Blood samples were collected for biochemical analysis of Tumor necrosis factor-α (TNF-α), Interleukin-1β (IL1β), Interleukin-6 (IL-6), Malondialdehyde (MDA), Catalase (CAT) and Superoxide dismutase (SOD) Results: γ-rays produced epidermal thinning and dermal inflammatory cells together with dermal endothelial proliferation and new vessels formation around the hair follicle compared with control group as demonstrated by CD31 staining. Furthermore, there was a significant elevation of TNF-α, IL-1β and IL-6 levels compared to control group. Moreover, MDA levels increased significantly in γ-rays group and decreased significantly in control group, whereas CAT and SOD activities decreased in the γ-rays compared with the control group. Conclusion: The early effects of γ -rays on the skin could be beneficial and stimulatory to hair growth.


INTRODUCTION
Angiogenesis is the growth of new capillaries from pre-existing .
arises in wound healing, and in association with tumors. Hair follicles represent a unique feature of cyclical transformations from growth phase (anagen) followed by regression phase (catagen), and then relative phase "quiescence" (telogen).
Radiation can affect vessel growth through photon stimulation of vessel growth and increases expression of angiogenic factors; γ-rays promote angiogenesis and increase metastasis (often by causing an increase in the expression of pro-angiogenic factors such as VEGF, HIF-1α, IL-6, and basic fibroblast growth factor (b-FGF) in the irradiated tissue) (3)(4)(5) .
Ionizing radiation is also known to cause oxidative stress through a generation of reactive oxygen species (ROS) resulting in an inequity of the pro-oxidant and antioxidant in the cells (6) . High level of nitric oxide (NO) appears to enhance oxidative stress through its oxidative and apoptotic influence. In addition to these influences, there are also some studies that show that NO participates in angiogenesis. It was reported that angiogenesis induced by VEGF can be slowed in vivo by NO-synthetase inhibition. It seems that NO participates in the angiogenic activity of VEGF (7) .
The current study aims to evaluate the histopathological and microcirculatory skin alteration induced by γ-rays in rats.

Animals
Fifty adult male Swiss albino rats (120-130g) were obtained from the Egyptian organization for biological product and vaccines Giza, Egypt. Animals received the standard requirements of food and water ad libitum and maintained under environmental conditions of humidity, temperature (20-22°C), and 12-h light-dark cycle. All procedures performed in this study were in accordance with the ethical standards of the REC-NCRRT, Egyptian Atomic Energy Authority, Egypt following the guidelines of NIH at which the studies were conducted (National Research Ethics Committees REC-NCRRT, Egypt + permit number: 11A /18).

Irradiation
It was done using Gamma-cell-40 (Cesium-137) located at NCRRT, Nasr City, Cairo, Egypt. Animals were exposed to a single dose of 10 Gy γ -rays delivered at a rate of 0.42Gy/min at the time of experimentation.

Experimental design and sample collection
All rats were divided into 2 groups (n=25); control and γ-rays group (animals were 694 irradiated with an acute single dose of 10 Gy). After the 3 rd day post irradiation, and an overnight fast, blood samples were collected by retro-orbital puncture from each rat using blood capillary tubes. Blood samples were collected in dry sterile tubes then, serum was separated using a refrigerated centrifuge at 3000 r.p.m. for 15 min, stored frozen at -20° C till biochemical analysis. Skin biopsies were taken from the dorsum of each rat and fixed in 10% buffered formalin solution followed by dehydration, cleated and embedded in paraffin. Paraffin sections of 4-micron thickness were prepared and stained with both of Haematoxylin and Eosin (H&E) according to Suvarna et al. (8) and stained immunohistochemically for CD31 (9) . At the end of the experiment, rats were sacrificed on the 3 rd day post radiation exposure.

Immunohistochemical staining
CD31 also known as Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31); is obtained from (Thermo Fisher, MA-USA) with dilution 1:100. It is 130-kDa membrane glycoprotein that belongs to the immunoglobulin supergene family and is constitutively expressed on the surface of endothelial cells, platelets, and circulating leukocytes and is commonly used as an endothelium-specific marker (10) . Mounting evidence implicates its involvement in a number of cell adhesion processes that take place in the vasculature, such as leukocyte adhesion and transmigration, the adherence of platelets to the vascular wall at sites of endothelial cell damage, endothelial cell-cell adherence and migration, a process required for neovascularization that is essential for tissue growth and repair.

Int. J. Radiat. Res., Vol. 18 No. 4, October 2020
(Thermo Scientific Multiskan MK3, USA). Also, MDA level was evaluated following the method described by Buege and Aust (11) . CAT activity was assayed using the method of Sinha (12) and SOD was determined according to the method designated by Kakkar et al. (13) .

Statistical analysis
Data were investigated using SPSS software (version 19.0). One way analysis of variance (ANOVA) followed by LSD as Post Hoc test were used. The results obtained were calculated by mean ± standard deviation. P-values < 0.05 were considered to be statistically significant (14) .

RESULTS
Histopathological sections of irradiated rat skin revealed thinning of the epidermis layer with flattened rete ridges with large nuclei together with vacuolation and spongiosis. Absence of stratum corneum and vesicles formation were rarely seen. Collagen fibers were disorganized and appeared more eosinophilic and homogenous. Dermal elastic fibers were fragmented and clumped ( figure 1).
There was positive CD31 immunoreactivity involving the blood vessel endothelium in the control and γ-irradiated rats ( figure 2). Furthermore, single dose (10 Gy) of γ-rays resulted in strong positive CD31 immunoreactivity around hair follicles which was abundant in the control (figures 3 and 4). A significant rise in the level of inflammatory cytokines TNF-α, IL-1β and IL-6 was detected in γ-irradiated group compared to the control group (table 1). Moreover, γ irradiation induced significant down-regulation in the activity of CAT and SOD and significant up-regulation in the level of MDA compared to the control group (table 2).

DISCUSSION
Hair follicles have long been recognized as a potential marker for radiation injury. Moreover, hair follicle cycling is associated with significant changes in skin perfusion, the epithelial hair bulbs of anagen follicles and the follicular dermal papilla express angiogenic factors (15) .
Angiogenic growth factors such as VEGFs and b-FGFs induce the secretion of endothelial proteinases and plasminogen activators that cause the breakdown of the vessel basement membrane, allowing the cells to interrupt the adjoining matrix. Subsequently, the endothelial cells migrate, multiply and ultimately differentiate to give rise to a new, lumen-comprising vessel. Thereafter, the endothelial cells establish a new basement membrane and release additional factors such as platelet-derived growth factor (PDGF), which draws the supporting pericytes to interact externally with the endothelial cells in order to stabilize the newly formed vessels (16) .
Hair follicle stem cells (HFSCs) have a powerful expansion capability and multidifferentiation potential properties. Recently, Xu et al. (17) revealed an efficient strategy of hair follicle HFSCs differentiation into endothelial cells by stimulation with VEGF and PFGF. Moreover, HFSCs expressed endothelial cell (EC)
In the current study a single dose of a whole body gamma irradiation (10 Gy) induced a significant up-regulation in the level of TNF-α, IL-1β and IL-6 as shown in (table 1). Several in-vivo and in-vitro studies revealed that TNF-α and IL-1β are the most important pro-inflammatory cytokines that exhibit a principle role in acute and chronic inflammation and in a variety of human diseases including autoimmune disorders, atherosclerosis, and cancer (19) . Up-regulation of TNF-α and IL-1β expression was also found in lung and intestine after irradiation (20,21) . IL-6 is another multifunctional pro-inflammatory cytokine that plays a main task in the mediation of the inflammatory and immune reactions originated by infection or injury (22) . In 2012 Getz and her colleagues (23) demonstrated that IL-6 promotes STAT3 phosphorylation and early activation of angiogenesis-related gene transcription, which leads to increased angiogenesis. Recently, in 2019 Jin and his colleagues (24) revealed that hypoxia induces expression of TNF-α by endothelial cells (ECs), which represents an autocrine loop that activates the HIF pathway via an NF-κB-dependent process, which facilitate VEGF production by ECs and angiogenesis.
Our study revealed for the first time that single acute dose of 10 Gy γ-rays induced after 72 hours, a significant angiogenesis around hair follicles was demonstrated by strong CD31 immunoreactivity which could be due to the up-regulation of IL-6 and TNF-α leading to release of VEGF from hair follicles. So, it seems that γ-rays induced indirectly angiogenesis through expression of angiogenesis-related genes and pro-angiogenic factors. In 2014 Kinoshita and his (25) colleagues reported that radiation induced anagen, and new hairs were clearly visible after 1 week of single dose γ-rays (10 Gy).
On the other hand, exposure to γ-irradiation raises the production of ROS and directs the irradiated cells into a state of oxidative stress that has been concerned in a variety of natural and pathological processes (26) . Recently in 2018 Soni and his colleagues (27) added that skin antioxidants were sensitive to radiation even at a low dose, which can be used as an indicator of radiation injury and changed in a dose-dependent manner. The current study showed a significant decrease in CAT and SOD together with significant rise in the level of MDA related to control group (table 2). In 2011 Pande and his coworkers (28) revealed that VEGF and MDA levels increased simultaneously and were positively correlated, VEGF is up-regulated by conditions associated with the generation of free radicals and reactive oxygen intermediates.

CONCLUSION
Radiation induced angiogenesis which usually associated with anagen which offers an attractive model for identifying the physiologic control of cutaneous angiogenesis, and a potential use of anti-angiogenic drugs in-vivo.