Longitudinal Neuropathological Consequences of Extracranial Radiation Therapy in Mice.

Cancer-related cognitive impairment (CRCI) is a consequence of chemotherapy and extracranial radiation therapy (ECRT). Our prior work demonstrated gliosis in the brain following ECRT in SKH1 mice. The signals that induce gliosis were unclear. Right hindlimb skin from SKH1 mice was treated with 20 Gy or 30 Gy to induce subclinical or clinical dermatitis, respectively. Mice were euthanized at 6 h, 24 h, 5 days, 12 days, and 25 days post irradiation, and the brain, thoracic spinal cord, and skin were collected. The brains were harvested for spatial proteomics, immunohistochemistry, Nanostring nCounter® glial profiling, and neuroinflammation gene panels. The thoracic spinal cords were evaluated by immunohistochemistry. Radiation injury to the skin was evaluated by histology. The genes associated with neurotransmission, glial cell activation, innate immune signaling, cell signal transduction, and cancer were differentially expressed in the brains from mice treated with ECRT compared to the controls. Dose-dependent increases in neuroinflammatory-associated and neurodegenerative-disease-associated proteins were measured in the brains from ECRT-treated mice. Histologic changes in the ECRT-treated mice included acute dermatitis within the irradiated skin of the hindlimb and astrocyte activation within the thoracic spinal cord. Collectively, these findings highlight indirect neuronal transmission and glial cell activation in the pathogenesis of ECRT-related CRCI, providing possible signaling pathways for mitigation strategies.

The Distant Molecular Effects on the Brain by Cancer Treatment.

Cancer survivors experience cancer-related cognitive impairment (CRCI) secondary to treatment. Chemotherapy and radiation therapy independently contribute to cognitive dysfunction; however, the underlying mechanisms leading to dysfunction remain unclear. We characterized brain gene expression changes in a mouse model of CRCI to identify the mechanistic underpinnings. Eleven-to-twelve-week-old SKH1 mice were treated with doxorubicin (DOX), hindlimb radiation (RT), concurrent hindlimb radiation and doxorubicin (DOX-RT), or no treatment (control). Sixteen days following treatment, gene expression was measured from murine brains using the NanoString nCounter® glial profiling panel. Gene expression was normalized and compared between groups. No two groups shared the same expression pattern, and only Gnb1 and Srpr were upregulated in multiple treatment groups. Brains from DOX-treated mice had upregulated Atf2, Atp5b, Gnb1, Rad23b, and Srpr and downregulated Sirt5 expression compared to control brains. Brains from RT-treated mice demonstrated increased Abcg2 and Fgf2 and decreased C1qa and C1qb expression compared to control brains. Brains from DOX-RT-treated mice had upregulated Adar, E2f3, Erlec1, Gnb1, Srpr, Vim, and Pdgfra expression and downregulated Rock2 and Inpp5f expression compared to control brains. The gene expression changes demonstrated here highlight roles for neuronal transmission and oxidative stress in the pathogenesis of doxorubicin-related CRCI and inflammation in RT-related CRCI.

Study of the Effect of Photobiomodulation on a Skin Repair Model in SKH-1 Mice.

Background and objective: Laser applied at low power (400-1100 nm), currently named photobiomodulation (PBM), is a noninvasive therapy to speed up wound healing. The purpose of this study was whether two different laser PBM delivery protocols would impact the skin wound healing in a mouse model. Materials and methods: A total of 24 SKH-1 mice were divided into three groups: Group 1 (control: untreated ulcers), Group 2 (a single postsurgical laser application), and Group 3 (laser each other day for 10 days; total five applications). Laser parameters were 940 nm, 0.4 W, 10 mm spot size, 0.008 J/cm2, 300 sec/wound. Each animal received two skin wounds which were photographed on days 0, 5, and 10 to determine wound closure (ImageJ). Half of the animals in each group were sacrificed on day 5 and the other half on day 10. Samples were routinely processed for histological analysis (re-epithelization, angiogenesis, granulation tissue formation, inflammation, and collagen deposition). Results: The closure of the wounds at the end of the experiment in the animals photobiostimulated each other day was more advanced than in the controls and in those treated only once, in both the macroscopic and microscopic studies. Angiogenesis was higher in both treated groups than in the control in the first study time (day 5). However, inflammation, maturation of the granulation tissue, and collagen deposition only improved when the laser was applied each other day. Conclusions: In our study, with the parameters used, PBM improved the healing of skin wounds when applied every other day and not in a single dose.

Cancer treatment induces neuroinflammation and behavioral deficits in mice.

Introduction: Cancer survivors are increasingly diagnosed with a syndrome of neurocognitive dysfunction termed cancer-related cognitive impairment (CRCI). Chemotherapy and radiation therapy have been implicated in CRCI; however, its underlying pathogenesis remains unclear, hindering effective prevention or treatment. Methods: We used the hairless strain SKH1 (11-12-week-old) and treated the mice with radiation to the right hindlimb, doxorubicin (a chemotherapy agent), concurrent radiation, and doxorubicin, or no treatment (control). Neurocognition was evaluated via standardized behavioral testing following treatment. Mice were subsequently humanely euthanized, and plasma and brains were collected to identify inflammatory changes. Results: Mice treated with radiation, doxorubicin, or both radiation and doxorubicin demonstrated equivalent hippocampal dependent memory deficits and significant increases in activated microglia and astrocytes compared to control mice. Doxorubicin-treated mice had significantly increased plasma IL-6 and failed to gain weight compared to control mice over the study period. Discussion: This study demonstrates that non-brain directed radiation induces both gliosis and neurocognitive deficits. Moreover, this work presents the first characterization of SKH1 mice as a relevant and facile animal model of CRCI. This study provides a platform from which to build further studies to identify potential key targets that contribute to CRCI such that strategies can be developed to mitigate unintended neuropathologic consequences associated with anticancer treatment.

Comparison of Antibacterial Activity and Wound Healing in a Superficial Abrasion Mouse Model of Staphylococcus aureus Skin Infection Using Photodynamic Therapy Based on Methylene Blue or Mupirocin or Both.

Background: Antibiotic resistance and impaired wound healing are major concerns in S. aureus superficial skin infections, and new therapies are needed. Antimicrobial photodynamic therapy (aPDT) is a new therapeutic approach for infections, but it also improves healing in many wound models. Objective: To compare the antimicrobial activity and the effects on wound healing of aPDT based on Methylene Blue (MB-aPDT) with mupirocin treatment, either alone or in combination, in superficial skin wounds of S. aureus-infected mice. Additionally, to evaluate the clinical, microbiological, and cosmetic effects on wound healing. Materials and Methods: A superficial skin infection model of S. aureus was established in SKH-1 mice. Infected wounds were treated with MB-aPDT, MB-aPDT with a daily topical mupirocin or only with mupirocin. No treatment was carried out in control animals. Daily clinical and microbiological examinations were performed until complete clinical wound healing. Histopathological studies and statistical analysis were performed at the end of the study. Results: MB-aPDT treatment induced the best wound healing compared to mupirocin alone or to mupirocin plus MB-aPDT. Superficial contraction at 24 h and a greater reduction in size at 48 h, quicker detachment of the crust, less scaling, and absence of scars were observed. Histopathological studies correlated with clinical and gross findings. By contrast, mupirocin showed the highest logaritmic reduction of S. aureus. Conclusions: MB-aPDT and mupirocin treatments are effective in a murine superficial skin infection model of S. aureus. One session of MB-aPDT was the best option for clinical wound healing and cosmetic results. The addition of mupirocin to MB-aPDT treatment improved antimicrobial activity; however, it did not enhance wound healing. No synergistic antibacterial effects were detected.

Methotrexate reduces keratinocyte proliferation, migration and induces apoptosis in HaCaT keratinocytes in vitro and reduces wound closure in Skh1 mice in vivo.

The aim of the present work was to evaluate MTX treatment (0.1, 1 and 10 µg mL-1) in vitro in order to characterize its effects on cell proliferation alterations in cell cycle of HaCaT keratinocytes and wound healing in a Skh1 mice treated with MTX (low doses 30 mg kg-1, high doses 200 mg kg-1 and repeated doses at 1.5 mg kg-1). We analyzed the cytotoxic effect of methotrexate by a resazurin assay. The effects in the proliferation, cell cycle and apoptosis of HaCaT cells were analyzed by flow cytometry. The effects of MTX on wound healing in vivo were also analyzed. A trend toward reduction in the resazurin assay was found (p > 0.05). Reduced proliferation was also identified in a clonogenic assay and a CFSE assay (p < 0.05) due to the MTX treatment. A reduction in the G2/M and S phases was observed accompanied by apoptosis induction with increased sub G0 phase and annexin V FITC staining. Effect of MTX was evidenced in vivo on the wound closure process after day 10 (p < 0.05) with alterations in tissue architecture and remodeling. There is a marked effect of MTX on wound healing in vivo in Skh1 mice with implications for long-term therapy and surgical interventions.

Evaluation of Bacterial RNA Polymerase Inhibitors in a Staphylococcus aureus-Based Wound Infection Model in SKH1 Mice.

Chronic wounds infected with pathogens such as Staphylococcus aureus represent a worldwide health concern, especially in patients with a compromised immune system. As antimicrobial resistance has become an immense global problem, novel antibiotics are urgently needed. One strategy to overcome this threatening situation is the search for drugs targeting novel binding sites on essential and validated enzymes such as the bacterial RNA polymerase (RNAP). In this work, we describe the establishment of an in vivo wound infection model based on the pathogen S. aureus and hairless Crl:SKH1-Hrhr (SKH1) mice. The model proved to be a valuable preclinical tool to study selected RNAP inhibitors after topical application. While rifampicin showed a reduction in the loss of body weight induced by the bacteria, an acceleration of wound healing kinetics, and a reduced number of colony forming units in the wound, the ureidothiophene-2-carboxylic acid 1 was inactive under in vivo conditions, probably due to strong plasma protein binding. The cocrystal structure of compound 1 with RNAP, that we hereby also present, will be of great value for applying appropriate structural modifications to further optimize the compound, especially in terms of plasma protein binding.

Stable PEG-coated silver nanoparticles - A comprehensive toxicological profile.

The present study was purported to assess the toxicological profile of bare and polyethylene glycol (PEG) coated spherical silver nanoparticles (AgNPs) by means of in vitro (on human keratinocytes - HaCat cells) and in vivo non-invasive tests (after intraperitoneal - i.p. administration to mice). Bare and PEG-coated AgNPs were synthesized by applying Turkevich's method slightly modified. The physico-chemical characterization revealed the formation of stable, spherical AgNPs and PEG-AgNPs, with narrow size distributions and mean hydrodynamic sizes in the range of 19 nm and 50 nm, respectively. Toxicity data revealed a dose-dependent safe profile for low concentrations of test compounds (<10 µM) in terms of cell viability, whereas higher concentrations were associated with a high rate of cell mortality. In vivo acute/subacute toxicity data showed no denotive changes in mice health status after i.p. administration. Histological observations of internal organs and the biochemical parameters analyzed together with the other biological observations showed a low toxicity level with no major differences related to control, albeit at skin level a reduced number of mast cells was detected. All these observations provide strong support for the idea that coated silver nanoparticles could be applied as targeted nanocarriers for skin pathologies and diagnostic.

Spontaneous and Vaccine-Induced Clearance of Mus Musculus Papillomavirus 1 Infection.

Mus musculus papillomavirus 1 (MmuPV1/MusPV1) induces persistent papillomas in immunodeficient mice but not in common laboratory strains. To facilitate the study of immune control, we sought an outbred and immunocompetent laboratory mouse strain in which persistent papillomas could be established. We found that challenge of SKH1 mice (Crl:SKH1-Hrhr) with MmuPV1 by scarification on their tail resulted in three clinical outcomes: (i) persistent (>2-month) papillomas (∼20%); (ii) transient papillomas that spontaneously regress, typically within 2 months (∼15%); and (iii) no visible papillomas and viral clearance (∼65%). SKH1 mice with persistent papillomas were treated by using a candidate preventive/therapeutic naked-DNA vaccine that expresses human calreticulin (hCRT) fused in frame to MmuPV1 E6 (mE6) and mE7 early proteins and residues 11 to 200 of the late protein L2 (hCRTmE6/mE7/mL2). Three intramuscular DNA vaccinations were delivered biweekly via in vivo electroporation, and both humoral and CD8 T cell responses were mapped and measured. Previously persistent papillomas disappeared within 2 months after the final vaccination. Coincident virologic clearance was confirmed by in situ hybridization and a failure of disease to recur after CD3 T cell depletion. Vaccination induced strong mE6 and mE7 CD8+ T cell responses in all mice, although they were significantly weaker in mice that initially presented with persistent warts than in those that spontaneously cleared their infection. A human papillomavirus 16 (HPV16)-targeted version of the DNA vaccine also induced L2 antibodies and protected mice from vaginal challenge with an HPV16 pseudovirus. Thus, MmuPV1 challenge of SKH1 mice is a promising model of spontaneous and immunotherapy-directed clearances of HPV-related disease.IMPORTANCE High-risk-type human papillomaviruses (hrHPVs) cause 5% of all cancer cases worldwide, notably cervical, anogenital, and oropharyngeal cancers. Since preventative HPV vaccines have not been widely used in many countries and do not impact existing infections, there is considerable interest in the development of therapeutic vaccines to address existing disease and infections. The strict tropism of HPV requires the use of animal papillomavirus models for therapeutic vaccine development. However, MmuPV1 failed to grow in common laboratory strains of mice with an intact immune system. We show that MmuPV1 challenge of the outbred immunocompetent SKH1 strain produces both transient and persistent papillomas and that vaccination of the mice with a DNA expressing an MmuPV1 E6E7L2 fusion with calreticulin can rapidly clear persistent papillomas. Furthermore, an HPV16-targeted version of the DNA can protect against vaginal challenge with HPV16, suggesting the promise of this approach to both prevent and treat papillomavirus-related disease.

Cactus cladodes (Opuntia humifusa) extract minimizes the effects of UV irradiation on keratinocytes and hairless mice.

CONTEXT: Cactus cladodes [Opuntia humifusa (Raf.) Raf. (Cactaceae)] is one of the cactus genera, which has long been used as a folk medicine for skin disorders. OBJECTIVE: This study investigated the skincare potential of cactus cladodes extract (OHE), including its ability to regulate ultraviolet B (UVB)-induced hyaluronic acid (HA) production. MATERIALS AND METHODS: Gene expression levels of hyaluronic acid synthases (HASs) and hyaluronidase (HYAL) were measured in UVB-irradiated HaCaT cells with OHE treatment (10, 25, 50, 100 µg/mL) by real-time polymerase chain reaction (PCR). The HA content was analyzed in hairless mice (SKH-1, male, 6 weeks old) treated with OHE for 10 weeks by using enzyme-linked immunosorbent assay (ELISA). Haematoxylin and eosin (H&E) and immunohistological staining were performed to examine epidermal thickness and levels of CD44 and hyaluronic acid-binding protein (HABP). RESULTS: HA synthases (HAS,1 HAS2, HAS3) mRNA levels were increased by 1.9-, 2.2- and 1.6-fold, respectively, with OHE treatment (100 µg/mL), while UVB-induced increase of hyaluronidase mRNA significantly decreased by 35%. HA content in animal was decreased from 42.9 to 27.1 ng/mL by OHE treatment. HAS mRNA levels were decreased by 39%, but HYAL mRNA was increased by 50% in OHE group. CD44 and HABP levels, which were greatly increased by UVB-irradiation, were reduced by 64 and 60%, respectively. Epidermal thickness, transepidermal water loss (TEWL), and erythema formation was also decreased by 45 (45.7 to 24.2 µm), 48 (48.8 to 25 g/h/m2) and 33%, respectively. CONCLUSION: OHE protects skin from UVB-induced skin degeneration in HaCaT cells and hairless mice.

Dose-response on the chemopreventive effects of sarcophine-diol on UVB-induced skin tumor development in SKH-1 hairless mice.

Sarcophine-diol (SD) is a lactone ring-opened analogue of sarcophine. It has shown chemopreventive effects on chemically-induced skin tumor development in female CD-1 mice, as well as in a UVB-induced skin tumor development model in hairless SKH-1 mice at a dose of 30 µg SD applied topically and 180 mJ/cm(2) UVB. The objective of this study was to determine the dose-response on the chemopreventive effects of SD on SKH-1 hairless mice when exposed to a UVB radiation dose of 30 mJ/cm(2). This UVB dose better represents chronic human skin exposure to sunlight leading to skin cancer than previous studies applying much higher UVB doses. Carcinogenesis was initiated and promoted by UVB radiation. Female hairless SKH-1 mice were divided into five groups. The control group was topically treated with 200 µL of acetone (vehicle), and the SD treatment groups were topically treated with SD (30 µg, 45 µg, and 60 µg dissolved in 200 µL of acetone) 1 h before UVB radiation (30 mJ/cm(2)). The last group of animals received 60 µg SD/200 µL acetone without UVB exposure. These treatments were continued for 27 weeks. Tumor multiplicity and tumor volumes were recorded on a weekly basis for 27 weeks. Weight gain and any signs of toxicity were also closely monitored. Histological characteristics and the proliferating cell nuclear antigen (PCNA) were evaluated in the mice skin collected at the end of the experiment. The dose-response study proved a modest increase in chemopreventive effects with the increase in SD dose. SD reduced the number of cells positively stained with PCNA proliferation marker in mice skin. The study also showed that SD application without UVB exposure has no effect on the structure of skin. The results from this study suggest that broader range doses of SD are necessary to improve the chemopreventive effects.