Exposure to hyperbaric O2 levels leads to blood-brain barrier breakdown in rodents.

INTRODUCTION: Hyperbaric oxygen has been used as a medical treatment tool in hyperbaric chambers and is an integral part of professional and combat divers' activity. In extreme cases, exposure to hyperbaric oxygen can develop central nervous system oxygen toxicity (CNS-OT), which leads to seizures and eventually death. CNS-OT is caused by neuronal hyperactivity due to high oxygen levels, potentially damaging brain cells including the blood-brain barrier (BBB). However, the effect of hyperbaric oxygen levels on the healthy BBB has not been characterized directly yet. METHODS: Six or three different groups of ~ eight rats or mice, respectively, were exposed to increasing levels of partial pressure of oxygen (0.21 to 5 ATA) in a hyperbaric chamber, followed by MRI scanning with gadolinium. Statistical significance (adjusted p-value ≤ 0.05) was assessed using linear regression and ordinary one-way (rats) or two-way (mice) ANOVA with correction of multiple comparison tests. In rats, the effect of 100% oxygen at 5 ATA was independently validated using FITC-Dextran (5 kDa). Statistical significance (p-value ≤ 0.05) was assessed using Welch's t-test and effect size was calculated by Cohen's D. RESULTS: In rats, analyzed MRI scans showed a significant trend of increase in the % gadolinium in brain tissues as a result of hyperbaric oxygen pressures (p-value = 0.0079). The most significant increase was measured at 4 ATA compared to air (adjusted p-value = 0.0461). Significant increased FITC-Dextran levels were measured in the rats' brains under 100% oxygen at 5 ATA versus air (p-value = 0.0327; Effect size = 2.0). In mice, a significant increase in gadolinium penetration into the hippocampus and frontal cortex was measured over time (adjusted p-value < 0.05) under 100% oxygen at 3 and 5 ATA versus air, and between the treatments (adjusted p-value < 0.0001). CONCLUSIONS: The BBB is increasingly disrupted due to higher levels of hyperbaric oxygen in rodents, indicating a direct relation between hyperbaric oxygen and BBB dysregulation for the first time. We suggest considering this risk in different diving activities, and protocols using a hyperbaric chamber. On the other hand, this study highlights the potential therapeutic usage of hyperbaric oxygen for controlled drug delivery through the BBB into brain tissues in different brain-related diseases.

CNS-oxygen toxicity and blood glucose levels in MnSOD enzyme knockdown mice.

Many studies have been conducted in the search for the mechanism underlying CNS-oxygen toxicity (OT), which may be fatal when diving with a closed-circuit apparatus. We investigated the influence of hyperbaric oxygen (HBO) on blood glucose level (BGL) in Mn-superoxide dismutase (SOD2) knockdown mice regarding CNS-OT in particular under stress conditions such as hypoglycemia or hyperglycemia. Two groups of mice were used: SOD2 knockdown (Heterozygous, HET) mice and their WT family littermates. Animals were exposed to HBO from 2 up to 5 atmosphere absolute (ATA). Blood samples were drawn before and after each exposure for measurement of BGL. The mice were sacrificed following the final exposure, which was at 5 ATA. We used RT-PCR and Western blot to measure levels of glucose transporter 1 (GLUT1) and hypoxia inducible factor (HIF)1a in the cortex and hippocampus. In the hypoglycemic condition, the HET mice were more sensitive to oxidative stress than the WT. In addition, following exposure to sub-toxic HBO, which does not induce CNS-OT, BGL were higher in the HET mice compared with the WT. The expression of mRNA of GLUT1 and HIF-1a decreased in the hippocampus in the HET mice, while the protein level decreased in the HET and WT following HBO exposure. The results suggest that the higher BGL following HBO exposure especially at SOD2 HET mice is in part due to reduction in GLUT1 as a consequence of lower HIF-1a expression. This may add part to the puzzle of the understanding the mechanism leading to CNS-OT.

Self-regulating novel iron oxide nanoparticle-based magnetic hyperthermia in swine: biocompatibility, biodistribution, and safety assessments.

Studies demonstrating the successful and safe application of magnetic hyperthermia in large animals are scarce. A therapeutic approach for advanced cancer comprising multicore encapsulated iron oxide (IO) Sarah Nanoparticles (SaNPs), that uniquely self-regulate their temperature, was developed thus overcoming the safety challenges of hyperthermia. SaNPs are intravenously injected and accumulate in tumor tissue, leading to selective heating upon exposure to an external alternating magnetic field (AMF). A series of studies were conducted in healthy swine to assess SaNPs' safety, alone or combined with AMF application. Administration of single high (up to 22 mg IO/kg) or low (3.6 mg IO/kg) SaNP doses had no adverse effects, including no infusion reactions. Vital signs remained stable with no significant clinical pathology changes, and no treatment-associated toxicities. Biodistribution analysis indicated that SaNPs predominantly accumulate in the lungs and clear in a dose- and time-dependent manner. In minipigs that received a single SaNP no-observed-adverse-effect-level (NOAEL)-based dose (3.6 mg IO/kg) with AMF, the average percentage remaining in vital organs after 90 days was 13.7%. No noticeable clinical signs were noted during the 87 to 92-day observation period following irradiation, and no inflammation, necrosis, nor thermal damage were found in the histopathology evaluation. In another minipig, ~ 90 days after three recurrent high doses (14 mg IO/kg), without AMF, almost half of the injected SaNPs were cleared with no residual detrimental effects. We demonstrate that the approach is safe and well tolerated in swine, opening potential avenues as a novel therapeutic modality for cancer patients.

Novel Nanoparticle-Based Cancer Treatment, Effectively Inhibits Lung Metastases and Improves Survival in a Murine Breast Cancer Model.

Sarah Nanoparticles (SaNPs) are unique multicore iron oxide-based nanoparticles, developed for the treatment of advanced cancer, following standard care, through the selective delivery of thermal energy to malignant cells upon exposure to an alternating magnetic field. For their therapeutic effect, SaNPs need to accumulate in the tumor. Since the potential accumulation and associated toxicity in normal tissues are an important risk consideration, biodistribution and toxicity were assessed in naïve BALB/c mice. Therapeutic efficacy and the effect on survival were investigated in the 4T1 murine model of metastatic breast cancer. Toxicity evaluation at various timepoints did not reveal any abnormal clinical signs, evidence of alterations in organ function, nor histopathologic adverse target organ toxicity, even after a follow up period of 25 weeks, confirming the safety of SaNP use. The biodistribution evaluation, following SaNP administration, indicated that SaNPs accumulate mainly in the liver and spleen. A comprehensive pharmacokinetics evaluation, demonstrated that the total percentage of SaNPs that accumulated in the blood and vital organs was ~78%, 46%, and 36% after 4, 13, and 25 weeks, respectively, suggesting a time-dependent clearance from the body. Efficacy studies in mice bearing 4T1 metastatic tumors revealed a 49.6% and 70% reduction in the number of lung metastases and their relative size, respectively, in treated vs. control mice, accompanied by a decrease in tumor cell viability in response to treatment. Moreover, SaNP treatment followed by alternating magnetic field exposure significantly improved the survival rate of treated mice compared to the controls. The median survival time was 29 ± 3.8 days in the treated group vs. 21.6 ± 4.9 days in the control, p-value 0.029. These assessments open new avenues for generating SaNPs and alternating magnetic field application as a potential novel therapeutic modality for metastatic cancer patients.