Selective β-adrenergic Receptor Expression on Human Memory CD8+ T Lymphocyte Subsets Regulates Mobilization and INF-y Production

Introduction: CD8⁺ T lymphocytes (CD8TLs) express β-adrenergic receptors (βAR), which bind the neurotransmitter norepinephrine and stress hormone epinephrine released during inflammation, trauma, and psychological stress. Little is known about the functions of this βAR expression on CD8TLs. Methods: Volunteers were exposed to a psychological stressor (N=24). Flow cytometry identified CD8TL subsets by CCR7, CD27, CD28 and CD45RA expression. Adrenergic receptor subtype expression was determined by micro-array. The effects of βPAR stimulation on IFN-γ production in activated CD8TLs was tested in vitro using PMA/Ionomycin. Results: Stress caused selective migration of effector-memory (CCR7⁻CD27⁻CD28⁻) CD8TLs into the blood (+148%, p<.001). An 8-fold up-regulation of the β₂AR was demonstrated in effector-memory cells as compared to naïve CD8TLs. Stimulation of the β₂AR subtype completely inhibited IFN-γ production. Conclusion: These results show that β₂AR stimulation enhances peripheral immune surveillance in a highly selective manner, and might protect against excessive cytokine release during inflammation and stress.

     

Effects of Oxygen Supplementation on Injectable and Inhalant Anesthesia in C57BL/6 Mice

Oxygen supplementation is rarely considered when anesthetizing laboratory mice, despite reports that mice become profoundly hypoxic under anesthesia. Little is known about the effects of hypoxia on anesthetic performance. This article focuses on the effects of oxygen supplementation on physiologic parameters and depth of anesthesia in male and female C57BL/6 mice. Anesthesia was performed via common injectable anesthetic protocols and with isoflurane. Mice anesthetized with injectable anesthesia received one of 3 drug protocols. Low-dose ketamine/xylazine (100/8 mg/kg) was chosen to provide immobilization of mice, suitable for imaging procedures. Medium-dose ketamine/xylazine/acepromazine (100/10/1 mg/kg) was chosen as a dose that has been recommended for surgical procedures. High-dose ketamine/xylazine/acepromazine (150/12/3 mg/kg) was chosen after pilot studies to provide a long duration of a deep plane of anesthesia. We also tested the effects of oxygen supplementation on the minimum alveolar concentration (MAC) of isoflurane in mice. Mice breathed supplemental 100% oxygen, room air, or medical air with 21% oxygen. Anesthetized mice that did not receive supplemental oxygen all became hypoxic, while hypoxia was prevented in mice that received oxygen. Oxygen supplementation did not affect the MAC of isoflurane. At the high injectable dose, all mice not receiving oxygen supplementation died while all mice receiving oxygen supplementation survived. At low and medium doses, supplemental oxygen reduced the duration of the surgical plane of anesthesia (low dose with oxygen: 22 ± 14 min; low dose without supplementation: 29 ± 18 min; medium dose with oxygen: 43 ± 18 min; medium dose without supplementation: 61 ± 27 min). These results suggest that mice anesthetized with injectable and inhalant anesthesia without supplemental oxygen are routinely hypoxic. This hypoxia prolongs the duration of anesthesia with injectable drug protocols and affects survival at high doses of injectable anesthetics. Because of variable responses to injectable anesthetics in mice, oxygen supplementation is recommended for all anesthetized mice.

Anesthesia is frequently required for mice used in biomedical research, but anecdotal communications suggest that mice receive significantly less anesthetic monitoring and supportive care than do other research species. Monitoring of anesthetized mice is often minimal due to lack of specialized monitoring equipment, and the fact that many rodent surgeries are performed by a single person who acts as both surgeon and anesthetist. Supportive care during anesthesia is limited by a lack of supporting experimental evidence. The lack of monitoring and supportive care may increase the mortality rate in anesthetized mice.Previous studies have shown that mice anesthetized with both inhalant and injectable anesthetics without supplemental oxygen become profoundly hypoxic.^{1,6,8,9,19,26,39,41} While mice in these studies appear to recover normally from anesthesia, little is known about the effects of hypoxia on physiologic parameters, anesthetic depth, and perioperative mortality. Respiratory complications, including hypoxia and hypoventilation, are second only to cardiovascular complications as a cause of perioperative mortality in veterinary species, and in humans, hypoxemia accounts for over 50% of deaths under anesthesia.⁴ To mitigate the risk of hypoxia under anesthesia, oxygen supplementation is commonly provided to anesthetized humans and animals, but is rarely provided to mice in research settings.^6,19All anesthetics affect respiratory function; ketamine and isoflurane are particularly known to cause respiratory depression in mice and rats by impairing the normal physiologic responses to hypoxemia and hypercapnia.^{9,12,20,23,28} The peripheral chemoreceptors, primarily in the carotid body, normally sense dropping arterial partial pressure of oxygen (PaO₂) while central chemoreceptors located in the medulla sense changes in pH and rising partial pressure of carbon dioxide (PaCO₂).^22,23,29,40 Both sets of chemoreceptors compensate by initiating increases in respiratory rate and tidal volume.^{23,28,31,34,40} Injectable and inhalant anesthetic agents depress the function of these chemoreceptors, preventing the increases in respiration that compensate for hypoxia and hypoventilation.^22,29Pulse oximetry is commonly used to monitor peripheral oxygen saturation and detect the presence of hypoxia. Pulse oximeters use the difference in light absorption of oxygenated hemoglobin and deoxygenated hemoglobin in arterial blood to provide an estimate of arterial oxygen content, abbreviated as SpO₂.¹⁷ An SpO₂ of less than 90% to 95% generally corresponds to a PaO₂ of less than 60 to 80 mm Hg, which is considered hypoxic in most species of mammals.^7,17 Because of the small size of mice, species-specific pulse oximetry equipment is necessary to obtain this measurement. Therefore, measurement of SpO₂ in anesthetized mice is not routinely performed, meaning that hypoxia under anesthesia generally goes unrecognized, and is likely more common than is appreciated by our field.The purpose of this study was to confirm that mice become hypoxic after receiving a ketamine/xylazine based anesthetic admixture or isoflurane, which are commonly used anesthetics in mice and to investigate the effects of oxygen supplementation on anesthetic depth, physiologic values, and anesthetic requirements in these mice.^9,35 We hypothesized that mice not receiving supplemental oxygen would be hypoxic, as indicated by lower SpO₂ while anesthetized, and that supplemental oxygen would correct this hypoxia. We also hypothesized that oxygen supplementation would increase the doses of injectable and inhalant anesthesia necessary to maintain mice at a surgical plane of anesthesia.     

Evaluation of smear layer removal by bicarbonate soda,ethylenediamine tetraacetic acid with cetrimide and sodium hypochlorite with a new model

Various methods are used to evaluate irrigants. The primary aim was to develop a model for preliminary testing of potential irrigants. The second aim was to investigate the effect of bicarbonate soda on smear layer by comparing it with ethylenediamine tetraacetic acid with cetrimide (EDTAC) and sodium hypochlorite (NaOCl). Extracted human single‐canal teeth were halved, and a uniform filing method was used to create smear layer. The following solutions were then applied – distilled water (control), 1% NaOCl, 17% EDTAC and bicarbonate soda at concentrations of 1%, 5%, 10% and 15%. Some samples had multiple solutions in different sequences. Samples were examined by scanning electron microscopy. Representative images were scored based on the degree of smear layer remaining. Results were analysed with the SAS system, using the GENMOD procedure. Complete smear layer was found in samples treated with all solutions except EDTAC used alone. There were no significant differences between the sequences, EDTAC/NaOCl/EDTAC and NaOCl/EDTAC/NaOCl. There were no significant differences between groups with and without bicarbonate soda. In conclusion, the model was effective for testing chemical effects on solutions on smear layer. Bicarbonate soda did not remove smear layer and provided no additional cleaning effects after EDTAC and NaOCl.