S. Samuels

Neuroglial ATP release through innexin channels controls microglial cell movement to a nerve injury

Microglia, the immune cells of the central nervous system, are attracted to sites of injury. The injury releases adenosine triphosphate (ATP) into the extracellular space, activating the microglia, but the full mechanism of release is not known. In glial cells, a family of physiologically regulated unpaired gap junction channels called innexons (invertebrates) or pannexons (vertebrates) located in the cell membrane is permeable to ATP. Innexons, but not pannexons, also pair to make gap junctions. Glial calcium waves, triggered by injury or mechanical stimulation, open pannexon/innexon channels and cause the release of ATP. It has been hypothesized that a glial calcium wave that triggers the release of ATP causes rapid microglial migration to distant lesions. In the present study in the leech, in which a single giant glial cell ensheathes each connective, hydrolysis of ATP with 10 U/ml apyrase or block of innexons with 10 µM carbenoxolone (CBX), which decreased injury-induced ATP release, reduced both movement of microglia and their accumulation at lesions. Directed movement and accumulation were restored in CBX by adding ATP, consistent with separate actions of ATP and nitric oxide, which is required for directed movement but does not activate glia. Injection of glia with innexin2 (Hminx2) RNAi inhibited release of carboxyfluorescein dye and microglial migration, whereas injection of innexin1 (Hminx1) RNAi did not when measured 2 days after injection, indicating that glial cells’ ATP release through innexons was required for microglial migration after nerve injury. Focal stimulation either mechanically or with ATP generated a calcium wave in the glial cell; injury caused a large, persistent intracellular calcium response. Neither the calcium wave nor the persistent response required ATP or its release. Thus, in the leech, innexin membrane channels releasing ATP from glia are required for migration and accumulation of microglia after nerve injury.

Innexins form two types of channels

Injury to the central nervous system triggers glial calcium waves in both vertebrates and invertebrates. In vertebrates the pannexin1 ATP‐release channel appears to provide for calcium wave initiation and propagation. The innexins, which form invertebrate gap junctions and have sequence similarity with the pannexins, are candidates to form non‐junctional membrane channels. Two leech innexins previously demonstrated in glia were expressed in frog oocytes. In addition to making gap junctions, innexins also formed non‐junctional membrane channels with properties similar to those of pannexons. In addition, carbenoxolone reversibly blocked the loss of carboxyfluorescein dye into the bath from the giant glial cells in the connectives of the leech nerve cord, which are known to express the innexins we assayed.

Arachidonic acid closes innexin/pannexin channels and thereby inhibits microglia cell movement to a nerve injury

Pannexons are membrane channels formed by pannexins and are permeable to ATP. They have been implicated in various physiological and pathophysiological processes. Innexins, the invertebrate homologues of the pannexins, form innexons. Nerve injury induces calcium waves in glial cells, releasing ATP through glial pannexon/innexon channels. The ATP then activates microglia. More slowly, injury releases arachidonic acid (ArA). The present experiments show that ArA itself reduced the macroscopic membrane currents of innexin‐ and of pannexin‐injected oocytes; ArA also blocked K+‐induced release of ATP. In leeches, whose large glial cells have been favorable for studying control of microglia migration, ArA blocked glial dye‐release and, evidently, ATP‐release. A physiological consequence in the leech was block of microglial migration to nerve injuries. Exogenous ATP (100 µM) reversed the effect, for ATP causes activation and movement of microglia after nerve injury, but nitric oxide directs microglia to the lesion. It was not excluded that metabolites of ArA may also inhibit the channels. But for all these effects, ArA and its non‐metabolizable analog eicosatetraynoic acid (ETYA) were indistinguishable. Therefore, ArA itself is an endogenous regulator of pannexons and innexons. ArA thus blocks release of ATP from glia after nerve injury and thereby, at least in leeches, stops microglia at lesions.

Assessment of Oropharyngeal and Laryngeal Cancer Treatment Delay in a Private and Safety Net Hospital System

Objective To examine the impact of treatment setting and demographic factors on oropharyngeal and laryngeal cancer time to treatment initiation (TTI). Study Design Retrospective case series. Setting Safety net hospital and adjacent private academic hospital. Subjects and Methods Demographic, staging, and treatment details were retrospectively collected for 239 patients treated from January 1, 2014, to June 30, 2016. TTI was defined as days between diagnostic biopsy and initiation of curative treatment (defined as first day of radiotherapy [RT], surgery, or chemotherapy). Results On multivariable analysis, safety net hospital treatment (vs private academic hospital treatment), initial diagnosis at outside hospital, and oropharyngeal cancer (vs laryngeal cancer) were all associated with increased TTI. Surgical treatment, severe comorbidity, and both N1 and N2 status were associated with decreased TTI. Conclusion Safety net hospital treatment was associated with increased TTI. No differences in TTI were found when language spoken and socioeconomic status were examined in the overall cohort.

Impact of Performance Status and Comorbidity on Palliative Radiation Treatment Tolerance and End-Of-Life Decision-Making

Purpose Previous studies have indicated a relationship between functional status and comorbidity on overall survival when treating patients with bone and brain metastases. However, the degree to which these findings have been integrated into modern-day practice remains unknown. This study examines the impact of performance measures, including Karnofsky Performance Status (KPS) and comorbidity, on palliative radiation therapy treatment tolerance and fractionation schedule. The relationship between a shorter fractionation schedule (SFx) and pending mortality is examined. Methods and materials This study included patients who were treated with palliative intent to the brain or bone between January 1, 2016 and June 30, 2016. Demographic and medical characteristics collected included KPS score (stratified as good [90-100], fair [70-80], and poor (≤60]), socioeconomic status, comorbidity (binary measure using the Adult Comorbidity Evaluation-27 scale), site of metastatic disease, and treatment facility. Univariable analyses were performed using the Cox proportional hazards regression model to assess the impact of the variables on the prescribed number of fractions (binary measure, ≥10 [long fractionation schedule], and <10 [SFx]), and major treatment interruptions (MTIs; defined as missing ≥3 radiation therapy treatment days or ending treatment prematurely). Results A total of 145 patients were eligible for study inclusion, including 95 patients who were treated for bony metastatic disease and 50 patients for brain metastases. High comorbidity (P = .029) and both fair (P = .051) and poor (P = .065) functional status were associated with more frequent MTIs. However, high comorbidity and low KPS score were not associated with shorter treatment plans. In addition, patients with an earlier time to death were not more likely to receive an SFx (P = .871). Conclusions Low KPS and elevated comorbidity scores predict for a poorer prognosis and more frequent MTIs; however, there was no indication that physicians incorporated this information in the fractionation scheduling.