Helmut Acker

The functional near infrared spectroscopy (fNIRS) technique was introduced for use in general medical practice to measure brain blood oxygenation along with physiological parameters such as 4-channel EEG, heart rate, blood oxygenation, blood volume changes and autonomic nerve activity. An artificial neuronal network was used to adjust the brain blood oxygenation measurements to evaluate changes in these physiological parameters. Early detection of cerebral blood flow disorders related to cognitive impairments such as Alzheimer´s disease appears to be feasible. Acupuncture with Hegu (LI4) was applied to stimulate brain neuronal networks in 5 control patients (CPs) and 5 brain disorder patients (BDPs). The following conclusions were drawn: 1. fNIRS recordings of brain hemoglobin oxygenation indicate the efficacy of brain microcirculation and brain oxygen supply 2. Central brain acupuncture stimulation reveals deficits in the brain microcirculation and oxygen supply of BDPs 3. A 20-second period of acupuncture stimulation results in brain hypoxia in BDPs but not in CPs, primarily due to mismatching of arterial and venous microcirculation 4. fNIRS combined with subsequent ANN analysis of brain oxygen supply could be very effective and user friendly for recording early signs of brain microcirculation dysregulation and therapeutic progress.

Carotid body (CB) type I cells sense the blood Po₂ and generate a nervous signal for stimulating ventilation and circulation when blood oxygen levels decline. Three oxygen-sensing enzyme complexes may be used for this purpose: 1) mitochondrial electron transport chain metabolism, 2) heme oxygenase 2 (HO-2)-generating CO, and/or 3) an NAD(P)H oxidase (NOX). We hypothesize that intracellular redox changes are the link between the sensor and nervous signals. To test this hypothesis type I cell autofluorescence of flavoproteins (Fp) and NAD(P)H within the mouse CB ex vivo was recorded as Fp/(Fp+NAD(P)H) redox ratio. CB type I cell redox ratio transiently declined with the onset of hypoxia. Upon reoxygenation, CB type I cells showed a significantly increased redox ratio. As a control organ, the non-oxygen-sensing sympathetic superior cervical ganglion (SCG) showed a continuously reduced redox ratio upon hypoxia. CN⁻, diphenyleneiodonium, or reactive oxygen species influenced chemoreceptor discharge (CND) with subsequent loss of O₂ sensitivity and inhibited hypoxic Fp reduction only in the CB but not in SCG Fp, indicating a specific role of Fp in the oxygen-sensing process. Hypoxia-induced changes in CB type I cell redox ratio affected peptidyl prolyl isomerase Pin1, which is believed to colocalize with the NADPH oxidase subunit p47_phox in the cell membrane to trigger the opening of potassium channels. We postulate that hypoxia-induced changes in the Fp-mediated redox ratio of the CB regulate the Pin1/p47_phox tandem to alter type I cell potassium channels and therewith CND.

Reactive oxygen species (ROS) generated by the NADPH oxidase have been proposed to play an important role in the carotid body (CB) oxygen sensing process (Cross et al. 1990). Up to now it remains unclear whether hypoxia causes an increase or decrease of CB ROS levels. We transfected CBs with the ROS sensitive HSP-FRET construct and subsequently measured the intracellular redox state by means of Förster resonance energy transfer (FRET) microscopy. In a previous study we found both increasing and decreasing ROS levels under hypoxic conditions. The transition from decreasing to increasing ROS levels coincided with the change of the caging system from ambient environment caging (AEC) to individually ventilated caging (IVC) (Bernardini A, Brockmeier U, Metzen E, Berchner-Pfannschmidt U, Harde E, Acker-Palmer A, Papkovsky D, Acker H, Fandrey J, Type I cell ROS kinetics under hypoxia in the intact mouse carotid body ex vivo: a FRET based study. Am J Physiol Cell Physiol. doi: 10.1152/ajpcell.00370.2013 , 2014). In this work we analyze hypoxia induced ROS reaction of animals from an IVC system that had been exposed to AEC conditions for 5 days. The results further support the hypothesis of an important impact of the caging system on CB ROS reaction.

Reactive oxygen species (ROS) mainly originating from NADPH oxidases have been shown to be involved in the carotid body (CB) oxygen-sensing cascade. For measuring ROS kinetics, type I cells of the mouse CB in an ex vivo preparation were transfected with the ROS sensor construct FRET-HSP33. After 2 days of tissue culture, type I cells expressed FRET-HSP33 as shown by immunohistochemistry. In one population of CBs, 5 min of hypoxia induced a significant and reversible decrease of type I cell ROS levels ( n = 9 CBs; P < 0.015), which could be inhibited by 4-(2-aminoethyl)benzensulfonylfluorid (AEBSF), a highly specific inhibitor of the NADPH oxidase subunits p47_phox and p67_phox. In another population of CBs, however, 5 min of hypoxia induced a significant and reversible increase of ROS levels in type I cells ( n = 8 CBs; P < 0.05), which was slightly enhanced by administration of 3 mM AEBSF. These different ROS kinetics seemed to coincide with different mice breeding conditions. Type I cells of both populations showed a typical hypoxia-induced membrane potential (MP) depolarization, which could be inhibited by 3 mM AEBSF. ROS and MP closely followed the hypoxic decrease in CB tissue oxygen as measured with an O₂-sensitive dye. We conclude that attenuated p47_phox subunit activity of the NADPH oxidase under hypoxia is the physiological trigger for type I cell MP depolarization probably due to ROS decrease, whereas the observed ROS increase has no influence on type I cell MP kinetics under hypoxia.

Carotid body (CB) type I cell hypoxia-sensing function is assumed to be based on potassium channel inhibition. Subsequent membrane depolarization initiates an intracellular calcium increase followed by transmitter release for excitation of synapses with linked nerve endings. Several reports, however, contradict this generally accepted concept by showing that type I cell oxygen-sensing properties vary significantly depending on the method of their isolation. We report therefore for the first time noninvasive mapping of the oxygen-sensing properties of type I cells within the intact adult mouse CB ex vivo by using multifocal Nipkow disk-based imaging of oxygen-, calcium- and potential-sensitive cellular dyes. Characteristic type I cell clusters were identified in the compact tissue by immunohistochemistry because of their large cell nuclei combined with positive tyrosine hydroxylase staining. The cellular calcium concentrations in these cell clusters either increased or decreased in response to reduced tissue oxygen concentrations. Under control conditions, cellular potential oscillations were uniform at ∼0.02 Hz. Under hypoxia-induced membrane depolarization, these oscillations ceased. Simultaneous increases and decreases in potential of these cell clusters resulted from spontaneous burstlike activities lasting ∼1.5 s. type I cells, identified during the experiments by cluster formation in combination with large cell nuclei, seem to respond to hypoxia with heterogeneous kinetics.

The cellular oxygen sensing system of the body ensures appropriate adaptation of cellular functions toward hypoxia by regulating gene expression and ion channel activity. Two-photon laser microscopy is an ideal tool to study and prove the relevance of the molecular mechanisms within oxygen sensing pathways on the cellular and complex tissue or organ level. Images of hypoxia inducible factor 1 (HIF-1) subunit nuclear mobility and protein-protein interaction in living cells, of hypoxia-induced changes in membrane potential and intracellular calcium of live ex vivo carotid bodies as well as of rat kidney proximal tubulus function in vivo, will be shown.

Autofluorescence (AF) originating from the cytoplasmic region of mammalian cells has been thoroughly investigated; however, AF from plasma membranes of viable intact cells is less well known, and has been mentioned only in a few older publications. Herein, we report results describing single‐ and two‐photon spectral properties of a strong yellowish‐green AF confined to the plasma‐membrane region of transformed human hepatocytes (HepG2) grown in vitro as small three‐dimensional aggregates or as monolayers. The excitation–emission characteristics of the membrane AF indicate that it may originate from a flavin derivative. Furthermore, the AF was closely associated with the plasma membranes of HepG2 cells, and its presence and intensity were dependent on cell metabolic state, membrane integrity and presence of reducing agents. This AF could be detected both in live intact cells and in formaldehyde‐fixed cells. Microsc. Res. Tech., 2007.

Hypoxia‐inducible factor‐1 (HIF‐1) coordinates the cellular response to a lack of oxygen by controlling the expression of hypoxia‐inducible genes that ensure an adequate energy supply. Assembly of the HIF‐1 complex by its oxygen‐regulated subunit HIF‐1a and its constitutive β subunit also known as ARNT is the key event of the cellular genetic response to hyp‐oxia. By two‐photon microscopy, we studied HIF‐1 assembly in living cells and the mobility of fluorophore‐labeled HIF‐1 subunits by fluorescence recovery after photobleaching. We found a significantly slower nuclear migration of HIF‐1α than of HIF‐1β, indicating that each subunit can move independently. We applied fluorescence resonance energy transfer to calculate the nanometer distance between α and β subunits of the transcriptionally active HIF‐1 complex bound to DNA. Both N termini of the fluorophore‐labeled HIF‐1 sub‐units were localized as close as 6.2 nm, but even the N and C terminus of the HIF‐1 complex were not further apart than 7.4 nm. Our data suggest a more compact 3‐dimensional organization of the HIF complex than described so far by 2‐dimensional models.—Wotzlaw, C., Otto, T., Berchner‐Pfannschmidt, U., Metzen, E., Acker, H., Fandrey, J. Optical analysis of the HIF‐1 complex in living cells by FRET and FRAP. FASEB J. 21, 700–707 (2007)

Current concepts of cellular oxygen-sensing include an isoform of the neutrophil NADPH oxidase, different electron carrier units of the mitochondrial electron transport chain (ETC), heme oxygenase-2 (HO-2), and a subfamily of 2-oxoglutarate dependent dioxygenases termed HIF (hypoxia inducible factor) prolyl hydroxylases (PHDs) and HIF asparagyl hydroxylase FIH-1 (factor-inhibiting HIF). Different oxygen sensitivities, cell-specific distribution and subcellular localization of specific oxygen-sensing cascades involving reactive oxygen species (ROS) as second messengers may help to tailor various adaptive responses according to differences in tissue oxygen availability. Herein, we propose an integrated model for these various oxygen-sensing mechanisms that very efficiently regulate HIF-alpha activity and plasma membrane potassium-channel (PMPC) conductivity.