Entwicklung von in vivo NMR-Techniken zur Untersuchung der Auswirkung von Ozeanversauerung und -erwärmung auf die Neurophysiologie Antarktischer Fische

Ocean acidification and warming as a consequence of climate change affect marine organisms. Thus, for example, neurological changes could be observed in polar cod, which were induced by ocean acidification and warming. As a potential cause, a connection between the neurological impairments and changes in intracellular pH (pHi) has been postulated as it was described for animal models in preclinical research. Therefore, the non-invasive determination of pHi with high spatial and temporal resolution is of great interest. A promising tool for non-invasive pH measurements is the CEST contrast (Chemical Exchange Saturation Transfer), which enables the indirect detection of endogenous or exogenous molecules with exchangeable protons using changes in the NMR signal of the water pool. The CEST effect mainly depends on the concentration of a metabolite and the exchange rate between this metabolite and water, which in turn is influenced by physical and physiological parameters. These properties allow for CEST to be used for in vivo determination of changes in metabolite concentration and pH. The present thesis describes the development and adaption of the measurement method CEST MRI (Magnetic Resonance Imaging) and its application for determining changes in pHi with a high temporal and spatial resolution in the brain of marine fish. CEST from glutamate to water (GluCEST) was experimentally investigated in a broad temperature and pH range. The applicability of GluCEST to determine relative changes in pH even at low temperatures depends on glutamate concentration and on the parameters used in the experiments. Localized 1H NMR spectroscopy in combination with the application of quantification algorithms is an established method for the quantification of metabolite concentrations. In most cases, these algorithms use model functions based on chemical shifts at 37AdegreeC. Therefore, the temperature dependency of the chemical shifts for important metabolites has been examined in order to avoid systematic quantification errors caused by the use of incorrect prior knowledge for spectra recorded at temperatures different from 37AdegreeC. The exchange rates show an exponential behaviour as a function of temperature, thus offering a completely new picture of metabolites whose exchange regime offers the possibility to determine a CEST effect at temperatures near the freezing point. In this context, the taurine based TauCEST effect was investigated in a broad pH and temperature range. Investigations showed that the specificity of TauCEST offers the possibility to use this method as a suitable tool to detect pHi changes in the brain of polar cod. The first in vivo application of the TauCEST effect in the brain of polar cod showed an increase of about 1.5-3% under different CO2 concentrations. Localized 1H NMR spectroscopy, which was successfully adapted for the in vivo application in the brain of marine fish at 9.4 T, indicated no significant changes in concentration for those metabolites that mainly determine the CEST effect. Thus, the in vivo application of TauCEST proves to be an adequate method for determining non-invasively relative changes in pHi with high spatial and temporal resolution in the brain of polar cod during exposure to elevated CO2 concentrations

Characterization of amine proton exchange for analyzing the specificity and intensity of the CEST effect: from humans to fish

Chemical exchange saturation transfer (CEST) at about 2.8 ppm downfield from water is characterized besides other compounds by exchanging amine protons of relatively high concentration amino acids and is determined by several physiological (pH, T) and experimental (B0, B1, tsat) parameters. Although the weighting of the CEST effect observed in vivo can be attributed mainly to one compound depending on the organism and organ, there are still several other amino acids, proteins and molecules that also contribute. These contributions in turn exhibit dependences and thus can lead to possible misinterpretation of the measured changes in the CEST effect. With this in mind, this work aimed to determine the exchange rates of six important amino acids as a function of pH and temperature, and thus to create multi-pool models that allow the accurate analysis of the CEST effect concerning different physiological and experimental parameters for a wide variety of organisms. The results show that small changes in the above parameters have a significant impact on the CEST effect at about 2.8 ppm for the chosen organisms, i.e. the human brain (37 °C) and the brain of polar cod (1.5 °C), furthermore, the specificity of the CEST effect observed in vivo can be significantly affected. Based on the exchange rates ksw(pH, T) determined for six metabolites in this study, it is possible to optimize the intensity and the specificity for the CEST effect of amino acids at about 2.8 ppm for different organisms with their specific physiological characteristics. By adjusting experimental parameters accordingly, this optimization will help to avoid possible misinterpretations of CEST measurements. Furthermore, the multi-pool models can be utilized to further optimize the saturation

pH-Bildgebung am Gehirn von polaren Fischen: eine TauCEST Anwendung

Chemical exchange saturation transfer (CEST) ist ein Bildkontrast, der die indirekte Detektion von Änderungen im pH ermöglicht. CEST bietet daher die Möglichkeit, die Säure-Basen-Regulation im Fischgehirn unter CO2-Konzentrationen, wie sie durch den Klimawandel bewirkt werden, zu verfolgen. Ziel dieser Studie war es, einen geeigneten Metaboliten zu finden, um Änderungen im intrazellulären pH-Wert mit hoher zeitlicher und räumlicher Auflösung im Fischgehirn bei 1.5°C zu detektieren. Der TauCEST-Effekt erwies sich als geeignet und wurde zum ersten Mal in vivo angewendet

MRI and MRS on preserved samples as a tool in fish ecology

Magnetic Resonance Imaging (MRI) andMagnetic Resonance Spectroscopy (MRS) gain increasing attention and importance as a tool in marine ecology. So far, studies were largely limited to morphological studies, e.g. for the creation of digital libraries. Here, the utility ofMRI andMRS for ecologists is tested and exemplified using formalin preserved samples of the Antarctic silverfish, Pleuragramma antarctica. As this species lacks a swim bladder, buoyancy is attained by the deposition of large amounts of lipids that are mainly stored in subcutaneous and intermuscular lipid sacs. In this study MRI andMRS are not only used to study internal morphology, but additionally to investigate functional morphology and to measure parameters of high ecological interest. The data are compared with literature data obtained by means of traditional ecological methods. The results fromthis study show that MR scans are not only an alternative to histological sections (as shown before),but even allow the visualization of particular features in delicate soft tissues, such as Pleuragramma's lipid sacs. 3D rendering techniques proved to be a useful tool to study organ volumes and lipid content,which usually requires laborious chemical lipid extraction and analysis. Moreover, the application of MRS even allows for an analysis of lipids and fatty acids within lipid sacs, which wouldn't be possible using destructive methods. MRI and MRS, in particular when used in combination, have the capacity to provide useful data on parameters of high ecological relevance and thus have proven to be a highly valuable addition, if not alternative, to the classical methods

Tracking gonadal development in fish: An in vivo MRI study on polar cod, Boreogadus saida (Lepechin, 1774)

AbstractMagnetic resonance imaging (MRI) was applied to determine the sex of polar cod (Boreogadus saida Lepechin, 1774) (Actinopterygii: Gadidae) and to follow the gonadal development in individual animals over time. Individual unanaesthetised fish were transferred to a measurement chamber inside a preclinical 9.4 T MRI scanner and continuously perfused with aerated seawater. A screening procedure at an average of 3.5 h, consisting of a set of MRI scans of different orientations, was repeated every 4 weeks on the same set of reproducing B. saida (n = 10) with a body length of about 20 cm. Adapted multi‐slice flow‐compensated fast low‐angle shot (FcFLASH) and rapid acquisition with relaxation enhancement (RARE) protocols with an in‐plane resolution of 313 μm and an acquisition time of 2.5 min were used to visualise the morphology of various organs, including the gonads within the field of view (FOV). The MR images provided high resolution, enabling specific sex determination, calculation of gonad volumes, and determination of oocyte sizes. Gonad maturation was followed over 4 months from November 2021 until shortly before spawning in February 2022. The gonad volume increased by 2.3–25.5% for males and by 11.5–760.7% for females during the observation period. From October to February, the oocyte diameter increased from 427 μm (n = 1) to 1346 ± 27 μm (n = 4). Interestingly, individual oocytes showed changes in MR contrast over time that can be attributed to the morphological development of the oocytes. The results fit well with previous literature data from classical invasive studies. The presented approach has great potential for various ecophysiological applications such as monitoring natural or delayed development of internal organs or sex determination under different environmental conditions.</jats:p

CO2 induced pHi changes in the brain of polar fish: a TauCEST application

Chemical exchange saturation transfer from taurine to water (TauCEST) is primarily detectable in the low temperature range. Since, TauCEST asymmetry is bijective in the physiological pH-range (6.8-7.5), TauCEST is a potential candidate for in vivo studies on brain of polar fish. The specificity of TauCEST MRI on the brain of polar cod at 1.5°C shows a taurine contribution of 65%. TauCEST in brain of polar cod significantly increased under elevated CO2 concentrations by about 1.34%-3.17% in comparison to control, reflecting pHi changes since localized 1H NMR spectra show no significant changes in metabolite concentration for the different treatments

Temperature dependence of 1H NMR chemical shifts and its influence on estimated metabolite concentrations

Objectives: Temperature dependent chemical shifts of important brain metabolites measured by localised 1H MRS were investigated to test how the use of incorrect prior knowledge on chemical shifts impairs the quantication of metabolite concentrations. Materials and methods: Phantom measurements on solutions containing 11 metabolites were performed on a 7 T scanner between 1 and 43 °C. The temperature dependence of the chemical shift differences was fitted by a linear model. Spectra were simulated for di erent temperatures and analysed by the AQSES program (jMRUI 5.2) using model functions with chemical shift values for 37 °C. Results: Large differences in the temperature dependence of the chemical shift differences were determined with a maximum slope of about ±7.5 × 10−4 ppm/K. For 32–40 °C, only minor quantification errors resulted from using incorrect chemical shifts, with the exception of Cr and PCr. For 1–10 °C considerable quantification errors occurred if the temperature dependence of the chemical shifts was neglected. Conclusion: If 1H MRS measurements are not performed at 37 °C, for which the published chemical shift values have been determined, the temperature dependence of chemical shifts should be considered to avoid systematic quantification errors, particularly for measurements on animal models at lower temperatures

In vivo 31P-MRS of muscle bioenergetics in marine invertebrates: Future ocean limits scallops' performance

Object: Dynamic in vivo 31P-NMR spectroscopy in combination with Magnetic Resonance Imaging (MRI) was used to study muscle bioenergetics of boreal and Arctic scallops (Pecten maximus and Chlamys islandica) to test the hypothesis that future Ocean Warming and Acidification (OWA) will impair the performance of marine invertebrates. Materials & methods: Experiments were conducted following the recommendations for studies of muscle bioenergetics in vertebrates. Animals were long-term incubated under different environmental conditions: controls at 0 °C for C. islandica and 15 °C for P. maximus under ambient PCO2 of 0.039 kPa, a warm exposure with +5 °C (5 °C and 20 °C, respectively) under ambient PCO2 (OW group), and a combined exposure to warmed acidified conditions (5 °C and 20 °C, 0.112 kPa PCO2, OWA group). Scallops were placed in a 4.7 T MR animal scanner and the energetic status of the adductor muscle was determined under resting conditions using in vivo 31P-NMR spectroscopy. The surplus oxidative flux (Qmax) was quantified by recording the recovery of arginine phosphate (PLA) directly after moderate swimming exercise of the scallops. Results: Measurements led to reproducible results within each experimental group. Under projected future conditions resting PLA levels (PLArest) were reduced, indicating reduced energy reserves in warming exposed scallops per se. In comparison to vertebrate muscle tissue surplus Qmax of scallop muscle was about one order of magnitude lower. This can be explained by lower mitochondrial contents and capacities in invertebrate than vertebrate muscle tissue. Warm exposed scallops showed a slower recovery rate of PLA levels (kPLA) and a reduced surplus Qmax. Elevated PCO2 did not affected PLA recovery further. Conclusion: Dynamic in vivo 31P-NMR spectroscopy revealed constrained residual aerobic power budgets in boreal and Arctic scallops under projected ocean warming and acidification indicating that scallops are susceptible to future climate change. The observed reduction in muscular PLArest of scallops coping with a warmer and acidified ocean may be linked to an enhanced energy demand and reduced oxygen partial pressures (PO2) in their body fluids. Delayed recovery from moderate swimming at elevated temperature is a result of reduced PLArest concentrations associated with a warm-induced reduction of a residual aerobic power budget

Regional differences in body condition of Pleuragramma antarcticum – causes and consequences

The Antarctic silverfish Pleuragramma antarcticum is a mesopelagic zooplankton feeding species with a circum-Antarctic distribution. P. antarcticum occurs in shoals and represents a major trophic link in the Antarctic marine food web. Due to the lack of a swim bladder neutral buoyancy is mainly attained by large amounts of lipids which are stored in lipid sacks. However, the functional role of lipids in P. antarcticum is not yet fully understood, i.e. it is not clear whether the function of lipids is limited to buoyancy or they serve as energy deposit as well. If the lipids are used as energy storage, differences in nutritional state should be reflected in the amount of body lipid content. During the RV Polarstern expedition ANT XXVII-3 in 2011 samples of P. antarcticum were taken by means of bottom trawls and bentho-pelagic nets from 5 different regions along a north-south gradient: The South Orkney Islands, King George Island, western Weddell Sea (Larsen A and B), and the southeastern Weddell Sea. Condition factors of P. antarcticum significantly differed between regions, indicating a decline in individual nutritional state from the north to the south (highest condition at South Orkneys, lowest in the south-eastern Weddell Sea). To get a better understanding for the observed differences Magnetic Resonance Imaging (MRI) was used to determine body composition of preserved individuals from all stations. High resolution morphological 2D- and 3D-MR images could be obtained from P. antarcticum for the first time. Digital image processing allowed determining the overall fat and muscle distribution and a calculation of individual lipid and water contents. Percentage overall lipid contents were in good correlation with literature data obtained from standard destructive techniques. The first few datasets showed substantial regional differences in %lipid content. Lipid content was relatively high in individuals from the South Orkneys and King George Island, and low in individuals from the south-eastern Weddell Sea, following the trend of the condition factors. However, individuals from Larsen A show extraordinary high lipid contents compared to all others, and individuals from Larsen B extraordinary low lipid contents. These differences might indicate extensive differences in nutrient availability in both Larsen areas. However, this will not explain the discrepancy in condition factor and lipid content. Analyses of lipid composition in muscle and liver tissues are currently under way to test for potential causes