Hardware and Experimental Setup  
Equipment
 
  All measurements are performed in a vertical 9.4 Tesla Bruker UltraShieldTM magnet (in the picture at the left). The bore of the magnet is 89 mm. However, this space is not completely available for the sample to be measured, since the probe head and – if required – also the gradient system have to fit in the bore somehow. Connected to the magnet is a Bruker AvanceIII console (not shown) which is controlled by a Linux PC (just at the right) via ParaVision 5.1 (imaging) and TopSpin 3.0 (high resolution spectroscopy), respectively.

For spatially-selective measurements two water-cooled microimaging systems with actively shielded gradients are at our disposal, which differ with regard to inner diameter (ID) and gradient strength (Micro2.5: ID 40 mm, 1.5 Tesla/m; Mini0.5: ID 57 mm, 0.2 Tesla/m). The available space is further restricted by the respective probe heads and the corresponding coils. For in vivo measurements resonators with 10, 25, 30, and 38 mm ID are available as well as a 8-mm solenoid and some surface coils with most of them capable of X-nuclei detection, i.e. coils are tuned not only for the 1H but also for the 19F and 31P nucleus, repectively.

However, even the reduced space within the resonator is not left to the laboratory animal only. As described in more detail elsewhere the recording of the ECG and the respiration are not only required for monitoring of the vital functions of mouse or rat but also indispensable for the accurate performance of heart measurements. That's why ECG electrodes, a respiraton sensor as well as tubings for inhalation anaesthesia have additionally to be kept within the probe head.

For measurements on perfused organs or cells with no volume selection required, we are equipped with a wide range of probe heads, by which all biological relevant nuclei can be selectively detected (1H, 13C, 17O, 19F, 23Na, 31P, 87Rb, 133Cs). Probe heads are available with inner diameters of 5, 10, 20, and 30 mm and are suitable for both high resolution spectroscopy of tissue extracts as well as for physiological studies of isolated hearts (mouse, rat, guinea pig).

 
 
 
In vivo measurements
 
 
  For in vivo studies the laboratory animal has to be positioned in the magnet within the area of the sensitive volume (scheme at the left) and to be convinced to stay calm at its place for the duration of the measurements – needless to say that this succeeds only, when the animal is maintained in anaesthesia. This is carried out by use of a home-made respiratory mask, by which the inhalation anesthetic isoflurane (1.5 % in air) is applied.

Isoflurane is also used for human anaesthesia and is well tolerated by rodents. Already about 1-2 minutes after termination of the MR investigation the animals briskly climb around in their cages. After inducing anaesthesia, the animal is placed with caution in the animal handling system of the probe head and accurately fixed within (pictures at the right), so that it can not shift during the measurements. Afterwards the complete setup is carried over into the magnet.

 
Within the magnet the vital functions (ECG, respiration) of the animal are continuously monitored by electrodes as well as sensors mounted in the probe head, and the body temperature is kept at 37 °C. In preceding control experiments we have assured ourself, that the vertical position of the animals in the magnet does not affect their normal physiology. Seeing how the mice usually scramble around headlong in their cages, this was not to expect either. Concerning more details to the MR measurements please refer to the respective pages of heart, vessels, and brain.
 
 
 
Isolated heart measurements (Langendorff model)
 
 
  Studies of isolated, perfused organs offer the advantage to have almost full control over each individual experimental parameter. For example, drugs can be applied under much more defined conditions directly to the object of interest, and disturbing interferences with the blood circulation or other organs can be ruled out.  
 
The heart is especially suitable for that kind of investigations, since it immediately starts beating by itself – even outside the body – when it is sufficiently supplied with nothing but oxygen, fuels (such as glucose or fatty acids), and some essential ions like Ca2+, K+, and Na+. However, for the preparation and the "hanging" of the small mouse heart at the perfusion cannula you have to get the right feeling (for comarison of dimensions just at the right a normal match). To the left of the heart a water-filled balloon is shown, which is introduced into the left cardiac chamber in order to assess the pump function of the heart.
  

The perfusion and the appropriate supply of the heart is carried out via the aorta (refer to the figures right at the top and at the left) with a special buffer solution (Krebs Henseleit buffer, KHB) at a physiological pressure of 100 mmHg. Then, the heart is transferred into a 10-mm NMR tube together with half a dozen of cables for monitoring of the functional parameters. Afterwards the whole setup is placed – in the same manner as the anesthetized mouse – within the sensitive volume of the magnet (see right at the top) at 37 °C. A long black trunk (adjoining picture) ensures that inside the magnet the heart is supplied according to its requirements. In the middle of the photo the buffer reservoir can be made out as well as a couple of pumps which prevent a buffer overflow in the NMR tube.

Finally, rightmost the measuring tower for determination of physiological variables can be seen. Besides the data accessible by NMR spectroscopy (cf. energy, metabolism, and myoglobin), routinely the following parameters are recorded: left ventricular developed pressure (LVDP), first derivative of LVDP (dP/dt), heart rate, coronary flow as well as the myocardial oxygen consumption. Furthermore, the investigation of the coronary effluate provides valuable information about compounds relased by the heart, such as second messengers, when it is stressed or challenged, e.g. under hypoxic conditions or β-adrenergic stimulation.

 
 
 
Measurements of perfused cells
 
 
  The restriction to the isolated organ level as decribed above simplifies the study of organ-specific mechanisms without interaction of the whole body circulation. However, due to the heterogeneity of the tissue this approach detects the sum of all metabolic processes by the different existing cell types (in the heart, for example endothelial and muscle cells). As a consequence, cells with individual functions – and with that often with a particular metabolic pattern – can not be distinguished. Compared with this, the use of perfused cell cultures allows the continuous monitoring and characterization of the specific metabolism of individual cell types.

In order to obtain a satisfying temporal resolution during acquisition of the NMR spectra, a relative large amount of cells (approximately 108) in a small volume (ca. 1 cm3) is required. This can be realized quite easy when studying cells that grow in suspension, such as lymphocytes. To get similar large cell quantities also for adherently growing cultures, like endothelial or muscle cells, it is necessary to maximize the growing and adhering area available in the sample volume. For this purpose microcarrier beads (small coated spheres), dialysis membranes, hollow fiber bioreactors (purpose-developed breeder reactors) as well as agarose and basement membrane gel threads can be used.

Very similar as described above in the section about the isolated heart, for NMR measurements the cells are transferred into a NMR tube and are supplied within the magnet at 37 °C with a perfusion system. Also in this setup exhaustion pumps prevent a flooding of the probe head. Since the cell are not "hanged", a porous disc takes care that the cells are retained within the sensitive volume during perfusion (see the adjoining figure).

 
 
 
Highly resolved measurements of tissue extracts
 
 
  What actually is the evidence to perform NMR investigations on tissue extracts, when the main trumps of the MR technique – that is non-invasiveness and the possibility of repetitive studies of the same object – are thrown overboard?

Sacrificing temporal dynamics allows measurements of perfect homogenous samples over a much longer period of time and, thus, enables experiments which otherwise would almost be impossible to realize:

  • highly resolved spectra
  • fingerprint analysis
  • 13C labelling experiments (see also metabolism)
  • metabolomics via 1H and 13C
  • sophisticated NMR experiments (e.g. 2D correlations etc.)

The adjoining figure gives an example for the enhanced spectral resolution gained when changing from the isolated organ (bottom) to the tissue extract (top) – obviously, a lot of details remain hidden in the bottom spectrum.

  Despite the reduced line width of the signals in tissue extract spectra, there may still occur signal overlapping which complicates assignment and analysis of spectra. This may be overcome by 2D correlation experiments (e.g. H-H, C-H). The figure at the left shows an example of a 2D H,H-TOCSY (Total Correlation Spectroscopy) recorded from a heart extract. Direct H-H couplings (theory) appear as cross peaks at the F1 and F2 axis, whereas the normal 1D spectrum can be found along the diagonal. Altogether, high resolution spectroscopy of tissue extracts yields a wealth of information, which profoundly complement the in vivo measurements.

It should be mentioned, that for this an appropriate preparation of the samples is required. Immediately after excision the tissue has to be snap-frozen with liquid nitrogen, in order to suddenly stop all metabolic processes. Subsequently, the tissue is extracted with strong acids (e.g. perchloric acid), which makes the cytosolic components accessible and, concomitantly, denaturizes all proteins.