The Use of Pharmacological-challenge fMRI in Pre-clinical Research: Application to the 5-HT System

The overall goal of the following experiment is to assess serotonin or five HT neurotransmitter function and free breathing animal with pharmacological magnetic resonance imaging and an intravenous challenge with a selective serotonin re-uptake inhibitor fluoxetine. This is achieved by combining functional MRI with a drug challenge after initial baseline measures, which is used to visualize the brain hemodynamic response to the specific drug before the start of and during the experiment. Extensive monitoring and maintenance of the animal's physiological responses is needed to ensure that the brain response visualized with FMRI is solely attributable to the pharmacological challenge given.

Finally, statistical data analysis is performed on the MRI data in order to determine the brain's voxels, which exhibit signal variants compared to baseline values and are thus attributable to the serotonergic challenge. Results are obtained that give an indication about the brain serotonin function based on the brain's response to a serotonergic challenge as measured with functional MRI. The main advantage of this technique over existing imaging methods like PET or SPECT, is that it is less invasive since no radio tracers are used.

It's therefore a very suitable technique for translational research as it can also be used in vulnerable human subjects so such as children or patients. Though this method can provide insight into the functioning of the serotonin system, it can also be applied to other neurotransmitter systems when using a different drug challenge such as the dopaminergic system using it, for example, an amphetamine challenge To begin this experiment, first fully anesthetize the rat, then cannulate the femoral artery and vein for blood gas and blood pressure measurements, as well as administration of the challenge medication. After completing the surgery, lower the anesthesia level to 2%for maintenance.

Depending on the arterial blood gas values. Use a warm air heating system to maintain body temperature within 1.5 degrees of 37 degrees Celsius and be aware that MR.Imaging may affect temperature. It is also possible to place a warm water bed inside the stereotactic bed.

Next, place the animal in an MR compatible stereotactic bed in a prone position using ear and tooth bars to maintain head position. Place a respiratory cuff coupled to a pressure sensor under the animal to monitor and record the animal's respiration rate. During imaging.

Also connect the arterial line to a pressure transducer to monitor arterial blood pressure. Here, a 4.7 Tesla small animal MRI system is used with 72 millimeter inner diameter and a cylindrical quadrature transmit receive RF coil. Then place the bed in the MRI scanner bore.

Once the animal has been placed in the scanner, physiological responses should be constantly monitored to assure the animal remains stable, fully anesthetized, and free breathing throughout the procedure. For in vivo imaging, it is important to avoid any adjustments in the anesthesia regime during the pH MRI scan. As this could affect the MR signal arterial blood gas values should be stable and within the ranges displayed here.

Before starting the pH MRI scan, if not slightly, adjust the level of anesthesia to allow deeper or shallower respiration and check values. Again, after a short while, the respiration rate should be stable at 45 to 75 breaths per minute, blood pressure should be constant and between a hundred to hundred and 50 millimeters of mercury. The pharmacological challenge will induce changes in these measures as described.

Subsequently in the protocol, begin imaging with a three plane scout image to correctly position the brain in the middle of the MRI field of view. Then use localized shim correction to improve magnetic field homogeneity. Next, acquire a T two weighted anatomical image volume for registration and segmentation purposes.

30 coronal slices of one millimeter thickness centered eight millimeters coddled to the posterior edge of the olfactory bulb are recommended. See the manuscript for specific image parameters assuring all physiological responses are constant. Continue with the pH MRI scan here.

A T two weighted turbos spin echo is used and 32 volumes are acquired. With a total scan time of 84 minutes. Acquire a number of baseline volumes before administering the challenge medication, at least for 10 minutes.

Be sure to start the infusion at exactly the same time for all animals within a study. In this protocol administration was started at the start of the ninth volume after approximately 21 minutes Baseline scanning. Note that the pharmacological challenge with fluoxetine will induce a short 15 to 20%rise in respiration rate, as well as a short but steep drop of about 20%in arterial blood pressure.

This should recover within five to 10 minutes. Make sure the post infusion imaging period is long enough to visualize signal changes and to reach steady state or recovery of the MR signal depending on the research question and choice of drug challenge. When imaging is finished, remove the animal from the scanner and perform a final blood gas measurement to ensure stability of the blood gas parameters during image acquisition described.

Here are several steps in the pre-processing of the MR data in order to optimize the data for statistical analysis. The tools mentioned are suggested many different tools are available. First, convert all raw images to the file format compatible with the MRI analysis software to be used.

Then in order to ensure compatibility with analysis algorithms designed for use with human data, foxhole size should be multiplied by a factor of 10. Be sure to visually check your images for irregularities and orientation artifacts, signal to noise ratio and motion discard scans with clear artifacts or excessive motion as they will distort your results. Orientation of all scans should be similar between the anatomical and functional images and in concordance with the used reference brain to correct.

For any motion artifacts in the four D time series, use a motion correction tool such as fsls flt, which uses a fine intermodal brain image registration. Next, perform brain segmentation for both the four D time series and the 3D anatomical image. For this, the FSL tool, BET can be used.

Default settings are developed for use with human brains. Therefore, be sure to optimize the values for the rat brain. The goal of the statistical analysis is to determine the voxels which exhibit additional variants attributable to the drug challenge.

Various approaches and software packages are available for this. Here, a suggested method is given prior to analysis. A general linear model to which your data can be tested must first be determined.

This can be a simple square on off model or a specific model based upon the data. For a data-driven model, perform a two sample T-test on all baseline volumes versus all post challenge volumes. The first volume and the volume during which the challenge is given should be left out as these may not represent steady state imaging subsequently discriminate all voxels with more than a certain percent change from baseline, such as a 1%change average the time course in all the discriminated voxels to provide an impression of the shape of the model.

This can determine if the challenge has had an immediate or delayed effect if the effect reaches a plateau or peak, and if the effect declines again during the time course of the scan. This can be done for all animals and then again average to get the mean signal change in all animals. This will be the model for your data-driven GLM.

Next, within the analysis tool, a first level analysis must be set up. Set the TR and note there is no need to set a high or lowpass filter spatially smooth the data using a full width half maximum kernel of eight millimeters. Now statistically test the raw four D time series image of each animal against the previously established general linear model.

This is your explanatory variable, IE, the waveform or design matrix you are testing your data against. Next, apply a threshold to the resulting statistic image to indicate which voxels or clusters of voxels are activated at a particular significance level. Multiple comparisons, corrections should be used due to the large number of voxels tested.

Finally, spatially normalize the data to a reference in order to perform group statistics. First, register the functional data to the animal's brain extracted anatomical image, and then to the reference image. Then run the analysis by pressing go and check the outcome.

After this, the first level analysis of all animals can be combined in higher level statistical analysis. This is highly dependent on your own study, design and research questions. Recorded physiological responses can also be coupled to the MR signal if needed.

When the challenging drug enters the vascular system, a clear physiological response should be visible. Fluoxetine causes an increase in respiration rate as well as a decrease in blood pressure. These responses normalize on average within five to 10 minutes.

Here, this drop in blood pressure is clearly visible. A good example of a positive activated voxel and its accompanying signal time course can be seen here. The average signal time course in activated voxels should show a relatively stable baseline and a clear effect of the challenge.

Preferably, there should be no challenge in dependent drift in the signal artifacts such as respiration, depression, or failure or changes in anesthesia are often clearly visible in the signal. An example of general signal loss due to respiration depression is seen here after first level analysis. The activation pattern is expected to be mainly positive and located in specific serotonin related regions only, such as the cortical areas, the hippocampus, the hypothalamus, and thalamus.

If the entire brain shows a linear decrease of the MR signal, this is often indicative of more systemic effects. For example, two deep anesthesia or respiration depression Also was watching this video. You should have a good understanding of how to assess serotonin neurotransmitter function, the life and free breathing animal with pharmacological magnetic resonance imaging, and an intravenous challenge with a selective serotonin re-uptake Inhibitor.

Don't forget that working with high magnetic fields can be extremely hazardous, and precautions should always be taken to avoid any magnetic metal objects being inside the magnetic field while performing this procedure. This means that all equipment used to monitor the animal during image acquisition should be a more compatible.