The overall goal of this procedure is to monitor structural and functional alterations along the visual pathway in mice in vivo. This is accomplished first with an intravitreal injection of paramagnetic manganese ions, which serve intracellularly as MR.Sensitive tracers and are applied as manganese chloride solution 24 hours before the scan. The second step is to arrange the anesthetized mouse inside the three T MR scanner using appropriate hardware for image acquisition and monitoring of vital functions.
Next, the mouse adopted and tracer sensitive MEMR protocol is driven and multiplanar 3D images of the intra cerebral signal enhancement along the rettino temporal pathway are reconstructed. The final step is to remove the mouse from the MRI scanner and to support its vital functions for rapid recovery from anesthesia, which allows for repetitive EMRI studies of the same animal or subsequent behavioral or histological examinations. Ultimately manganese enhanced MRI of the tactile projection is used to show acute or progressive neuro pathologies in experimentally or genetically modified mice mimicking neurodegenerative CNS diseases.
The main advantage of this technique over existing methods like postmortem histology, is that structural alterations directly can be monitored in vivo or by repetitive studies on individual animals. This method can help to answer key questions in the neuros scientific field, such as on structural pathologies in Alzheimer's or Parkinson's diseases after CNS drama or even in poly characterized metabolic diseases. So this method can provide insights into special neuro anatomic features of the visual projections, but it can also be applied to other systems such as the auditory or factory projections or even to aspects of the motor system researchers, Which are new to this field.
Phase two problems. One is to get these micro injections of the manganese solutions in the rbs. Exactly right.
So the MR imaging enhancement. The manganese enhancement in effect is consistent from animal to animal and the other is to get the complex task of MR Imaging right and as it requires some dedicated small animal hardware like the received coral we are using To begin, prepare the contrast agent by diluting one microliter of one molar manganese chloride stock solution in 132 microliters of water under sterile conditions. Load five microliters of the final solution into a five microliter syringe connected to a 34 gauge small hub removable needle.
24 hours prior to the MRI scan, anesthetize the mouse and confirm deep sedation by checking the pedal reflux. Once sedated, apply one drop of liquid 0.4%oxy butane hydrochloride, a topical anesthetic to the cornea. Next, position the mouse on its left side under a binocular microscope and gently open and fix the right eye between the thumb and forefinger of your left hand.
Hold the syringe with your right hand resting the needle close to the tip. Carefully insert the needle into the vitreous body at the in frontotemporal circumference approximately one millimeter distal to the limbus to avoid damaging any scleral vessels while an assistance slowly applies the total volume of two microliters. Monitor optimal needle placement under the microscope to avoid puncturing the lens or spilling any liquid to minimize any liquid leakage from the injection site.
Keep the needle inserted after injection for an additional 30 seconds before slowly withdrawing it for bilateral signal enhancement. Repeat the injection procedure for the left eye after the procedure apply eyedrops containing three milligrams per milliliter of a floxacin and an ointment containing panol to prevent infections and trying of the eye. Return the mice to their cages until the start of the MR scan, the custom made mouse holder, shown here as a bite bar for head fixation and sensors for respiratory and temperature monitoring.
Following inhalational anesthesia, mount the mouse on the mouse holder in an almost horizontal untwisted position with the animal in its final position. Insert the animal and holder into a dedicated signal to noise efficient linearly polarized LITS coil for small animals with an effective field of view of 35 millimeters by 38 millimeters in diameter. Operate the coil in transmit receive mode.
Insert the holder in the shield tube. Adjust the tune end match of the coil with the aid of a vector impedance analyzer. Position the coil in the three Tesla MR Scanner and begin monitoring respiration and heart rate during the scan supply anesthesia using a continuous insufflated mixture of 1.5%isof fluorine, 98.5%oxygen gas with an evaporator connected to the mouse head holder by an integrated tube.
Maintain a stable respiration rate of about 40 breaths per minute and a body temperature above 35 degrees Celsius. Manually check the frequency adjustment, shim currents and transmitter reference voltage to optimize image homogeneity and quality. Next, acquire T one weighted two dimensional turbo spin images with a resolution of 0.5 millimeters by 0.5 millimeters by two millimeters in both the sagittal and transversal view for planning.
Using the planning, MR scans acquire the manganese enhanced MR images in the coronal measurement direction. Rotated to be parallel to the animal's head with phasing coating along the left right direction. To minimize acquisition time, use a tight field of view of 54 millimeters and apply a rectangular field of view of 93.8%Apply a spoiled 3D fast flow angle shot or flash sequence with a base matrix of 256.
Use 128 slices of 0.11 millimeter thickness with slice resolution set to 61%to create a final isotropic interpolated 3D volume data set. Use an echo time of 6.51 milliseconds repetition time of 16 milliseconds. Flip angle of 22 degrees and bandwidth of 160 hertz per pixel.
Apply two averages and three repetitions to achieve a total acquisition time of approximately 30 minutes. After the scan average the acquired three image sets to get the final image. Remove the mouse from the holder and supply pure oxygen.
To accelerate recovery from anesthesia, use a red light heating source to keep body temperature stable. Analyze the data using single fast view or similar software. Select defined regions of interest in two dimensional planar MRI Recordings and determine signal intensities of the manganese enhanced structure and tissue background here shown as mean as well as the standard deviation of the noise st.
For each sample, quantify three consecutive images for the mean contrast to noise ratio in bilaterally injected animals analyze each hemisphere independently. When necessary, use a mouse brain atlas for neuro anatomical orientation of the lateral genus, nucleus and superior colus. Next, calculate the contrast to noise ratio by taking the difference main intensity of the manganese enhanced structures from the tissue background and dividing by the standard deviation of the noise SD for horizontal coronal and sagittal images generate multiplanar reconstructions from the original 3D MRI dataset.
These processed images are not recommended for quantitative analysis. Use angiography post-processing software to create animated 3D reconstructions of the retal projection manganese injected Retin eye show strong signal enhancement in T one weighted MRI compared to non enhanced brain areas and controls. However, at 150 nanomolar manganese injections produce signal saturation, the contrast to noise ratio or CNR was greater for the dark adapted retina compared to the light stimulated condition.
However, signal for the light and dark conditions are no different in the lateral gen nucleus and superior colus when looked at by age mice between three and 26 months show no effective age in signal enhancement. Optic nerve crush injury causes breakage of the axon sles and blocks retile transport of manganese. One week after the optic nerve was lesioned unilaterally, the crushed side showed complete loss of signal enhancement in contralateral LGN and SC compared to the ipsilateral intact side.
The location and severity of an injury can be detected by assessing signal intensity along the optic nerve before and after injury. One day after injury, signal intensity drops to background levels at locations along the optic nerve beyond the lesion site. NF Kappa BP 50 knockout mice, which suffer from a spontaneous atrophy of the retile projection at 10 months of age show a reduced manganese dependent signal enhancement in the LGN 24 hours after injection compared to wild type mice over the course of 72 hours.
Wild type and mutant mice show a similar time course of decreasing signal intensity in the retina and the LGN such reduced rather than simply delayed. Manganese transport indicates fiber atrophy. While attempting this procedure, it's important to remember to carefully perform micro injections and to continuously monitor vital functions during and after the procedure, especially when longitudinal studies are desired.
Following this procedure, other methods like histological or behavioral studies can be performed in order to address additional questions concerning the ultra structure or functionality of the visual system. After Introduction of this technique to neuroscientific research, it helped to explore the path mechanisms in a multitude of mouse models that mimic inherited or acquired CNS diseases. Don't forget, if you're Working with our eye scanners, the strong magnetic field poses a serious hazard and you cannot bring any equipment which is magnetic close to the scanner.
