Functional Interrogation of Adult Hypothalamic Neurogenesis with Focal Radiological Inhibition

The overall goal of this procedure is to describe an advanced radiological technique, which allows one to perform focal irradiation on small animal models and subsequently inhibit proliferation with anatomic specific resolution. This is accomplished by first employing computer tomography guided three dimensional volumetric imaging for localization of the region of interest. The second step of the procedure is to calculate the delivery and duration of radiological treatment to the region of interest.

The third step is to perform film-based calibration for the radiation dose. The final step is to determine radiation beam accuracy by direct visualization of the radiation beam in the tissue. Ultimately, results from these studies can demonstrate the potential functional role of select proliferating neuro progenitor populations by post-treatment analysis of physiology and behavior.

The main advantage of this technique over existing methods for suppressing neurogenesis such as broad irradiation of the brain, is that this uses radiation beams as small as 0.5 millimeters in diameter to target specific neurogenerative populations. This allows one to unambiguously associate behavioral or physiological defects with the specific functions of neurogenerative populations. Visual demonstration of this method is critical as the radiation beam set up steps are difficult to learn initially because of the numerous steps involved in using both the hardware and software for prop localization and dosage, Prepare an ISO fluorine gas anesthesia chamber, then add a single mouse into the chamber.

In parallel, prepare a heating pad on the low setting for post-treatment animal care. When the mouse no longer responds to a footpad compression, bring it to the radiological platform and place it on the immobilization bed of the robotic stage. Place its mouth into the nose cone anesthesia cup and its teeth into the bite guard.

Lay the mouse flat on the immobilization bed. If it remains unresponsive, secure the mouse with gauze tape. Make sure the head is level to a horizontal plane by pulling up on the ears.

Once the mouse is in the correct position, close the lead protective shield. Now acquire a computer tomography scan using the onboard software of the radiological platform to gain a three dimensional anatomical structural scan of the mouse subject from the images. Check that the head is level to the horizontal plane.

If not, continue adjusting the mouse's head until it is so. Now identify the ROI from the CT images to visualize the ventral basal hypothalamus with CT imaging. Operate the x-ray tube as listed.

Calculate the distance from the ROI to the surface of the skull using a 45 degree angle to the horizontal plane. Using onboard software, take an x-ray of the mouse subject from above. Then remove the mouse from the radiological platform.

Place it on the heating pad and monitor it until it is active. From the coronal CT images, calculate the average ROI anatomical depth of at least three mice in order to determine delivery dosing. As an example from a previous study where 10 gray of I radiation was administered to the ventra basal hypothalamus, the depth of the ROI from the skull from a 45 degree angle is 0.66 centimeters.

Once the value is known, use dose planning software to calculate the appropriate rotation, speed, and length of treatment for the desired dose to the ROI. Next measure the dose distributions of the calculated parameters. Embed three GAF chromic radiation sensitive films into a water equivalent plastic mock mouse.

Position them between four vertically stacked water equivalent plastic blocks. Place the mock mouse on the robotic stage and run the focal irradiation beam with the newly calculated parameters. In this example, a 10 grade dose of radiation to the ventral basal hypothalamus is tested Following the irradiation check the pattern and intensity of the dosage on the films for a 360 degree angle rotation, targeting the ventral basal hypothalamus, the cone beam.

A radiation produces a dark ring in the film above the ISO center, A small crisp spot at the ISO center film and a lighter ring in the film below. The ISO center now superimposed the ISO center GA FCH Chromic film over the x-rays. The irradiated focal point at ISO center should overlap with the desired ROI later.

The accuracy of the irradiation beam can be assessed in the tissue by visualizing a marker of double stranded DNA breaks. Once satisfied with the calibration and targeting of the irradiation beam, proceed with the experiment. In this example, five week old C 57 black six J female mice were given a high fat diet for one week prior to the treatment.

The day before the treatment, the mice were weighed and split into two cohorts with no significant difference in weight between the cohorts. On the day of the treatment, the mice were re weighed and then gently transported to the radiological platform and anesthetized two mice, one in the experimental group and the other a control. Also set the heating pad to the low setting for postoperative treatment.

Set the mouse up to receive irradiation as previously described. While the treatment is going on, keep the sham mouse in the anesthesia chamber near the CFIR platform, so any effects on ambient radiation are factored in. After the target is identified on the ct, move the mouse subject under the robotic control to align the target with the beam.

Then input the calculated dose setting and deliver the radiation After the irradiation. Place both the mice on the heating pad and monitor them until they wake up. After all the mice are irradiated, monitor them daily and weigh them twice a week.

Three days after treatment To confirm irradiation, administer intraperitoneal injections of BRDU one month later. Identify neurogenesis in cells with BRDU and a neuronal marker. Dose distributions were measured with GAF chromic.

Radiation sensitive films embedded in a mock mouse. The cone of radiation had a full width at a half maximum of 2.31 millimeters. The focal beam demonstrates the precision alignment of beams from different directions.

This film can be superimposed on top of an x-ray of the real mouse subject demonstrating beam position and precision. Should targeting of the ROI be insufficient with the anatomical landmarks, an injection of intrathecal iodine contrast can be used to enhance the targeting. Lateral and third ventricles are clearly visualized in CT scans acquired on A-C-F-I-R radiological platform to further confirm the CT guided targeting of the hypothalamic me.

The location of the beam in the tissue was visualized from the resulting double stranded DNA breaks gamma H two A x staining demonstrated precise targeting with an extremely sharp edge to the beam. The stereotactic like arc treatment consisting of an arc at 45 degrees from the vertical effectively targeted the ventral basal hypothalamus. Without irradiating other neurogenic niches.

The effect of irradiation on neurogenesis was investigated with high fat diet fed adult mice. There was approximately 85%inhibition of me neurogenesis compared with sham treated controls. Whereas in an adjacent structure bordering the irradiation site, there was no statistically significant difference between irradiated animals and sham controls.

Irradiated mice fed high fat diets prior to treatment had decreased weight gain post-treatment compared to the sham treated group. In contrast, normal chow fed control mice, which had significantly lower levels of me neurogenesis than their high fat fed counterparts did not show a significant difference in weight between the sham versus irradiated groups. Moreover, this reduced weight gain in irradiated high fat fed mice is accompanied by changes in metabolism and activity Once mastered.

This technique can be done in approximately 15 minutes per mouse if performed properly. Just don't forget that working with radiation can be extremely hazardous and precautions such as the use of lead shielding, radiation detectors, and proper radiation usage training should always be taken prior to and while performing this procedure.