Autoradiographic Measurements of [¹⁴C]-Iodoantipyrine in Rat Brain Following Central Post-Stroke Pain

The overall goal of this experimental procedure is to assess the involvement of neural substrates of thalamic lesion induced central post-stroke pain using the radio isotope tracer method. This method can help answer key questions in which brain area involved in central post-stroke pain. The main advantage of this tactic is that it is a less expensive and more efficient to perform compared with the other brain mapping techniques.

In this procedure, perform the von Frey and Plantar tests to establish baselines by placing the rat in an acrylic enclosure for 30 minutes. Obtain von Frey filaments that have the same length but varying diameters to provide a range of forces of two to 100 grams. Next, use the filaments to stimulate the center of the animal's hindpaw through a net-like port on the acrylic plate.

When the rats exhibit a paw-withdrawal response to the stimulation, record the filament number. Then use ascending filaments and determine the lowest value for the withdrawal response. Repeat the test for a total of three times in succession on the same rat until the maximum applied pressure is recorded.

Using a reference list, convert the filament number to the corresponding force, and then average the values. Use an infrared beam to stimulate the center of the rat's hindpaw through a glass plate. Press the red bottom of the Plantar device twice to measure the withdrawal response latency.

Set the inter-trial interval to at least five minutes to avoid successive stimulation. Record the duration of the infrared light when the rat exhibits a paw-withdrawal response. The longest duration should not exceed 20 seconds in each trial to avoid tissue damage.

Repeat the rest with three trials for the left and right hindpaws and calculate the average of the withdrawal responses for each hindpaw. In this step, transfer the anesthetized rat to a stereotaxic device with a heating pad to maintain its body temperature. Apply ointment to its eyes to prevent dryness while under anesthesia.

Subsequently, shave the animal's head with a sterilized electric clipper. Make an incision with a scalpel along the mid-line of the scalp. Then, clean the skin and skull with alternating alcohol and povidone iodine.

Afterward, sterile gloves and instruments are used. Drill a small hole with a diameter of three millimeters in the skull over the ventral basal nucleus of the thalamus. Lower the needle and micro-inject 0.5 microliters of normal saline or 0.125 units of type four collagenase solution.

When it is completed, keep the injection needle in place so that the skull landmark with a dark line has been marked for identification. The injection needle allows for drug diffusion for an additional five minutes. Then, use dental cement to fill the hole in the skill and suture the incision.

In this procedure, place the rat in a resting cage for five to 10 minutes for adaptation. Using a splitter, connect to two one milliliter syringes. Fill one syringe with normal saline, and the other with IAP solution.

Connect a PE-50 tubing to the splitter. Then, inject the radiotracer in the external jugular vein of the animal. After that, fill the syringe with three molar potassium chloride.

10 seconds after the radiotracer injection, inject potassium chloride under an overdose of isoflurane before sacrificing the animal. After one minute, remove the brain and use OCT compound to freeze it in dry ice and methylbutane. Subsequently, store the brain tissue in the freezer.

To prepare brain sections, orient the brain in a microtome with the hindbrain facing down. Using a cryostat, slice the brain into 20 micrometer thick sections. Next, place the brain slices on microscope slides at negative 20 degrees Celsius.

Subsequently, place the microscope slides and five standard filter papers with graded radioactivity in the exposure cassettes. According to this sequence of brain slices, arrange the microscope slides from top to bottom. Then, place the filter places in the bottom of the cassettes.

Afterward, remove the phosphor screen from the exposure cassettes. Use a variable mode imager to read the phosphor screen and generate images to show the IAP uptake for the brain slices. Shown here are the regions of interest in an anatomical atlas.

The ROI analysis showed that activation of infralimbic cortex, prelimbic cortex, and cingulate cortex area one was significantly higher in the CPS-P group in the right hemisphere with the exception of the ventral-basal nucleus. Differences in inter-regional correlations of rCBF were observed between the CPSP and sham groups in the right hemisphere. And pain-related neural substrates were determined by analyzing the differences in the inter-regional correlations of rCBF.

The red lines indicate significant positive correlations, and the blue lines indicate significant negative correlations. this technique can be done in two hours if it is performed properly. After its development, this technique paved the way for researcher in the field of brain mapping to expose central post-stroke brain in hemorrhagic stroke.

Following this procedure, other masses like FMRI can be performed in order to answer additional questions like real time neural activity and neural network. After watching this video, you should have a good understanding of how to exam which brain areas involve in the central post-stroke pain.