The overall goal of this methodology is to refine a protocol for brain microinjection using personalized, reusable quartz needle which does not produce detectable tissue damage and ensures reliable delivery even in deep regions. This method can help answer key questions in the field of neuroscience such as the pathophysiology of neurodegenerative and psychiatric diseases and effects of gene and cell therapy via viral vectors and cell graft. The main advantage is that these needles do not produce any detectable tissue damage, which ensured a reliable delivery in deep brain regions.
They are cost-effective as they can be cleaned and reused. To begin this procedure, clean and sterilize the quartz capillaries by placing them for five minutes in distilled water. Five minutes in 99%ethanol, followed by five minutes in diethyl ether.
Then leave the capillaries under the hood for at least one hour to dry completely. In the meantime, prepare the long and thin needles using a laser puller. Then place the needles in a petri dish previously cleaned with 70%ethanol.
Cover the dish with a plastic paraffin film. Next, cut the capillaries at the micro dissector. Firmly fix the needle on the microscope table and position its tip at the center of the computer screen.
On the menu bar, click on the pencil icon and use it to draw a mark for the needle to be cut. On the side bar function, select the flag containing the Start Cut button to allow the laser to cut the previously marked spot. If needed, cut the side holes by drawing a mark and repeating the cutting procedures without repositioning the needle.
After cutting the needle, connect it to a void pump through a plastic tube. Next, turn the pump on and manually set the forward flow rate to three microliters per minute. Then aspirate the 99%ethanol followed by the distilled water to remove impurities due to cutting.
After that, aspirate the 99%ethanol again to ease drying. Subsequently, store the needles in a properly covered petri dish until the day of surgery. To reuse the needles, at the end of the experiment, clean them with the appropriate detergents and 70%ethanol to remove the injected solution and possible tissue residue.
If needed, these needles can even be boiled in devoted glass jars filled with distilled water. In this step, select the settings menu to calibrate the pump according to the diameter per volume of the microinjection syringe. And set the flow rate at 0.3 microliters per minute.
Then fill a 10 milliliter syringe with sterile water. And use it to fill the microinjections syringe and the injection assembly parts kit for the manual microninjection pump. After that, connect the needle to the manual microinjection pump kit.
Disconnect the 10 milliliter syringe from the microinjection syringe while slowly pushing the piston, in order to leave drop of water at the top of the microinjection syringe. Then, insert the piston into the microinjection syringe. Now, connect the microinjection syringe to the pump and set the flow rate at 0.3 microliters per minute.
Subsequently, change the flow rate to 0.1 microliters per minute and start the surgery procedure. To perform the surgery, shave the animal's head and clean it with an alcohol wipe. Next, position the rat in the stereotaxic frame.
Make a longitudinal cut over the skull by using a sterilized scalpel. Be gentle to avoid excessive bleeding. Following that, open the scalp using a flat spatula, and apply surgical clips on the flaps of the skin to keep it open.
Very gently, detach the periosteum to expose the bregma and the area of skull through which the needle will be inserted. Subsequently, mount the arm on the stereotaxic frame and move the tip of the needle right above the bregma, paying attention not to break its tip. Annotate the anteroposterior and medial lateral coordinates of the bregma and move the needle to the desired coordinates.
Then, lower the needle until its tip almost touches the skull, marking the drilling spot after raising the stereotaxic arm a little. Drill the skull on the marked spot, being careful not to damage the meninges and the brain. Next, cut a small piece of plastic paraffin film and place it on the skull.
Using the 10 microliter pipet, make a drop of the solution to apply on the plastic paraffin film. Stop the pump, slightly retrieve the pusher of the pump and create a little air bubble in the capillary by gently pulling the piston of the microinjection syringe. Lower the tip of the needle until it reaches the drop.
Draw the sample by pulling the piston of the microinjection syringe very slowly, avoiding any bubble formation. Replace the pump pusher in contact with the piston and turn on the pump at a flow rate of 0.3 microliters per minute. Then, wait until the formation of a drop before proceeding to the next step.
Afterward, lower the flow to 0.1 microliters per minute. Nick the dura with a stainless steel bevel tip needle and slowly lower the needle to enter into the brain tissue. When the desired position is reached, go down an additional 0.1 millimeters, then stop the pump.
Raise the stereostatic arm by 0.1 millimeters and start the pump again at 0.3 microliters per minute. After that, start the timer and the injection. Monitor the bubble movement while injecting.
At the end, stop the pump and wait five minutes before slowly retrieving the needle. When the quartz needle is completely extracted, start the pump again and check for the formation of a drop at the tip of the needle. Next, seal the opening in the skull surface with bone wax.
Remove the homeostatic clips and suture the scalp. Coat the area around the wound with an antibiotic cream and return the animal to the home cage. These are the eight micrometer coronal sections of the rat brains at the striatum level.
This image shows the damages produced by the injection of two microliters of ACSF, using a 26-gauge blunt end stainless steel needle. Conversely, the striatum injected with a quartz needle, did not show any detectable damage. These other images show the damages induced by the two needles at the level of the dorsal hippocampus.
But the quartz needle did not produce any detectable damage. Successful injections with both needles were shown by the distribution of Trypan blue. Once mastered, this technique can be done in two hours, if it is performed properly.
Following these procedures, all that mattered, like additional surgeries, can be performed in order to answer different questions like drug induced effect. After this development, this technique paved the way for researchers in the field of neuroscience to explore the effect of toxin and drugs and more recently, gene and cell therapy products in animal models. After watching this video, you should have a good understanding on how to perform microinjections in deeper brain regions without any detectable lesions using personalized quartz needles.
Don't forget that using laser-dissected reagents and/or pathogens can be extremely hazardous. And precautions such as laser safety procedures and personal protective equipment should always be taken when performing this procedure.
