The overall goal of this procedure is to perform highly precise micro injections into deep brain structures for delivery of drugs, viral vectors, or cell transplants. This is accomplished by first producing glass capillary needles with a 50 micrometer lumen. The second step of the procedure is to fill up 50%of the needle with mineral oil by capillary and insert the plunger by the widest end of the tube.
The third step of the procedure is to load the needle with the substance or the cell suspension that you want to inject. The final step of the procedure is to introduce the needle tip and inject the substance at very low rate flow into a deep brain structure. Ultimately, results can be obtained that show very precise targeting of substances into the brain through micro injections with a glass capillary tube.
Hi, I am Oscar Gonzalez from the Laboratory of Neuroscience in the School of Psychology at the University of Colima, Mexico. Today, we will show you a procedure for performing microinjection with a glass capillary tube to deliver drugs into the mouse brain. We use this procedure in enough laboratory to study adult neural stem cells.
So let's get started. Use of glass capillary needles with a 50 micrometer lumen for micro injections into the brain significantly reduces brain damage, minimizes passive diffusion of drugs, and allows for precise targeting into the rodent brain. Before micro injections can be performed, the glass needles must be constructed.
Begin by placing the capillary glass tube into a pulling high resistance electrode of a micro pipette puller. Next turn on the electrode of the micro pipette puller. To heat the middle of the glass tube to locally soften the glass tube, stretch the glass tube along its longitudinal axis by an initial distance of three to five millimeters, sufficient to cause a reduction in the diameter of the glass tube in the localized area.
Continue stretching the glass tube until it breaks. Repeat these steps to create two identical single barrel needles. Now place the glass needles into a micro forge.
A 30 degree angle is enough to bevel the tip. Finally, place the needles under the microscope and check that they both have the correct inner diameter. For most hydrophilic drugs, a 30 to 50 micrometer inner diameter is ideal with the needles constructed.
Let's see how to use them for micro injections. To begin fill the constructed needle with mineral oil from the widest end. Let the mineral oil enter by capillary until it reaches approximately 50%of the length of the capillary tube.
Put high vacuum grease around the widest end of the needle and insert the plunger through it. To gently push the mineral oil until a small drop of scene going out of the tip of the glass needle. Now secure the needle in the holder of the micro injector and begin preparing the animal for the injection.
First, put a heating pad on the stereotactic animal holder to keep the animal's body temperature. At 37 degrees Celsius, shave the anesthetized mouse's head, then place and secure it into the stereotactic device. Using ear bars and a teeth holder, use a surgical blade to incise the skin from the ears to the Lambda skull Line.
Polish the skull with a cotton swab to dry it out and pull the skin out of the surgical field. Next, use the tip of the glass needle to point at the vertex of the bgma suture and set the initial position of injection there. Now set the point on the skull that is to be drilled by moving the x and Y axis of the stereotactic device over the skull.
Using a research micro drill, carefully drill holes at the selected coordinates with fine forceps. Carefully remove the deepest layer of bone and break the dura modern membrane. You will see some cerebral spinal fluid leaking out.
To confirm that the needle can go through the drilled holes, slowly lower the glass needle using the Z axis of the stereotactic device. Now pipette one microliter of the drug to be injected and place it onto a small piece of parfum. Place the parfum onto the skull.
Suck up the drug by spinning the micro injector wheel while you check through the microscope that the fluid is going up into the glass needle. Remove the para film and reset the zero coordinate. Now move the needle to the selected coordinates and lower the holder until the tip of the glass needle gently touches the brain surface and set the Z.Coordinate at zero.
Introduce the glass needle into the brain parenchyma at the desired depth. Inject the drug slowly at a rate of about one nanoliter per second. When the desired volume is injected, let the drug diffuse into the parenchyma for two minutes, and then proceed to remove the needle smoothly and slowly.
Finally glue the surgical wound and remove the animal from the stereotactic device. Place the animal into a pre-war cage for anesthesia recovery. Now we'll show some microinjection results.
Lysophosphatidylcholine or lyin was injected into the corpus cossum that produces demyelination of white matter tracts to minimize the brain injury produced by the glass needle. Only 20 liters of lyin was injected. Demyelination is represented by the absence of myelin basic protein expression in the white matter Tracts, a very precise injection is obtained and a very narrow needle tract minimizes brain injury.
We've just shown you how to construct glass needles to perform micro injections into deep brain structures. When doing this procedure, it's important to remember to have all the equipments and materials ready to be used in the surgical room. So that's it.
Thanks for watching and good luck with your experiments.
