The overall goal of this experimental procedure is to utilize a novel application of enzyme-based microelectrode array technology to measure neurotransmitters in neonatal piglets. In this example, in vivo glutamate activity is examined to study anesthesia-induced neurotoxicity. The main advantage of this technique is that it can measure in vivo neurotransmitter activity with exceptional spatial and temporal resolution in a clinically-relevant animal model of anesthesia-induced neurotoxicity.
Although this method can provide insight into mechanisms of anesthesia-induced neurotoxicity, it can also be applied to other pathologic states, such as pediatric brain trauma, epilepsy, and stroke. Generally, individuals new to this method will struggle because use of the piglet model requires experience and practice in implementation. In addition, the use of microelectrode arrays requires a specialized skill set.
Visual demonstration of this method is critical, as the surgical and microelectrode placement steps are difficult to learn, because of their delicate nature. For this experiment, use piglets during their peak brain growth time, when they are three to five days old. Allow them to acclimate for at least 24 hours before the experiment.
Trained staff must care for the piglets. They should be provided ad lib access to nutrition, blankets, and some toys for stimuli. At least three hours prior to anesthesia, remove the milk replacer from the cage to ensure the piglet's stomach is empty.
Follow ARRIVE guidelines to eliminate any potential sex-based confounders. Later, at an anesthesia workstation equipped with a pediatric ventilator and appropriate monitoring devices, intubate and mechanically ventilate the piglet. Then, administer sevoflurane anesthesia at 1 MAC for 3.5 hours of anesthesia.
Now, use a toe pinch to confirm an adequate depth of anesthesia, then secure the piglet to a piglet-specific stereotaxic frame that has adequate padding. Position the teeth of the maxilla over the tooth bar. Next, fix and tighten the two penetrating ear bars with the piglet centered at midline.
Insert the ear bars firmly enough to hear the tympanic membranes pop. Start a rocuronium loading dose and an infusion to prevent movements while the piglet is secured in the frame. It is vital that the piglet stay warm, and that its vital signs are monitored.
Use a heat lamp, and/or a blanket, to maintain normal thermia. Make sure that the heat lamp isn't so close that it burns. If piglet survival is desired, take additional preparations to keep the surgical field sterile.
Now, proceed with implantation of the microelectrode array. To begin, create a four-to six-centimeter midline incision along the skull, using caution to avoid scoring the skull with the scalpel. Once the incision is made, use gentle retraction and blunt dissection to elevate the scalp from the skull.
Next, gently scrub the skull with a gauze pad to remove any connective tissue and expose the suture lines. Then determine the intended location for the craniotomy. If the area of interest remains obscured, further reflect the scalp.
Now, use a surgical drill to create a craniotomy window that is about 0.25 square centimeters overlying the structure of interest. Be careful not to injure the dura or the underlying brain. As needed, use fine surgical tools to excise the dura overlying the brain tissue.
Use extreme care to avoid damaging the brain. This experiment utilizes a previously described enzyme-based microelectrode array pre-coated with glutamate oxidase, and electroplated with mPD. The microelectrode arrays have a 40 millimeter rigid shaft, customized for use with piglets.
Secure the metal arm to the micro-manipulator, and then position the microelectrode array as vertically as possible over bregma. Then, carefully lower the array as low as possible without touching the surface of the skull, noting the coordinates of bregma. Now use a piglet brain atlas to determine the exact stereotaxic coordinates of the structure of interest, then reposition the microelectrode accordingly.
Next, place the pseudo-reference electrode under the scalp, ensuring contact with the animal. Now slowly lower the microelectrode array into the brain to nearly the appropriate depth. For the final two millimeters of travel, use a hydraulic micro-drive to gently lower the array into the structure of interest with minimal tissue trauma.
After the microelectrode array is positioned, wait 30 minutes to allow the electrodes to reach baseline. Then, take measurements for about three hours. If the piglet is to survive the experiment, close the incision after collecting data.
Real time in vivo glutamate measurements were taken in the hippocampi of three-to four-day old piglets under sevoflurane anesthesia, as described. The recording sessions exceeded three hours. Amperometry measurements were recorded at 4 hertz, and converted to concentration using a linear regression based on calibration parameters.
For each time point, the signals from the two glutamate-sensitive sites were averaged before subtracting the averaged sentinel signal, to yield a corrected glutamate signal. The mean basal glutamate concentration was approximately 4.6 micromoles, and remained relatively stable over the course of the anesthetic exposure. Transient glutamatergic activity was identified by analyzing peaks in the signal that were not correlated with the sentinel signal, and had a signal-to-noise ratio greater than three.
A total of 116 transient peaks were detected over the experimental period. The amplitude of the resulting transient peaks was generally observed to be within the 1 micromole range. In order to quantify the duration of each transient, the time required for each maximum peak value to decay 80%was obtained, and found to be about 4 to 5.5 seconds.
Once mastered, this technique can be done in four hours, if methodically and carefully performed. While attempting this procedure, it's important to remember to minimize any unintentional tissue damage that can confound the experimental data. Following this procedure, other methods, like measurement of other electrochemical analytes, can be performed in order to answer additional questions.
