Ex vivo electrochemical measurement of glutamate release during spinal cord injury

Graphical abstract


Value of the Protocol
Glutamate biosensor measures in real time with better spatial and temporal resolution than microdialysis Measuring from the spinal cord ex vivo removes processes and variables, such as hemorrhage and ischemia, that may obscure glutamate release The glutamate biosensors used were made by direct writing of nanocomposite ink, which is an easy and fast fabrication method in comparison to conventional micro-fabrication

Description of protocol
This protocol records extracellular glutamate with high spatial (100 mm) and temporal (1 s) resolution from a spinal cord segment during injury [1,2]. Glutamate release following traumatic spinal cord injury (SCI) exacerbates the extent of SCI [3], yet the mechanism behind sustained high levels of extracellular glutamate has remained unclear. This protocol can be used to study the relationship between extracellular glutamate and other molecules, such as acrolein, and develop therapeutic interventions [4].  (Figs. 2 and 3a).

Materials
a Place the spinal cord segment across the central compartment, sucrose gap compartments and outside wells.  b Continuously perfuse central compartment with 2 mL/min 37 C oxygenated Krebs solution using peristaltic pump. 8 Mount a pseudo-Ag/AgCl reference electrode [1] and Pt auxiliary electrode to the sidewalls of the central compartment of the recording chamber, so they sick into the Krebs solution as shown in Fig. 3a. Notes: 9 Information on making a pseudo-Ag/AgCl electrode is included in the supplementary material section. 10 We measured À76 mV as the potential of the pseudo Ag/AgCl electrode vs. the BASi RE-5B Ag/AgCl reference electrode. 11 Polyimide tape was used to secure the pseudo-Ag/AgCl reference and auxiliary electrodes to the recording chamber. 12 Attach glutamate biosensor (working electrode) to the Plexiglass arm above the recording chamber with tape. 13 Using a micromanipulator, lower the Plexiglass arm with the glutamate biosensor attached (Fig. 2), so the glutamate biosensor sticks 1-1.5 mm into the spinal cord segment. Note: Although the 50-mm thick liquid crystal polymer biosensor shank is flexible compared to silicon and ceramic, we were able to insert these shanks 1-1.5 mm into the spinal cord segment. We tested implantation before ex-vivo implantation with 0.6% agarose gel, a model for device insertion into brain tissue [7]. 14 Connect working, reference and counter electrodes to the potentiostat with test hook clips. 15 Apply +0.5 V to the working electrode versus the pseudo-Ag/AgCl reference electrode and record current.
Note: The choice of holding potential depends on the electrochemical sensor used. Applying +0.5 V vs. Ag/AgCl is an adequate holding potential oxidase/Pt-based electrochemical biosensors [1,8,9]. Another holding potential typically used for this class of biosensors is +0.7 V vs. Ag/AgCl. 16 Wait at least 20 min (1200 s) after applying the 0.5 V potential for non-Faradaic current to decrease. 17 At 20 min (1200 s), simulate spinal cord injury by compressing about 70 N with forceps for 5 s at the part of the spinal cord segment immediately in front of where the glutamate biosensor is inserted (Fig. 3b). Notes: 18 70 N corresponds to about 70% of one's maximum pinching force [10]. 19 Compressing the spinal cord for more than 10 s risks breaking it in two. 20 For comparison, at 40 min (2400s) use a micropipette to inject 100 mL 50 mM glutamate at the site of injury.

Protocol validation
Using this method, we measured spikes in glutamate concentration following injury of half segment of rat spinal cord ex vivo. Fig. 4 shows these measurements.