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. 2018 Dec 5;8(1):17656.
doi: 10.1038/s41598-018-36330-2.

The Potential of Substance P to Initiate and Perpetuate Cortical Spreading Depression (CSD) in Rat in Vivo

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Free PMC article

The Potential of Substance P to Initiate and Perpetuate Cortical Spreading Depression (CSD) in Rat in Vivo

Frank Richter et al. Sci Rep. .
Free PMC article

Abstract

The tachykinin substance P (SP) increases neuronal excitability, participates in homeostatic control, but induces brain oedema after stroke or trauma. We asked whether SP is able to induce cortical spreading depression (CSD) which often aggravates stroke-induced pathology. In anesthetized rats we applied SP (10-5, 10-6, 10-7, or 10-8 mol/L) to a restricted cortical area and recorded CSDs there and in remote non-treated areas using microelectrodes. SP was either applied in artificial cerebrospinal fluid (ACSF), or in aqua to perform a preconditioning. Plasma extravasation in cortical grey matter was assessed with Evans Blue. Only SP dissolved in aqua induced self-regenerating CSDs. SP dissolved in ACSF did not ignite CSDs even when excitability was increased by acetate-preconditioning. Aqua alone elicited as few CSDs as the lowest concentration of SP. Local pretreatment with 250 nmol/L of a neurokinin 1 receptor antagonist prevented the SP-induced plasma extravasation, the initiation of CSDs by 10-5 mol/L SP diluted in aqua, and the initiation of CSDs by aqua alone, but did not suppress KCl-induced CSD. Thus neurokinin 1 receptor antagonists may be used to explore the involvement of SP in CSDs in clinical studies.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Experimental design and typical samples of SP-induced CSDs. Schematic top view (a) on the skull of the rat with the position of the recording electrodes. CSDs after either application of SP at 10−5 mol/L (b) or 10−8 mol/L (c) dissolved in aqua. The separated traces in the left sides of each panel show the control CSDs elicited by KCl microinjection. Note the propagation from electrode 1 to 4 when KCl is injected versus propagation from electrode 4 to 1 when SP is applied. The propagation direction of the CSDs is accentuated by oblique thin dotted lines.
Figure 2
Figure 2
Occurrence of CSD events after topical application of either SP dissolved in aqua at different concentrations or aqua alone to the treatment area. The left panel shows the CSDs in the treated area, the right panel shows the respective CSDs in the untreated area. Each row represents the data from one animal, each bar represents the peak time of one single CSD wave. Thin bars show classical CSDs, thick bars sustained CSD events. Note that no sustained CSDs occurred in the untreated area.
Figure 3
Figure 3
Original intracortical DC-ECoG recordings and filtering of the DC-ECoG recordings to obtain the AC-ECoG during KCl- and SP-evoked CSDs. Single KCl-induced CSDs (application marked by arrows) started at DC electrode 1 (accentuated by blue dotted lines) and moved to electrodes 3/4. Self-regenerating CSDs ignited by SP at 10−5 mol/L started at DC electrode 3 or 4 (accentuated by red dotted lines) and moved to electrodes 1 and 2. Analysis revealed that in both cases real “spreading depressions of EEG activity” occurred: A Recordings of spreading depression-related negative shifts in the DC recordings, negative shifts are facing upwards; B high-pass filtered ECoG data with a lower frequency limit of 0.5 Hz of the respective recordings, C power of bandpass filtered recordings (0.5–45 Hz), and D integral of power of bandpass filtered recordings C (0.5–45 Hz) respectively, which allow the visualization of the spreading depressions in the ECoG recordings. Note that the application of the hypoosmolar solution (indicated by the beginning of the red bar, the thin dotted line and the small positive shifts in traces A of electrodes 3 and 4) had no effect on the AC-ECoG pattern.
Figure 4
Figure 4
Traces of a complete experiment with simultaneous recording of the regional cerebral blood flow (rCBF) in the SP treated brain area. Far left the control period is shown in which the cortex was superfused with ACSF and CSDs were elicited by KCl. Then SP at 10−5 mol/L dissolved in aqua was applied and evoked a series of three CSD waves at the DC electrodes 4, 3, and 1, only one typical wave at electrode 2, but four events in the rCBF recording thus further underlining that not all CSD events were picked up at all recording sites.
Figure 5
Figure 5
Sustained negative shift observed at DC electrode 4 after 10−6 mol/L of SP dissolved in aqua. The positive shift of the EEG baseline was initiated when SP was applied. Note the sequence of five negative DC deflections at electrode 4 starting about 8 min after application of SP that did not completely return to baseline whereas in the untreated brain area six CSD waves can be clearly distinguished. The last CSD far right is elicited by KCl.
Figure 6
Figure 6
Pretreatment with the NK-1RA L-703,606 prevented the occurrence of CSDs ignited by SP or by aqua alone. (a) The traces show from left to right the control CSD by KCl in the native cortex, as well as a CSD by KCl after one hour pretreatment with the NK-1RA and the two hour observation phase when 10−5 mol/L of SP dissolved in aqua was applied to the treated area. The CSD far right is elicited by KCl. (b) The traces show from left to right the control CSD by KCl in the native cortex, the beginning and the end of one hour of blocking the NK-1R and the complete phase when aqua was applied. No CSDs occurred in the time range when the NK-1R was blocked. The CSD far right is elicited by KCl.
Figure 7
Figure 7
Laser scanning photomicrographs of cortical slices after injection with Evans Blue displaying plasma extravasation. (a) Plasma extravasation in the superficial layers of the cerebral cortex after 2 hours of topical application of 10−5 mol/L SP dissolved in aqua that induced self-regenerating CSD waves. (b) No plasma extravasation in the remote (untreated) cortical areas of the same animal. (c) No plasma extravasation occurred when the NK-1R were blocked in a 1 hour pretreatment before application of 10−5 mol/L SP dissolved in aqua. (d) The application of aqua bidest. alone did not induce any plasma extravasation.

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