Electrophysiological and molecular changes following neuroprotective placental protein administration on tinnitus-induced rats

Abstract

Objective: Despite 6%-20% of the adult population suffering from tinnitus, there is no standard treatment for it. Placenta extract has been used for various therapeutic purposes, including hearing loss. Here, we evaluate the effect of a novel neuroprotective protein composition (NPPC) extract on electrophysiological and molecular changes in the medial geniculate body (MGB) of tinnitus-induced rats.

Methods: To evaluate the protein analysis by western blot, the rats were divided into three groups: (1) saline group (intraperitoneal injection of 200 mg/kg saline twice a day for 28 consecutive days, (2) chronic Na-Sal group received sodium salicylate as in the first group, and (3) chronic treatment group (received salicylate 200 mg/kg twice daily for 2 weeks, followed by 0.4 mg NPPC daily from day 14 to day 28). Single-unit recordings were performed on a separate group that was treated as in group 4. Gap-prepulse inhibition of the acoustic startle (GPIAS) and pre-pulse inhibition (PPI) was performed to confirm tinnitus in all groups at the baseline, 14th and 28th days.

Results: Western blot analysis showed that the expression of γ-Aminobutyric acid Aα1 subunit (GABA Aα1), N-methyl-d-aspartate receptor subtype 2B (NR2B or NMDAR2B), α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors subunit GluR1 (GluR1), and α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors subunit GluR2 (GluR2) decreased after Na-Sal injection, while NPPC upregulated their expression. MGB units in rats with tinnitus showed decreased spontaneous firing rate, burst per minute, and a spike in a burst. After NPPC administration, neural activity patterns showed a significant positive effect of NPPC on tinnitus.

Conclusion: NPPC can play an effective role in the treatment of tinnitus in salicylate-induced rats, and MGB is one of the brain areas involved in these processes.

Level of evidence: NA.

Keywords: placenta extract; single unit recording; sodium salicylate; startle reflex; tinnitus.

FIGURE 1

Schematic diagram of the study's method.

FIGURE 2

Schematic picture of the GPIAS and PPI paradigms is shown to illustrate the stimulus conditions and presumption results for the three conditions no‐gap, gap, and tinnitus.

FIGURE 3

Characteristic frequencies (CFs) and MGB unit thresholds at baseline, 14th and 28th day: scatter plot of individual MGB unit thresholds at their CFs from the base (green) and 14th (red) and 28th days (blue) rats. The average threshold at CFs of MGB unit recording in three situations (Base, 14th and 28th days) did not exhibit any significant difference (p > .05).

FIGURE 4

An example of a rat in the stereotaxic system with electrodes and a microphone. The arrows indicate the electrode and insertion phone place.

FIGURE 5

PPI and GPIAS test at baseline and after Na‐Sal and NPPC administration. (A) Chronic effects of Na‐Sal and NPPC on pre‐pulse inhibition (PPI) values as a criterion for the hearing evaluation. The animal in the Na‐Sal group received only Na‐Sal (200 mg/kg) in all over 28 days. While the rats in the treatment group received Na‐Sal in the first 14 days, and Na‐Sal (200 mg/kg) and also NPPC (0.4 mg/kg) in the second 14 days. There were no significant differences in the PPI values neither among the three groups (Saline, Na‐Sal and treatment) nor intra groups (baseline, 14 days and 28 days) (n = 7, p < .05). (B) Chronic effects of Na‐Sal and NPPC on Gap pre pulse inhibition of acoustic startle (GPIAS) values as a criterion for tinnitus confirmation. Saline injection had no effect on GPIAS performance (n = 7, p < .05). In contrast, injection of Na‐Sal at a dose of 200 mg/kg caused a significant reduction in GPIAS performance on both the 14th day and the 28th day in the Na‐Sal group, compared with the baseline and the two comparison groups (n = 7, p < .05). The same matter happened in the treatment group on the 14th day. While the injection of Na‐Sal (200 mg/kg) with NPPC at a dose of 0.4 mg/kg caused a significant increase in GPIAS performance on the 28th day (n = 7, p < .05). Data are the mean ± SEM (two‐way repeated measures analysis of variance‐post hoc Tukey's test).

FIGURE 6

Western blots' analysis of inhibitory and excitatory subunits in Medial Geniculate Body (MGB) tissue. (A) Western blots' analysis of GABAARα1 subunit in MGB tissue immunoreactivity against the GABAARα1 subunit in Medial Geniculate Body (MGB) tissue from the saline group (Control), Na‐Sal group (Tinnitus) and the treatment group; the plot showing immunoreactivity against the GABAARα1 subunit in the MGB. It can be observed that the level of the GABAARa1 in MGB tissue in the Na‐Sal group significantly decreased, while it increased in the treatment group and the control group. (B) Immunoreactivity against the NR2B subunit in MGB tissue from the saline group (Control), Na‐Sal group (Tinnitus) and the treatment group; the plot showing immunoreactivity against the NR2B subunit in the MGB. It can be observed that the level of the NR2B subunit in MGB tissue decreased in the Na‐Sal group, while it increased in the treatment group, compared with the control group. (C) Immunoreactivity against the GluR2 subunit in MGB tissue from the saline group (Control), Na‐Sal group (Tinnitus) and the treatment group; the plot showing immunoreactivity against the GluR1 subunit in the MGB. It can be observed that the level of the GluR2 subunit in MGB tissue decreased in the NA‐Sal group, while it increased in the treatment group, compared with the control group. (D) Immunoreactivity against the GluR1 subunit in MGB tissue from the saline group (Control), Na‐Sal group (Tinnitus) and the treatment group; the plot showing immunoreactivity against the GluR1 subunit in the MGB. It can be observed that the level of the GluR1 subunit in MGB tissue decreased in the Na‐Sal group, while it increased in the treatment group, compared with the control group. Data are the mean ± SEM (one‐way repeated measures analysis of variance‐post hoc Tukey's test).

FIGURE 7

Chronic effects of Na‐Sal and NPPC on MGB unit recordings. (A) photomicrograph shows histological verification of recording site (MGB) and the schematic equivalent is taken from the atlas of Paxinos, 6th Edn. Amsterdam, published online, 2007. (B, E, F) The analysis of MGB Units recordings revealed that there was a significant decrease in the mean number of Burst firing‐per‐minute, Intra‐Burst‐Frequency and Firing Rate on the 14th day condition compared to the baseline and 28th day, whereas there was no significant difference between the baseline and 28th day (one‐way analysis of variance). (C) But there was no significant difference in the mean number of the spike in a burst between every three conditions (one‐way analysis of variance). (D) In addition, there was a significant increase in the mean of burst duration in seconds on the 14th‐day condition compared to baseline and the 28th day, whereas there was no significant difference between baseline and the 28th day (one‐way analysis of variance). The star indicates the significant difference value between the baseline and the 14th day, and the triangle indicates the significant difference value between the 14th day and the 28th day (p < .05).