cAMP-Epac1 signaling is activated in DDAVP-induced endolymphatic hydrops of guinea pigs

Abstract Objective To explore whether Cyclic Adenosine Monophosphate (cAMP)-Epac1 signaling is activated in 1-Desamino-8-D-arginine-Vasopressin-induced Endolymphatic Hydrops (DDAVP-induced EH) and to provide new insight for further in-depth study of DDAVP-induced EH. Methods Eighteen healthy, red-eyed guinea pigs (36 ears) weighing 200-350 g were randomly divided into three groups: the control group, which received intraperitoneal injection of sterile saline (same volume as that in the other two groups) for 7 consecutive days; the DDAVP-7d group, which received intraperitoneal injection of 10 mg/mL/kg DDAVP for 7 consecutive days; and the DDAVP-14d group, which received intraperitoneal injection of 10 μg/mL/kg DDAVP for 14 consecutive days. After successful modeling, all animals were sacrificed, and cochlea tissues were collected to detect the mRNA and protein expression of the exchange protein directly activated by cAMP-1 and 2 (Epac1, Epac2), and Repressor Activator Protein-1 (Rap1) by Reverse Transcription (RT)-PCR and western blotting, respectively. Results Compared to the control group, the relative mRNA expression of Epac1, Epac2, Rap1A, and Rap1B in the cochlea tissue of the DDAVP-7d group was significantly higher (p< 0.05), while no significant difference in Rap1 GTPase activating protein (Rap1gap) mRNA expression was found between the two groups. The relative mRNA expression of Epac1, Rap1A, Rap1B, and Rap1gap in the cochlea tissue of the DDAVP-14d group was significantly higher than that of the control group (p< 0.05), while no significant difference in Epac2 mRNA expression was found between the DDAVP-14d and control groups. Comparison between the DDAVP-14d and DDAVP-7d groups showed that the DDAVP-14d group had significantly lower Epac2 and Rap1A (p< 0.05) and higher Rap1gap (p < 0.05) mRNA expression in the cochlea tissue than that of the DDAVP-7d group, while no significant differences in Epac1 and Rap1B mRNA expression were found between the two groups. Western blotting showed that Epac1 protein expression in the cochlea tissue was the highest in the DDAVP-14d group, followed by that in the DDAVP-7d group, and was the lowest in the control group, showing significant differences between groups (p< 0.05); Rap1 protein expression in the cochlea tissue was the highest in the DDAVP-7d group, followed by the DDAVP-14d group, and was the lowest in the control group, showing significant differences between groups (p< 0.05); no significant differences in Epac2 protein expression in the cochlea tissue were found among the three groups. Conclusion DDAVP upregulated Epac1 protein expression in the guinea pig cochlea, leading to activation of the inner ear cAMP-Epac1 signaling pathway. This may be an important mechanism by which DDAVP regulates endolymphatic metabolism to induce EH and affect inner ear function. Oxford Centre for Evidence-Based Medicine 2011 Levels of Evidence Level 5.

cAMP-Epac1 signaling is activated in DDAVP-induced endolymphatic hydrops of guinea pigs.

OBJECTIVE: To explore whether Cyclic Adenosine Monophosphate (cAMP)-Epac1 signaling is activated in 1-Desamino-8-D-arginine-Vasopressin-induced Endolymphatic Hydrops (DDAVP-induced EH) and to provide new insight for further in-depth study of DDAVP-induced EH. METHODS: Eighteen healthy, red-eyed guinea pigs (36 ears) weighing 200-350 g were randomly divided into three groups: the control group, which received intraperitoneal injection of sterile saline (same volume as that in the other two groups) for 7 consecutive days; the DDAVP-7d group, which received intraperitoneal injection of 10 mg/mL/kg DDAVP for 7 consecutive days; and the DDAVP-14d group, which received intraperitoneal injection of 10 µg/mL/kg DDAVP for 14 consecutive days. After successful modeling, all animals were sacrificed, and cochlea tissues were collected to detect the mRNA and protein expression of the exchange protein directly activated by cAMP-1 and 2 (Epac1, Epac2), and Repressor Activator Protein-1 (Rap1) by Reverse Transcription (RT)-PCR and western blotting, respectively. RESULTS: Compared to the control group, the relative mRNA expression of Epac1, Epac2, Rap1A, and Rap1B in the cochlea tissue of the DDAVP-7d group was significantly higher (p <  0.05), while no significant difference in Rap1 GTPase activating protein (Rap1gap) mRNA expression was found between the two groups. The relative mRNA expression of Epac1, Rap1A, Rap1B, and Rap1gap in the cochlea tissue of the DDAVP-14d group was significantly higher than that of the control group (p <  0.05), while no significant difference in Epac2 mRNA expression was found between the DDAVP-14d and control groups. Comparison between the DDAVP-14d and DDAVP-7d groups showed that the DDAVP-14d group had significantly lower Epac2 and Rap1A (p <  0.05) and higher Rap1gap (p < 0.05) mRNA expression in the cochlea tissue than that of the DDAVP-7d group, while no significant differences in Epac1 and Rap1B mRNA expression were found between the two groups. Western blotting showed that Epac1 protein expression in the cochlea tissue was the highest in the DDAVP-14d group, followed by that in the DDAVP-7d group, and was the lowest in the control group, showing significant differences between groups (p <  0.05); Rap1 protein expression in the cochlea tissue was the highest in the DDAVP-7d group, followed by the DDAVP-14d group, and was the lowest in the control group, showing significant differences between groups (p <  0.05); no significant differences in Epac2 protein expression in the cochlea tissue were found among the three groups. CONCLUSION: DDAVP upregulated Epac1 protein expression in the guinea pig cochlea, leading to activation of the inner ear cAMP-Epac1 signaling pathway. This may be an important mechanism by which DDAVP regulates endolymphatic metabolism to induce EH and affect inner ear function. OXFORD CENTRE FOR EVIDENCE-BASED MEDICINE 2011 LEVELS OF EVIDENCE: Level 5.

Vestibular-evoked myogenic potentials recorded from miniature pigs and rats.

OBJECTIVE: To report vestibular evoked myogenic potentials from different recording sites (neck extensor or masseter muscles) in miniature pigs and rats. METHODS: Potentials were recorded using 1000 Hz tone bursts from the neck extensor muscle or masseter muscle in normal adult Bama miniature pigs and rats anesthetized with 3% pentobarbital sodium and Sumianxin II. RESULTS: At 80 dB SPL, the first positive wave (P wave) of VEMPs was recognizable in 58% of rats with a latency of 6.45 ± 0.23 ms and an amplitude of 1.45 ± 0.49 µV when recorded from the neck extensor muscle, and in 50% of rats with a latency of 6.38 ± 0.34 ms and an amplitude of 1.57 ± 0.35 µV when recorded from the masseter muscle. In miniature pigs, at the same stimulus intensity, P wave was recognizable in 58% of the animals with a latency of 7.65 ± 0.64 ms and an amplitude of 1.66 ± 0.34 µV when recorded from the neck extensor muscle, and in 50% of the animals with a latency of 7.65 ± 0.64 ms and an amplitude of 0.31 ± 0.28 µV when recorded from the masseter muscle. CONCLUSION: VEMP can be induced from both neck extensor and masseter muscles in the miniature pig and rat. For a given species, the site of recording affects P wave induction rate and amplitude but not latency. Consistency and repeatability analysis suggests that the masseter muscle is a better recording site in miniature pigs while the cervical extensor is a better recording site in rats. For a given recording site, both latency and amplitude of the P wave are slightly greater in miniature pigs than in rats.

Inflammatory lesions of cochlea in murine cytomegalovirus-infected mice with hearing loss.

We sought to determine the distribution of tumor necrosis factor (TNF)-α, interleukin (IL)-6 and lymphocytes in the cochlea of mice infected with murine cytomegalovirus (MCMV). For this purpose, 16 newborn mice were divided equally into model and control (uninfected) groups. In model group, 10 µl of MCMV was injected into the brain of each mouse whereas in control group, 10 µl of physiological saline was injected. Fourteen days after the injection of MCMV, the auditory brainstem response audiometry was performed. Later, the mice were killed and the acoustic capsule samples were collected for polymerase chain reaction analysis, histopathological and immunohistochemical studies. Compared with control mice, the incubation period was longer (F = 13.797; P = 0.003) and the amplitude was lower (F = 5.095; P = 0.043) in model group mice. It indicated that the intracerebral injection of MCMV caused hearing loss in mice. Histopathological examination of cochleas revealed increased levels of lymphocytic infiltration in the membrana vestibularis. Higher levels of TNF-α and IL-6 in scala tympani were detected by immunohistochemical staining. Taken together, the hearing loss in mice could be related with the inflammatory changes occurring in cochlea after inception of MCMV infection.