BACKGROUND‐ Salicylate and cochlear damage (by noise, drugs or surgery) are the most studied tinnitus inducers in animal models. However, salicylate‐induced tinnitus is reversible, whereas cochlear damage‐induced tinnitus is not, suggesting some key plastic phenomena differ between the two. Although tinnitus origins are still elusive, hyperactivation of cochlear nuclei (especially DCN) is consistently observed in its early stages, presumably as a result of signals from an unbalanced/damaged periphery; upon chronicization, cochlear nuclei inputs trigger plasticity at higher CNS structures, which eventually become independent from them. Microglia, the resident macrophage of nervous tissue, has been found to contribute to synaptic plasticity in healthy brains, and to modify its localization and activation state in response to inflammation and tissue damage, regulating the excitability of surrounding neural circuits in several pathological states. In chronic pain, microglialdependent maladaptive plastic remodeling of the first central station follows peripheral damage. In cochlear nuclei, cochlear destruction rapidly activates and recruits microglia (1). Salicylate is a tinnitus inducer, but also an anti‐inflammatory drug which counteracts microglia activation. In this work we started testing the possible role of microglia activation in cochlear nuclei as a trigger for permanent circuital changes. METHODS‐ Wistar rats (age: 25 days) either received three salycilate injections (300 mg/kg, i.p, daily), and were sacrificed 2h, 24h and 48h from last treatment (n=18 plus 6 saline‐injected controls) or underwent unilateral surgical cochlear destruction (under etherdiazepam anesthesia) by removing cochlear turns with fine forceps, and were sacrificed 15, 30 and 45 days after surgery. After sacrificing, rat heads were placed in carbogen‐bubbled Krebs' solution, brains were extracted and placed in 4% PFA. Care was taken not to exceed 6 min between sacrifice and fixation, because times longer than 10 min have been reported to induce tissue damage and microglial activation. After fixation and paraffin inclusion, brains were sliced at 3‐10 micron and immunohistochemistry for Iba‐1 was performed using DAB staining. The number of microglial cell processes and cochlear nuclei areas were measured using imageJ, by experimenters not aware of the nature (treated or control) of the samples observed. RESULTS AND CONCLUSIONS‐ In control conditions, microglial density in cochlear nuclei was 430±262 processes/mm2. After salicylate treatment,density was not significantly different from control (483±236 processes/mm2); no differences were noted between 2h, 24h and 48h samples. Microglial morphology also appeared similar in control and salicylate samples. Aftercochlear destruction, on the other hand, microglia displayed clear morphological activation signs (thicker processes, larger soma) in the ipsilateral cochlear nuclei, whereas no activated microglia was observed in contralateral nuclei or control animals. We suggest that the anti‐inflammatory action of salicylate, by blocking microglial activation, counteracts plastic remodeling of CN neurons, thus keeping tinnitus dependent on peripheral input. 1. Fuentes‐Santamaria V, Alvarado JC, Juiz JM J Comp Neurol 2012;520:2974‐90.

MICROGLIA IN THE RAT COCHLEAR NUCLEI: A PLAYER IN TINNITUS-RELATED CIRCUIT REORGANIZATION?

BOSELLI, CINZIA;PIZZALA, ROBERTO;PERIN, PAOLA
2013-01-01

Abstract

BACKGROUND‐ Salicylate and cochlear damage (by noise, drugs or surgery) are the most studied tinnitus inducers in animal models. However, salicylate‐induced tinnitus is reversible, whereas cochlear damage‐induced tinnitus is not, suggesting some key plastic phenomena differ between the two. Although tinnitus origins are still elusive, hyperactivation of cochlear nuclei (especially DCN) is consistently observed in its early stages, presumably as a result of signals from an unbalanced/damaged periphery; upon chronicization, cochlear nuclei inputs trigger plasticity at higher CNS structures, which eventually become independent from them. Microglia, the resident macrophage of nervous tissue, has been found to contribute to synaptic plasticity in healthy brains, and to modify its localization and activation state in response to inflammation and tissue damage, regulating the excitability of surrounding neural circuits in several pathological states. In chronic pain, microglialdependent maladaptive plastic remodeling of the first central station follows peripheral damage. In cochlear nuclei, cochlear destruction rapidly activates and recruits microglia (1). Salicylate is a tinnitus inducer, but also an anti‐inflammatory drug which counteracts microglia activation. In this work we started testing the possible role of microglia activation in cochlear nuclei as a trigger for permanent circuital changes. METHODS‐ Wistar rats (age: 25 days) either received three salycilate injections (300 mg/kg, i.p, daily), and were sacrificed 2h, 24h and 48h from last treatment (n=18 plus 6 saline‐injected controls) or underwent unilateral surgical cochlear destruction (under etherdiazepam anesthesia) by removing cochlear turns with fine forceps, and were sacrificed 15, 30 and 45 days after surgery. After sacrificing, rat heads were placed in carbogen‐bubbled Krebs' solution, brains were extracted and placed in 4% PFA. Care was taken not to exceed 6 min between sacrifice and fixation, because times longer than 10 min have been reported to induce tissue damage and microglial activation. After fixation and paraffin inclusion, brains were sliced at 3‐10 micron and immunohistochemistry for Iba‐1 was performed using DAB staining. The number of microglial cell processes and cochlear nuclei areas were measured using imageJ, by experimenters not aware of the nature (treated or control) of the samples observed. RESULTS AND CONCLUSIONS‐ In control conditions, microglial density in cochlear nuclei was 430±262 processes/mm2. After salicylate treatment,density was not significantly different from control (483±236 processes/mm2); no differences were noted between 2h, 24h and 48h samples. Microglial morphology also appeared similar in control and salicylate samples. Aftercochlear destruction, on the other hand, microglia displayed clear morphological activation signs (thicker processes, larger soma) in the ipsilateral cochlear nuclei, whereas no activated microglia was observed in contralateral nuclei or control animals. We suggest that the anti‐inflammatory action of salicylate, by blocking microglial activation, counteracts plastic remodeling of CN neurons, thus keeping tinnitus dependent on peripheral input. 1. Fuentes‐Santamaria V, Alvarado JC, Juiz JM J Comp Neurol 2012;520:2974‐90.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11571/694619
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