Dexmedetomidine Stereotaxic Injection Alleviates Neuronal Loss Following Bilateral Common Carotid Artery Occlusion via Up-Regulation of BDNF Expression.

BACKGROUND/AIM: Neurogenesis is an important process in the recovery from neurological damage caused by ischemic lesions. Endogenous neurogenesis is insufficient to restore neuronal damage following cerebral ischemia. Dexmedetomidine (DEX) exerts neuroprotective effects against cerebral ischemia and ischemia/reperfusion injury. DEX promotes neurogenesis, including neuronal proliferation and maturation in the hippocampus. In a previous study, we showed that early neurogenesis increased 3 days after bilateral common carotid artery occlusion (BCCAO). In this study, we investigated the effect of DEX on neurogenesis 3 days after BCCAO. MATERIALS AND METHODS: Male Sprague-Dawley (SD) rats (7-8 weeks old) were used as a BCCAO model. Right and left common carotid arteries of the rats were occluded using 4-0 silk sutures. Two hours after surgery, an intracranial DEX injection was administered to rats that underwent surgery using a stereotaxic injector. Brains were obtained from control and BCCAO rats 3 days after surgery. Immunohistochemistry was performed on the cortex and dentate gyrus of the hippocampus using a NeuN antibody. Western blot was performed with HIF1α and brain-derived neurotrophic factor (BDNF) antibodies. RESULTS: The number of mature neurons decreased 3 days after BCCAO, but DEX treatment alleviated neural loss in the parietal cortex and hippocampus. Up-regulation of BDNF was also observed after dexmedetomidine treatment. CONCLUSION: Stereotaxic injection of dexmedetomidine alleviates neural loss following BCCAO by up-regulating BDNF expression.

IGF-1 Protects Neurons in the Cortex and Subventricular Zone in a Periventricular Leucomalacia Model.

BACKGROUND/AIM: Chronic cerebral hypoperfusion affects early and mature neurons in the subventricular zone (SVZ) and cerebral cortex. Herein, we investigated the effects of insulin-like growth factor-1 (IGF-1), a neurogenesis-promoting agent, on neurons in these regions in periventricular leucomalacia (PVL) model rats. MATERIALS AND METHODS: Following right carotid artery ligation, the rats were placed in a hypoxia chamber and injected with recombinant IGF-1 (0.1 and 1 µg/µl). Their brain sections were immunohistochemically analysed using anti-nestin and anti-NeuN antibodies. RESULTS: The numbers of early-neuronal cells in the SVZ and mature neurons in the cerebral cortex were higher and lower, respectively, in the PVL group than in the control group. The number of NeuN-positive cells was significantly higher in the IGF-treated group than in the PVL group. CONCLUSION: PVL increased the number of early neuronal cells in the SVZ, reducing the survival of mature neurons in the cerebral cortex; IGF-1 reversed these effects.

Age-Related Hearing Loss in C57BL/6J Mice Is Associated with Mitophagy Impairment in the Central Auditory System.

Aging is associated with functional and morphological changes in the sensory organs, including the auditory system. Mitophagy, a process that regulates the turnover of dysfunctional mitochondria, is impaired with aging. This study aimed to investigate the effect of aging on mitophagy in the central auditory system using an age-related hearing loss mouse model. C57BL/6J mice were divided into the following four groups based on age: 1-, 6-, 12-, and 18-month groups. The hearing ability was evaluated by measuring the auditory brainstem response (ABR) thresholds. The mitochondrial DNA damage level and the expression of mitophagy-related genes, and proteins were investigated by real-time polymerase chain reaction and Western blot analyses. The colocalization of mitophagosomes and lysosomes in the mouse auditory cortex and inferior colliculus was analyzed by immunofluorescence analysis. The expression of genes involved in mitophagy, such as PINK1, Parkin, and BNIP3 in the mouse auditory cortex and inferior colliculus, was investigated by immunohistochemical staining. The ABR threshold increased with aging. In addition to the mitochondrial DNA integrity, the mRNA levels of PINK1, Parkin, NIX, and BNIP3, as well as the protein levels of PINK1, Parkin, BNIP3, COX4, LC3B, mitochondrial oxidative phosphorylation (OXPHOS) subunits I-IV in the mouse auditory cortex significantly decreased with aging. The immunofluorescence analysis revealed that the colocalization of mitophagosomes and lysosomes in the mouse auditory cortex and inferior colliculus decreased with aging. The immunohistochemical analysis revealed that the expression of PINK1, Parkin, and BNIP3 decreased in the mouse auditory cortex and inferior colliculus with aging. These findings indicate that aging-associated impaired mitophagy may contribute to the cellular changes observed in an aged central auditory system, which result in age-related hearing loss. Thus, the induction of mitophagy can be a potential therapeutic strategy for age-related hearing loss.

Reduced mitophagy in the cochlea of aged C57BL/6J mice.

An increase in mitochondrial damage has been associated with a decline in the ability to mitigate damage through mitophagy in age-related pathologies. The present study aimed to investigate the changes of mitophagy in a mouse model with age-related hearing loss. C57BL/6J mice were divided into two groups: young (1 month) and aged (12 months). Hearing tests were conducted through the measurement of auditory brainstem response (ABR). Mitochondrial DNA copy number, the level of mitochondrial DNA damage, mitochondrial biogenesis, and mitophagy-related genes and proteins were investigated using real-time PCR and western blot analysis. Coexpression of mitophagosomes and lysosomes in the cochlea was investigated through immunofluorescence imaging analysis. Major players of mitophagy, Parkin and BNIP3, were also investigated through immunohistochemical staining in the cochlea. Hearing thresholds were observed to have increased in the aged group. The mitochondrial DNA copy number, PGC-1α, and PGC-1ß significantly decreased in the cochlea of mice in the aged group. The mRNA levels of PINK1, Parkin, MUL1, Atg5, Atg12, Atg13, NIX, and BNIP3 significantly decreased in the cochlea of the mice in the aged group. The level of mitochondrial DNA damage significantly increased in the cochlea of mice in the aged group. Protein levels of PINK1, Parkin, BNIP3, COX4, LC3B, and all OXPHOS subunits significantly decreased in the cochlea of the mice in the aged group. Immunofluorescence imaging analysis of mitophagosomes and lysosomes revealed a decrease in the colocalization in the cochlea of mice in the aged group. Immunohistochemical imaging analysis of Parkin and BNIP3 revealed their decreased expression in aged cochlea. Our results indicate that reduced mitophagy with aging might be attributed to the cellular changes that occur in aged cochlea in the development of age-related hearing loss.

In vitro inhibitory activity of N-acetylcysteine on tympanostomy tube biofilms from methicillin-resistant Staphylococcus aureus and quinolone-resistant Pseudomonas aeruginosa.

OBJECTIVES: Biofilm formation in tympanostomy tubes causes persistent and refractory otorrhea. In the present study, we investigated the in vitro antibiofilm activity of N-acetylcysteine (NAC) against biofilm formation by methicillin-resistant Staphylococcus aureus (MRSA) and quinolone-resistant Pseudomonas aeruginosa (QRPA). METHODS: We examined the antibiofilm activity of NAC against biofilms produced by MRSA and QRPA strains using in vitro biofilm formation assay, adhesion assay, and biofilm eradication assay. Additionally, the antibiofilm activity of different concentrations of NAC against tympanostomy-tube biofilms from MRSA and QRPA strains was compared using a scanning electron microscope. RESULTS: The adhesion of MRSA and QRPA strains decreased significantly in a concentration-dependent manner after treatment with varying amounts of NAC. Treatment with NAC inhibited biofilm formation of both MRSA and QRPA strains and increased eradication of preformed mature biofilm produced by MRSA and QRPA. Besides, NAC exhibited significant eradication-activity against tympanostomy-tube biofilms produced by MRSA and QRPA strains. CONCLUSIONS: Our results show potent inhibition of MRSA and QRPA biofilm after treatment with NAC. NAC shows potential for the treatment of biofilms and refractory post-tympanostomy tube otorrhea resulting from MRSA and QRPA infection.

Hypoxia of Rats Subjected to Carotid Artery Ligation Results in Impaired Neurogenesis and Reduced Number of Cortical Neurons.

BACKGROUND/AIM: Cerebral ischemia is a major cause of abnormal brain development. In a cerebral ischemia model, periventricular leukomalacia (PVL), white matter lesion and a decrease in the number of subcortical neurons were observed. The aim of this study was to investigate the effect of hypoxia on neurogenesis and cell survival. MATERIALS AND METHODS: In seven-day postnatal rats, the right carotid artery was ligated. The rats were incubated either in a regular normoxic chamber (control group) or in a hypoxic chamber (PVL group, 8% 02 and 92% N2 at 37°C) for 2 h. Nestin- and NeuN-positive neurons were detected by immunohistochemistry. RESULTS: The densities of nestin-immunoreactivity (IR) cells in the cerebral parietal cortex and subventricular zone were increased with hypoxia. NeuN-IR cells in the cerebral cortex were significantly decreased in the PVL group. CONCLUSION: Perinatal white matter injury induced neurogenesis, while the survival of neurons was decreased in the cerebral cortex.

The protective role of ferulic acid against cisplatin-induced ototoxicity.

OBJECTIVES: While cisplatin is an effective chemotherapeutic agent, it can cause irreversible hearing loss. Ototoxicity leads to dose reduction during the cisplatin chemotherapy and results in inadequate treatment of malignant tumors. This study aimed to investigate the protective effects of ferulic acid on cisplatin-induced ototoxicity. METHODS: House Ear Institute-Organ of Corti 1 (HEI-OC1) cells were exposed to 30 µM of cisplatin for 24 h with or without pretreatment with ferulic acid. Cell viability was determined using the WST assay. Apoptotic cells were identified using TUNEL assay. Western blot analysis was performed to examine the change in expression of cleaved caspase, cleaved poly-ADP-ribose polymerase (PARP), nuclear factor erythroid 2-related factor 2 (Nrf2), and catalase. Intracellular reactive oxygen species (ROS) were determined by flow cytometry. Real-time PCR analyses were performed to examine the mRNA levels of antioxidant enzymes including glutamate-cysteine ligase catalytic subunit (Gclc), glutathione peroxidase 2 (Gpx2), catalase, and superoxide dismutase 2 (SOD2). Phalloidin staining of the organ of Corti was performed to determine hair cell survival or degeneration. RESULTS: Pretreatment with ferulic acid before cisplatin exposure significantly increased cell viability, levels of antioxidant enzymes, and hair cell survival. In addition, pretreatment with ferulic acid significantly reduced apoptotic cells, levels of cleaved caspase, levels of cleaved PARP, and intracellular ROS production. CONCLUSION: Our results demonstrated that ferulic acid inhibited cisplatin-induced cytotoxicity by preventing ROS formation and inducing the production of endogenous antioxidants and indicated that ferulic acid might be used as a protective agent against cisplatin-induced ototoxicity.

The effect of diet control on puberty onset and immunoreactivity of kisspeptin and neurokinin B in female rats.

Early onset puberty and irregular estrous cycles occur more frequently in rats which are fed a high-fat diet. Kisspeptin is an essential factor for the regulation of sexual maturation and is co-expressed with neurokinin B in neurons in the hypothalamic arcuate nucleus. However, the effects of a diet change on kisspeptin neuronal signaling are not well-understood. Therefore, in this study, we examined the immunoreactivity pattern of the kisspeptin/kiss1-receptor (KISS1R) and neurokinin B/neurokinin3-receptor (R). Pups born to high-fat diet rats were exposed to a high-fat diet until the onset of puberty. From puberty, the offspring originally exposed to a high-fat diet were fed a normal diet up to 85 postnatal days (PND 85). We examined kisspeptin/Kiss1-receptor and neurokinin B/neurokinin3-receptor immunoreactivity (IR) in the arcuate nucleus of the pups. The onset of puberty in the high-fat group was significantly earlier than the control group. At the onset of puberty, the densities of kisspeptin and neurokinin B IR cells were significantly higher in the high-fat diet group than in the control group; however, the densities of KISS1 and neurokinin 3-receptor IR cells did not differ between the two groups. At PND 85, the density of kisspeptin and neurokinin B IR cells did not differ between control and high fat group. The density of densities of KISS1 and neurokinin 3-receptor IR cells also did not differ between groups at this stage. These data suggest that a high-fat diet can influence puberty onset and the immunoreactivity of kisspeptin and neurokinin B. These effects can be modified by dietary control.

Regional Immunoreactivity of Pax6 in the Neurogenic Zone After Chronic Prenatal Hypoxia.

BACKGROUND/AIM: Neurogenesis is a complex process to generate new neurons from neural progenitor cells. Neural progenitor cells are observed in two principal neurogenic regions of the forebrain, the subventricular zone and the subgranular zone of the hippocampal dentate gyrus. The cerebral cortex also plays a role as the neurogenic zone under hypoxic conditions. Hypoxia has many effects on neurogenesis, but the effect of chronic prenatal hypoxia on paired box 6 (Pax6), a protein that plays an important role in neurogenesis, has not been studied in vivo. In the present study, we used a rat model to evaluate the effect of hypoxia on Pax6 immunoreactivity. MATERIALS AND METHODS: Hypoxia status was induced by unilateral uterine-artery ligation in pregnant rats. The fetuses were obtained from the uterine horn on the twenty-first day of pregnancy and immunohistochemistry of the fetal brain was examined regarding anti-hypoxia-induced factor 1α and Pax6 antibody. RESULTS: The density of HIF1α-IR cells in the hypoxia group was greater than the density of HIF1α-IR cells in the control group in the subventricular zone, subgranular zone, and cerebral cortex. The density of Pax6-IR cells in the hypoxic group was higher in both the subventricular zone and the subgranular zone than in the control group. However, the density of Pax6-IR cells in the cerebral cortex was lower in fetuses that experienced hypoxia than in control fetuses. CONCLUSION: These results suggest that Pax6 immunoreactivity showed diverse patterns in the neurogenic zone after prenatal hypoxia and Pax6 has important effects on neurogenesis.

The Immunoreactivity of PI3K/AKT Pathway After Prenatal Hypoxic Damage.

BACKGROUND/AIM: There is no consensus on the effect of hypoxia on neurogenesis. In this study, we investigated the immunoreactivity of BDNF and PI3K/Akt signaling after uterine artery ligation in pregnant rats. MATERIALS AND METHODS: Unilateral uterine artery ligation was performed at 16 days of gestation (dg). Fetuses from one horn with ligated artery were allocated to the hypoxic group. Immunohistochemistry was performed with primary antibodies; NeuN, BDNF, PI3K, Akt and phospho-Akt (pAkt). RESULTS: The densities of NeuN- and BDNF-immunoreactive (IR) cells in the cerebral cortex were lower in the hypoxic fetuses than in the controls at 21 dg. The density of PI3K and pAkt-IR cells in the cortex of the hypoxic group significantly decreased. The results in dentate gyrus were similar to the results in the cerebral cortex. CONCLUSION: Prenatal hypoxia reduced Akt phosphorylation, which affected neuronal survival in the cortex and dentate gyrus.