Search

The objective of this paper is to review current information regarding the treatment of age-related hearing loss by using cochlear hair cell regeneration. Recent advances in the regeneration of the inner ear, including the usefulness of stem cells, are also presented. Based on the current literature, cochlear cell regeneration may well be possible in the short term and cochlear gene therapy may also be useful for the treatment of hearing loss associated with ageing. The present review provide further insight into the pathogenesis of Inner Ear senescence and aged-related hearing loss and facilitate the development of therapeutic strategies to repair hair cells damaged by ageing. More research will be needed in order to translate them into an effective treatment for deafness linked to cochlear senescence in humans., (Copyright © 2017 Elsevier B.V. All rights reserved.)

We have evaluated the ability of the injured nigrostriatal dopaminergic system to produce highly reactive hydroxyl radicals ((*)OH) by the electrochemical detection of salicylate hydroxylation. Unilateral transection of the medial forebrain bundle transiently increased the formation of (*)OH in substantia nigra (SN) but not in striatum during the first 48 h postlesion, when most relevant changes in terms of oxidatively modified proteins take place. Short-term adaptive axotomy-induced changes in substantia nigra included downregulation of nigral tyrosine hydroxylase (TH) and dopamine transporter (DAT) mRNA expression and more intense TH immunoreactivity. Maintained inhibition of monoamine oxidase activity with deprenyl totally prevented the axotomy-induced formation of (*)OH, thus demonstrating the dopaminergic nature of these radicals. In contrast, deprenyl treatment, which is associated with a diminution in free radical production, failed to delay the onset of dopaminergic degeneration. This observation highlights the importance of being extremely cautious when analyzing parameters of oxidative stress and extrapolating them as a primary cause of cell death in the context of neurodegeneration. Long-term adaptive changes included a dramatic downregulation of DAT mRNA expression along with a moderate decrease in TH mRNA levels in SN. We anticipate a key regulatory role of the DAT to maximally optimize dopaminergic transmission in the synaptic cleft under conditions of degeneration.

We have recently characterized an animal model of status epilepticus induced by a single intraseptal injection of kainate. Under these conditions, there is a delayed expanding apoptotic hippocampal and amygdalar cell death. In order to further characterize this animal model, we have performed a detailed time-course analysis of the appearance of cell death, brain-derived neurotrophic factor messenger RNA expression and astroglial and microglial response in different brain areas related to the limbic system. We found a long-lasting delayed apoptotic cell death in the hippocampal formation, amygdala, medial thalamus, dorsal endopiriform nucleus and multiple cortical areas from two to 21 days post-injection. There was a spatiotemporal correlation between the appearance of cell death and induction of brain-derived neurotrophic factor messenger RNA expression in the areas studied, and interestingly this induction was found in non-degenerating cells. We conclude that our animal model of status epilepticus exhibits remarkable features of recurrent seizure activity and provides evidence for a neuroprotective role of brain-derived neurotrophic factor against seizure-induced apoptotic cell death.

We have previously studied changes in the serotoninergic and dopaminergic nigrostriatal systems following transection of the medial forebrain bundle and found a long-term axotomy-induced increase in the levels of serotonin and its main metabolite, 5-hydroxyindolacetic acid in substantia nigra [Venero et al. (1997) J. Neurochem. 68, 2458-2468]. In an attempt to find a rationale for this effect, we have performed an immunohistochemical study. Transection of the medial forebrain bundle of the rat interrupted most of the ascending serotoninergic pathways from the raphe nuclei as revealed by serotonin immunoreactivity. While serotonin immunostaining was almost absent in striatum, it doubled in the ventral mesencephalon at 21 days postlesion. This axotomy-induced increase was accompanied by an increased density of the serotonin nerve terminal network in the ipsilateral substantia nigra and ventral tegmental area. The increase in serotonin immunoreactivity was in line with the measured levels of serotonin and 5-hydroxyindolacetic acid in substantia nigra. In addition, the distribution pattern of glial fibrillary acidic protein-immunoreactive astrocytes and OX42-immunoreactive microglia correlated highly with the location of increased serotonin fibre density in the ventral mesencephalon, especially in ventral tegmental area and in the most medial part of substantia nigra. We suggest that a pruning effect may underly the axotomy-induced increase in serotonin immunoreactivity in the ventral mesencephalon, and further, that activated astroglia and microglia may play a role in directing serotoninergic axonal regeneration following axotomy.

We have performed a detailed time-course analysis of cell death in the hippocampal formation, basal forebrain and amygdala following a single intraseptal injection of kainate in adult rats. Acetylcholinesterase histochemistry revealed a profound loss of staining in the medial septum but not in the diagonal band, and cholinergic fiber density was highly reduced in the hippocampus and amygdala at 10 days postinjection. Terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphatebiotin nick end labeling (TUNEL) histochemistry was performed for precise location of apoptotic cells. Both the medial septum and amygdala exhibited numerous TUNEL-positive nuclei after the intraseptal injection of kainate, while the lateral septum exhibited a lower but significant incidence in terms of apoptotic cells. In the medial septum, the presence of apoptotic cells was at a location displaying acetylcholinesterase staining. TUNEL histochemistry revealed a time-dependent sequential apoptotic cell death in hippocampal pyramidal cells. During the first two days postinjection, apoptosis in the hippocampus was only evident in the CA3 region. At five days postinjection, the entire CA4 region became apoptotic. At 10 days postinjection, the whole extent of the CA1 pyramidal cell layer exhibited numerous TUNEL-positive nuclei. The time-course of kainate-induced apoptosis in Ammons's horn correlated with the disappearance of hippocampal pyramidal neurons as detected by Nissl staining, which is suggestive of a prominent apoptotic death for these cells. The temporal delayed distant damage to CA4 and CA1 hippocampal subfields after a single intraseptal kainate injection is not seen in other models employing kainate and may be a valuable tool for exploring the cellular mechanisms leading to cell death in conditions of status epilepticus.

1. The effects of 17 alpha-estradiol on KCl (60 mM), CaCl2 (30 microM to 10 mM) and vanadate (0.3 mM)-induced contractions in rat uterus have been assayed. Furthermore, the effect of 17 alpha-estradiol on calmodulin-stimulated cAMP-phosphodiesterase activity was also studied. 2. 17 alpha-estradiol relaxed the tonic contraction induced by KCl (60 mM) in a concentration-dependent way (IC50, 8.3 +/- 0.7 microM), and CaCl2 (0.1 to 10 mM) counteracted it. 3. CaCl2 (30 microM to 10 mM) produced concentration-dependent contraction of rat uterus in a calcium-free medium supplemented with 60 mM of KCl (EC50: 0.2 +/- 0.01 mM). 17 alpha-estradiol (8 microM) antagonized the contraction induced by CaCl2, increasing the EC50 value up to 0.7 +/- 0.1 mM (P < 0.01). 4. 17 alpha-estradiol (0.1 to 1 mM) relaxed in a concentration-dependent way the tonic contraction induced by vanadate in rat uterus incubated in a calcium-free medium and EDTA supplemented. The maximal relaxation achieved with 1 mM of 17 alpha-estradiol was 52.2 +/- 2.8%. 5. 17 alpha-estradiol (1 to 100 microM) did not modify the basal activity of cAMP-phosphodiesterase but inhibited the calcium plus calmodulin stimulated activity. The maximal inhibition achieved was 43 +/- 5.4%. 6. The relaxing effect of 17 alpha-estradiol on KCl (60 mM)-induced tonic contraction was unmodified with the antioestrogen tamoxifen (0.1 and 1 microM), the inhibitor of tirosine kinase (genistein, 10 microM) and the cAMP-dependent protein kinase inhibitor (Rp-adenosine 3',5'-monophosphothioate, triethylamine salt, 100 microM). However, the effect was antagonized with the inhibitor of transcription (actinomycin D, 5 micrograms/ml,), the inhibitor of protein synthesis (cycloheximide, 10 and 100 micrograms/ml), and the inhibitor of ornithine decarboxilase (alpha-difluoromethyl-ornithine, 10 mM). 7. Our results suggest that polyamines contribute to the relaxant effect of 17 alpha-estradiol in rat uterine smooth muscle behaving, presumably, as mediators of the transcriptional component involved in the effect of 17 alpha-estradiol.

1. The effects of the flavonoids genistein (3-60 microM), kaempferol (3-60 microM) and quercetin (1-100 microM) on KCl (60 mM)-induced tonic contraction in rat uterus and their modifications with the inhibitor of cAMP-dependent protein kinases (TPCK, 3 microM), the inhibitor of ornithine decarboxylase [alpha-difluoromethyl ornithine (DFMO), 10 mM] and the polyamine spermine (1 mM) have been assayed. The effects of the three flavonoids were also studied on the contraction elicited by CaCl2 (30 microM to 10 mM) on rat uterus incubated in medium lacking calcium and supplemented with 33, 60 or 90 mM of KCl. For comparison, the effects of the calcium channel blockers nifedipine and verapamil and the activator of adenylyl cyclase forskolin were assayed on contractions induced by KCl and CaCl2. 2. Genistein (IC50: 20.2 +/- 1.0 microM, n = 11), kaempferol (IC50: 10.1 +/- 0.8 microM, n = 8) and quercetin (IC50: 13.2 +/- 0.5 microM, n = 8) relaxed the tonic contraction induced by KCl (60 mM) in a concentration-dependent way. Verapamil (IC50: 70.1 +/- 5.8 nM, n = 7), nifedipine (IC50: 8.4 +/- 0.7 nM, n = 6) and forskolin (IC50: 0.62 +/- 0.08 microM, n = 14) also relaxed the KCl-induced contraction. TPCK (3 microM) significantly antagonized the effect of quercetin, kaempferol and forskolin (P < 0.01) but did not modify the effect of genistein. 3. Spermine (1 mM) increased the effects of genistein and verapamil and antagonized the effect of quercetin but did not modify those of kaempferol and forskolin. DFMO (10 mM) did not modify the effect of quercetin but increased that of genistein and antagonized those of kaempferol and forskolin. The addition of spermine (1 mM) plus DFMO (10 mM) antagonized the effect of quercetin. Spermine counteracted the effect of DFMO on forskolin but not on genistein. 4. KCl (33, 60 or 90 mM) did not produce contraction in calcium-free solution, but CaCl2 (30 microM to 10 mM) induced concentration-dependent contraction after depolarizing with KCl. The EC50 values for CaCl2 were: 0.74 +/- 0.08 (n = 12), 0.34 +/- 0.03 (n = 14) and 0.48 +/- 0.02 (n = 12) mM in a medium with 33, 60 or 90 mM of KCl, respectively. 5. Genistein (20 microM), kaempferol (10 microM), quercetin (15 microM), verapamil (70 nM), nifedipine (10 nM) and forskolin (0.5 microM) inhibited the concentration-response curve to CaCl2 in medium supplemented with 33, 60 or 90 mM of KCl. The effect of kaempferol was independent of the concentration of KCl in the incubation medium. However, the inhibitory effect of genistein on CaCl2-induced contraction was inversely related to the concentration of KCl in the medium. On the contrary, the effect of quercetin was directly related to the concentration of KCl in the medium. 6. The antagonism of verapamil, nifedipine and forskolin on CaCl2-induced contraction seems to be related to the degree of depolarization because increasing the KCl in the medium counteracted their effects. 7. Our results suggest that (1) cAMP contributes to the relaxant effects of quercetin and kaempferol on KCl (60 mM)-induced tonic contraction; (2) polyamines are involved in the relaxant effects of forskolin and kaempferol on KCl-induced tonic contraction but not on CaCl2-induced contraction in the depolarized uterus, and (3) the flavonoids assayed also possess a calcium antagonist action but show a different behavior toward the calcium channel blockers and the cAMP enhancer forskolin.

1. The effect of zeranol (3-100 microM) on rat uterus contractions induced by KCl (60 mM) and CaCl2 (30 microM-10 mM) has been assayed. 2. Zeranol relaxed the tonic contraction induced by KCl in a concentration-dependent manner (IC50 15.62 +/- 2.66 microM). CaCl2 (0.1-10 mM) did not counteract the relaxing effect of zeranol. 3. CaCl2 (30 microM -10 mM) produced a concentration-dependent contraction of rauuterus in medium lacking calcium plus KCl (60 mM) (EC50 0.34 +/- 0.03 mM). Zeranol (8 microM) displaced the CaCl2 concentration-response curve to the right and increased the EC50 to 1.27 +/- 0.57 mM (P < 0.05) without modifying the Emax. 4. The antiestrogen tamoxifen (1 microM) and the inhibitor of cAMP-dependent protein kinase TPCK (3 microM) did not modify the effect of zeranol. However, the inhibitors of transcription (actinomycin D, 4 microM), protein synthesis (cycloheximide, 100 microM), and ornithine-decarboxilase (alpha-difluoromethyl-ornithine, 10 mM)) antagonized the effect of zeranol, increasing the IC50 to 50.2 +/- 6.2 microM, 122 +/- 6.9 microM, and 23.51 +/- 1.14 microM, respectively. 5. Our results suggest that the relaxing effect of zeranol on rat uterus smooth muscle is produced by mechanisms unrelated to cAMP and estrogen receptors, but involves transcriptional effects and polyamine synthesis.

The effect of ascorbic acid depletion on the 1-methyl-4-phenylpyridinium ion (MPP+)-induced neurotoxicity in the dopaminergic system has been tested in guinea-pig striatal slices. Guinea-pigs were divided into three groups and fed on a control diet, ascorbic acid-free diet and ascorbic acid-supplemented diet, respectively. Diets were maintained during 30 days. Striatal slices from ascorbic acid-deficient animals showed the highest levels of dopamine following 25 microM MPP+ treatment; the results from animals under this treatment condition were statistically different from both control and ascorbic acid-supplemented animals under identical experimental conditions. In addition, neurochemical analysis demonstrated that the levels of ascorbic acid and dehydroascorbic acid were highly reduced in striatal tissue from ascorbic acid-deficient animals, thus proving scorbutic conditions in our experimental animals. In view of the higher resistance of the ascorbic acid-deficient animals to the neurotoxicity elicited by MPP+, additional dopaminergic parameters were also measured in striatal tissue from ascorbic acid-deficient animals in the absence of MPP+, including levels of dopamine and its metabolites, tyrosine hydroxylase activity and dopamine uptake, with the aim of finding an explanation for this unexpected result. While dopamine levels and tyrosine hydroxylase activity remained close to control levels, dopamine uptake was significantly reduced in striatal synaptosomes from ascorbic acid-deficient animals as compared with control animals. Since MPP+ is actively accumulated into dopaminergic nerve terminals via the high-affinity dopamine uptake system, this finding could explain the higher resistance of ascorbic acid-deficient animals to the dopamine-depleting effect induced by MPP+ toxicity assayed in striatal slices.

1. The effects of several gestagens (pregnenolone [1 to 30 microM], 20 alpha-hydroxy-pregnenolone [1 to 30 microM], and 20 beta-hydroxypregnenolone [1 to 30 micro M]) on rat uterine contraction induced by KCl (60 mM) and CaCl2 (30 microM to 6 mM) have been assayed. 2. The three drugs relaxed the tonic contraction induced by KCl in a concentration-dependent way. The respective EC50 values were: 27.6 +/- 1.58 microM (pregnenolone), 4.1 +/- 0.12 microM (20 alpha-hydroxy-pregnenolone), and 11.2 +/- 1.04 microM (20 beta-hydroxypregnenolone). CaCl2 (1 to 10 mM) totally counteracted the relaxing effect of pregnenolone but only partially compared to that of 20 alpha- or 20 beta-hydroxy-pregnenolone. 3. CaCl2 (30 microM to 6 mM) produced concentration-dependent contraction of rat uterus in medium lacking calcium plus 30, 60, or 90 mM of KCl. The EC50 values of CaCl2 were: 0.38 +/- 0.072, 0.183 +/- 0.015, and 0.183 +/- 0.015 mM in a medium with 30, 60, or 90 mM of KCl, respectively. 4. Pregnenolone (10 microM) did not significantly modify the EC50 of CaCl2 in a medium with 30, 60, or 90 mM of KCl. However, 20 beta-hydroxypregnenolone (10 microM) antagonized, in a noncompetitive manner, the concentration-response curve to CaCl2. 5. 20 alpha-Hydroxypregnenolone (4 microM) antagonized the concentration-response curve to CaCl2 in a competitive manner. This antagonism was directly related to the concentration of KCl in the medium. 6. Our results suggest a different calcium antagonist effect of the three gestagens assayed.