Effects of healthy ear compound of traditional Chinese medicine against age-induced apoptosis on cochlear spiral ganglion neurons in C57BL/6J mice and research on mechanisms by regulating caspase-3 / 中华老年医学杂志

Objective To explore the damage of spiral ganglion neurons(SGNs),the protective effects of different dosages of healthy ear compound (HEC)from traditional Chinese medicine(TCM) against age-induced SGNs degeneration and its possible mechanism in spiral ganglion neurons(SGNs)of C57BL/6J mice.Methods Totally 36 C57BL/6J mice just after ablactation were randomly divided into four groups.Normal control group (n =6)drank tap water daily from ablactation to 2 months old.Ageing-related SGNs apoptosis control group(n=12)drank tap water daily from ablactation to 7 months old.High dose TCM group(n=12)at drank 3.65 g/kg/d of HEC from ablactation to 7 months old.Low dose TCM group(n=6)drank 0.91 g/kg/d of HEC from ablactation to 7 months old.The animal cochleae were immediately removed at the termination of the experiment.In each group,the cochleae of 6 animals were used for paraffin embedding,slicing and toluidine blue staining to observe neuronal morphological changes.The caspase-3 mRNA expression study was performed by real-time PCR technique in 6 cochleae of High dose TCM group and ageing-related SGNs apoptosis control group.Results The morphological structure of cochlear SGNs represented healthy and normal density in normal control group at 2 months old.In contrast,amount or density of SGNs in cochlear basilar part was significantly damaged and reduced in ageing-related SGNs apoptosis control group at 7 months old(P＜ 0.001).But the high dose TCM group at 7 months of age was similar to the normal control group at 2 months old in morphological structure,amount or density of SGNs(P＞0.05).The low dose TCM group was significantly different from other 3 groups in amount or density of SGNs (P＜0.001).However,SGNs in the middle part and apical part showed integrity in each group.In addition,the expression level of caspase-3 in the cochlea of high dose TCM group was also obviously different with age-related SGNs apoptosis control group(P＜0.01) Conclusions Ageing-related damage of SGNs in C57BL/6J mice begins from the base of cochlea and progresses towards the apex.The HEC of TCM could significantly protect SGNs against age-induced apoptosis in SGNs.The efficacy of the high dose TCM is better than that of the low dose TCM.Its SGNs protective mechanisms might be related to involving the caspase-mediated cell apoptotic pathway.

Introspection of oxidant/antioxidant imbalance in the inner ear / 临床耳鼻咽喉头颈外科杂志

Free radicals are atoms, molecules or ions with unpaired electrons. In biological systems, free radicals can have a dual role, being beneficial in some situations and deleterious in others. Free radicals are required for normal cellular metabolism, but they lead to cellular degeneration if overproduced. To prevent the excessive buildup of free radicals, cells have developed an elaborate series of antioxidant enzymes that counteract oxidative stress and protect cells by maintaining the proper balance of oxidation and anti-oxidation. Therefore, when there is an oxidant/anti-oxidant imbalance, no matter what direction, cells are likely to be damaged. Numerous reports in the literature indicate that free radicals play important roles in diseases of the inner ear as a result of noise exposure, ototoxic drugs, aging, and other pathological conditions. Therefore, there have been many attempts to employ antioxidants treat inner ear damage. However, antioxidant therapy could be harmful if the improper compound or dose is employed. Effective antioxidant therapy requires prior knowledge of the type(s) of oxidative stress occurring in real time in the inner ear. Since most techniques for detecting free radicals in the inner ear are not clinically feasible, systemic anti-oxidant therapy is generally performed "blindly" and therefore likely to disrupt normal antioxidant levels in the inner ear or elsewhere in the body. If only a single anti-oxidant is used to treat a disease, it may disturb subsequent steps the oxidative/anti-oxidative chain reaction. An alternative approach, hydrogen therapy represents a promising therapeutic tool because it can selectively scavenge the strongest oxidant species, the hydroxyl radical and peroxynitrite anion, without disturbing normal oxidant/anti-oxidant cellular processes. In addition, hydrogen has no cytotoxic effects to cells so that it provides a near ideal therapy to eliminate toxic free radicals.

Cochlear organotypic cultures and histological examinations in rats / 中国耳鼻咽喉头颈外科

OBJECTIVE Exploration of project is to introduce a cochlear organotypic culture technique and the histological evaluation of cochlear hair cells, auditory nerve fibers, and spiral ganglion neurons from rat on post-natal day 3. METHODS The cochlear basilar membrane was placed on the surface of rattail collagen gel in culture medium. The cochlear specimens were double-labeled with FITC-conjugated phalloidin for showing the stereocilia and cuticular plate of the hair cells plus a monoclonal antibody against neurofilament 200 for showing the spiral ganglion neurons and their auditory nerve fibers. Specimens were observed under a Confocal Laser Scanning Microscope with appropriate filters. RESULTS Following the culturing period of 1 to 3 days, cochlear inner and outer hair cells presented normal without any contabescence or missing. The auditory nerve fibers were in an orderly arrangement and the morphology of spiral ganglion neurons were also in good shape. CONCLUSION The cochlear organotypic culture technique and the histological examinations will be helpful to evaluate the cochlear hair cells and the spiral ganglion neurons in various experimental models in vitro.

Experimental Study on Intervening Effects of CongEr Capsule in Age-induced Degeneration in Spiral Ganglion Neurons and Auditory Nerve Fibers in the Cochlea / 中华中医药杂志

Objective: A traditional Chinese medicine(TCM) transformed from Jian Er Ⅱ,Cong Er capsule was studied in protection against age-induced degeneration in cochlear spiral ganglion neurons and auditory nerve fibers in C57 BL /6J mice.A possible protective mechanism was also discussed.Methods : 40 neonatal C57 BL /6J mice were randomly divided into 2 groups with an average of 20 animals in each group.The animals in control group were fed with regular mouse feedstuff and regular water after ablactation,while animals in experimental group were fed the same mouse food after ablactation,but drink the medical solution of Cong Er capsule instead of regular water from two months of age.Animals were terminated at 7 months of age.The cochlea were removed and processed for a series of modiolus sections.The density of spiral ganglion neurons in Rosenthal's canal in different turns was counted,and the auditory nerve fibers in habenula perforata at basal turn and apical turn were also quantified respectively.Statistical comparative analysis was performed between the two groups.Results: Density of spiral ganglion neurons in cochlear basal turn in control group was significantly reduced.In survival ganglion neurons,an obvious degeneration was observed in control group.The number of auditory nerve fibers in cochlear basal turn also decreased.In contrast,Cong Er capsule treated animals had an increase in survival ganglions and auditory nerve fibers,especially,a significant difference was seen in density of spiral ganglion neurons in the basal turn between the two groups(P

Cochlear mitochondrial DNA3867bp deletion in aged mice / 中华医学杂志(英文版)

<p><b>OBJECTIVES</b>To study the status of cochlear mitochondrial DNA (mtDNA) and to determine the location of mtDNA deletion in aged mice.</p><p><b>METHODS</b>We detected cochlear mtDNA in 2, 7 - 10 and 17 - 19 month old mice by nested polymerase chain reaction (PCR) and DNA sequencing.</p><p><b>RESULTS</b>mtDNA3867bp deletions were found in the cochleae of aged mice. The deletion occurred within nt9103-nt12970 and were flanked by 15 base pair direct repeats. Comparing the incidence of mtDNA3867bp deletions, 17 - 19 month old mice (7/8) were significantly higher than 7 - 10 month old mice (4/16). The deletion was not observed in 2 month old mice (0/7). The ratio of deleted mtDNA/total mtDNA in 17 - 19 month old mice was higher than in 7 - 10 month old mice (P < 0.001).</p><p><b>CONCLUSION</b>Cochlear mtDNA 3867bp deletion in aged mice may be related to presbycusis.</p>

Deletions are easy detectable in cochlear mitochondrial DNA of Cu/Zn superoxide dismutase gene knockout mice / 中华医学杂志(英文版)

<p><b>OBJECTIVES</b>To investigate the tissue specificity of reactive oxygen species (ROS) damage to mitochondrial DNA (mtDNA) and to determine whether cochlear mtDNA is a sensitive target for ROS damage.</p><p><b>METHODS</b>10 Cu/ZnSOD gene (Cu/Zn superoxide dismutase gene, Sod1) knockout mice and 16 wild-type mice were analyzed by nested polymerase chain reaction (PCR).</p><p><b>RESULTS</b>Three deletions were detected in various tissues of Sod1 knockout mice. MtDNA3867bp and mtDNA3726bp deletions were the most visible, and mtDNA4236bp deletion was barely detected in these tissues. There were obvious differences in the ratio of deleted mtDNA/total mtDNA in different tissue. Deleted mtDNA was most abundant in the liver and kidney and less in cochlea, heart and brain. The lowest was in spleen and skin. The ratio in various tissues was 3 - 20 times in Sod1 knockout mice over wild-type mice. In cochlea, the ratio was about 15.</p><p><b>CONCLUSIONS</b>Without the protection of Sod1, ROS can lead to mtDNA deletions in various tissues with significant tissue specificity. Cochlear mtDNA is a sensitive target for ROS damage.</p>

Development of the guinea pig's eye during the stage of embryo / 眼科新进展

Objective　To investigate the development of the guinea pig's eye during the stage of embryo.Method　The guncotton grafts of the guinea pig's skull was observed on the 19th ,24th,28th,35th,40th,43th and the 45th day of embryo.Results　The optic vesicle and lens plate developed on the 19th day in embryo.The optic cup and lens fovea could be found on the 24th day.The eye of embryo of the guinea pig had formed on the 28th day.Conclusion　The results should be used for progressive study of the development of the eyes of embryo.

Accumulation of Gentamvcin in Perilymph of Guinea Pigs / 第二军医大学学报

According to the pharmacokinetic theory, the authors studied the accumulation of gentamycin in perilymph of guinea pigs by searching the drug concentration at various time with Fluorescene Polarization Immunoassay (FPIA). The results indicated that: (1) The drug concentration in perilymph increased with the augment of total doses administered and there was almost a linear correlation between them. (2) In animals of 7-day injectious group, the drug could be still detected during 72 hours after the last dose administration. The concentration was 1 37?0.95 ?g/ml which was close to the level of serum minimal inhibitory concentration (MIC). It suggested that the elimination of the drug from inner ear was too slow and evident accumulation of gentamycin was indeed. (3) The fact mentioned above implied that the drug ototoxities could still damage the ear even if the drug administered had ceased before. It is the reason by which we could explain the problem encountered in clinic, that is why in some patients the ototoxic syndrom may still presented or enhanced even though the treatment has been stopped a few days before.

A Transmission Electron Microscope Observation of Spiral Organ of Cochleae with Noise-induced Threshold Shift / 第二军医大学学报

Guinea pigs were exposed to either 110dB SPL white noise for 30min (ITS group )or 120 dB SPL for 150 nun (PTS group). At varying postexposure intervals, threshold shifts were assessed using auditory cortex evoked response to tone bursts and intracellular ultrastructural changes in the spiral organ of cochleae were exmained with a transmission electron microscope.. The threshold shifts induced by 110dB noise were reversible while those induced by 120dB noise were generally irreversible. In the TTS cochleae, damage was confined to the third row of OHCs where depolymerization of actin filaments within the stereocilia, slight celluar swelling and small vacuolization were found. The subnuclear area and nerve-endings were not involved. In the PTS cochleae, the inner hair cells (IHCs) and the first row of OHCs were affected. The abnormilities consisted of ruptures and holes in the cuticular plates, fusion of stereocilia, marked edema, tranclucence of subcuticular region, swelling of submembraneous cisterns and large vesiculation in the efferent nerve-endings blow OHCs. Based on the ultrastructural observations, structural bases in cochleae for TTS and PTS, sequence of pathological changes in hair cells as well as reversibility of specific pathologies were suggested.

Ultrastructural Changes in the Spiral Organ of Cochlea after Exposure to Impulse Noise / 第二军医大学学报

Ultrastructural changes in the spiral organ of cochlea at various intervals after exposure to impulse noise were investigated. Guinea pigs were exposed to 10 impulses noise of 166 dB SPL peak level which had duration of 0.1 ms. Thirteen of the exposed animals were used to systematically measure threshold shift at regular intervals from 30 min to 30d post-exposure. The other fourteen animals who had been exposed to the same impulse noise were killed for transmission electron microscopy (TEM) examination at the same intervals, respectively. The recovery pattern of threshold shift showed a nonmonotonic type. There was a progressive deterioration of changes in the hair cells between 30 min and 8h after exposure. Intracellular degeneration reached a peak at 8h and marked edema and swelling leading to deformation of the outer hair cells (OHCs), fused stereocilia, large vesicles in cytoplasm and swollen submembraneous cisterns were found. After that time the extent of degeneration in the hair cells reduced. The time sequence of changes in the spiral organ of cochlea in the present study was associated with the recovery pattern of threshold shift.