[Comparison between macroscopic identification and DNA barcoding identification of Amomi Fructus].

This study aims to explore the consistency between macroscopic identification and DNA barcoding identification of Amomi Fructus. With the DNA barcoding identification results, we evaluated the reliability of identifying Amomi Fructus quality by combining macroscopic traits with main volatile chemical components. Thirteen batches of Amomi Fructus samples were collected for identification. Firstly, the morphological and sensory characteristics of each sample were observed and recorded according to the standard in Chinese Pharmacopoeia(2020 edition). The 100-fruit weight, longitudinal diameter, transverse diameter, and longitudinal diameter-to-transverse diameter ratio were measured, which correspond to large, solid, and full kernel representing good quality in the sensory evaluation. The odor value detected by electronic nose and major volatile components(borneol, camphor, limonene, and borneol acetate) correspond to the sensory evaluation of strong odor representing good quality. Secondly, DNA barcoding was employed to identify the 13 batches of samples. Finally, clustering analysis was performed for the main volatile components and macroscopic traits, and the identification results were compared with those of DNA barcoding. Except two batches of samples(No.6 and No.10), the macroscopic identification showed the results consistent with those of DNA barcoding, with an identification rate of 84.62%. The clustering results of the content of four volatile chemical components and macroscopic traits were also consistent with the DNA barcoding identification results. DNA barcoding can verify the results of macroscopic identification and provide a scientific basis for the inheritance and development of macroscopic identification. Moreover, the combination of macroscopic traits and chemical components demonstrates higher accuracy in the quality evaluation of Chinese medicinal materials.

Decreased expression of TERT correlated with postnatal cochlear development and proliferation reduction of cochlear progenitor cells.

Cochlear progenitor cells are considered as one of the best candidates for hair cell regeneration, thus, the regulation of cochlear progenitor cell proliferation has become a focus in this field. Several genes expressed in the inner ear during postnatal development have been demonstrated to be involved in maintaining the proliferative potential of progenitor cells, but the mechanism for regulating the proliferation and differentiation of cochlear progenitor cells remains poorly understood. Telomerase reverse transcriptase (TERT) has rate limiting telomerase activity and the overexpression of TERT has been shown to promote cell proliferation in series of cell lines. The aim of the present study was to evaluate the expression of TERT in the postnatal development of the cochlea and progenitor cells. The results demonstrated that TERT was expressed in the basilar membranes during the first postnatal week. In vitro, TERT expression in progenitor cells reached a maximum at day 4 after culture and decreased as the culture time prolonged or the cell passage number increased. These results led us to hypothesize that TERT may be involved in the development of the cochlea and in maintaining the proliferation ability of progenitor cells.

Combinatorial enzymatic digestion with thermolysin and collagenase type I improved the isolation and culture effects of hair cell progenitors from rat cochleae.

The high incidence of hearing loss in human combined with the lack of hair cell regeneration in mammalian cochleae had got the attention to manipulate stem/progenitor cells to participate in hair cell regeneration for years. Cochlear progenitor cells are considered as the best candidate for hair cell regeneration. However, there is not any effective and feasible way to separate hair cell progenitors from rat cochleae, yet. In this study, we tried to isolate single epithelial cells from rat basilar membrane by combinatorial enzymatic digestion with thermolysin and collagenase type I. The results showed that the harvested single cells gave rise to otospheres with features of stem cells and could be induced to differentiate into hair cells. Significantly, more otospheres of epithelial origin were obtained by digesting with thermolysin and collagenase type I. The combinatorial enzymatic digestion would be a potential method for hair cell progenitor isolation and culture with broad applications.

[Rancidness of Armeniacae Semen Amarum involving Bianzhuang Lunzhi].

This article aims to compare the qualities of Armeniacae Semen Amarum before and after rancidness, in order to study the rancidness of Armeniacae Semen Amarum. In the experiment, content of fatty oil, acid value and peroxide value were determined before and after rancidness,respectively. Meanwhile, HPLC, GC-MS were utilized to analyze laetrile and fatty acid components. Besides, colorimeter and e-nose were introduced to quantify and compare "color and odor". A correlation analysis was conducted on the above results. The results showed that color of post-rancidness Armeniacae Semen Amarum changed from yellow to brown, with sour and lower content of laetrile. On the contrary, acid and peroxide values increased significantly, with changes in fatty acid component. There was a considerable correlation between appearance characteristics and changes in internal quality. The "sensory analysis-quality identification system" can provide a certain scientific basis for prediction of the content of chemical components in traditional Chinese medicine, preliminary judgment of quality of traditional Chinese medicine and real-time quality monitoring, which offers us novel ideas and reference for storage principles of traditional Chinese medicines of "pre-event prediction, during-event intervention and post-event identification".

Apoptosis-inducing factor and calpain upregulation in glutamate-induced injury of rat spiral ganglion neurons.

Spiral ganglion neuron (SGN) damage and apoptosis can lead to noise-induced hearing loss, age-associated hearing loss and, in certain cases, auditory neuropathy. The apoptosis-inducing factor (AIF)-associated pathway may be important in this process. The present study aimed to investigate the expression levels of AIF and calpain in damaged SGNs. Glutamate (Glu) perfusion and cell culture in different concentrations of Glu were performed to damage the SGNs of Sprague-Dawley (SD) rats, with saline water used as a control Different concentrations (5, 10, 20 and 40 mM) of Glu were injected into the cochlear tympanic canal of 18 SD rats, and 10, 20 and 40 mM Glu were added to SGN cultures. Auditory brainstem responses (ABR) were measured prior to and 2 days following the injection of Glu. Immunofluorescent staining was used to detect the SGN damage and the expression levels of AIF and calpain in vivo and in in vitro. Transmission electron microscopy (TEM) was used to measure cell apoptosis and reverse transcription-quantitative polymerase chain reaction was used to analyse the gene expression levels of AIF and calpain in the damaged SGNs. The TEM identified mitochondrial vacuolisation, swelling of the SGN and heterochromatin formation. Injection of Glu reduced the number of SGNs and induced apoptosis. AIF was observed to translocate into the nuclei of the SGNs in the 20 and 40 mM Glu groups, and the expression levels of AIF and calpain were markedly upregulated in the modiolus of the Glu-damaged SGNs. The upregulation of AIF and calpain may be important in the process of SGN damage and apoptosis.

Calpain inhibitor PD150606 attenuates glutamate induced spiral ganglion neuron apoptosis through apoptosis inducing factor pathway in vitro.

OBJECTIVE: This research aimed to investigate whether glutamate induced spiral ganglion neurons (SGNs) apoptosis through apoptosis inducing factor (AIF) pathway. And verify whether PD150606, a calpain inhibitor could prevent apoptosis by inhibiting cleaving and releasing AIF in mitochondrion. METHODS: SGNs of postnatal days 0-3 were harvested and cultured in dishes. 20 mM Glu, the caspase inhibitor Z-VAD-FMK and calpain inhibitor PD150606 were added into cultured dishes separately. We used optical microscope and immunofluoresence staining to observe cell morphology and AIF distribution, RT-PCR and Westernblot to analyse AIF and calpain expression in SGNs. TUNEL assay was used to test cell apoptosis. RESULTS: Cell morphology and nuclear translocation of AIF were altered in SGNs by 20 mM Glu treated in vitro. The axon of SGN shortened, more apoptosis SGN were observed and the expression of AIF and calpain were up-regulated in Glu-treated group than the normal one (P<0.05). The same experiments were conducted in 20 mM+PD150606 treated group and 20 mM+Z-VAD-FMK group. Obviously AIF were located from cytoplasm to the nuclear and the expressions of AIF and calpain were down-regulated by PD150606 (P<0.05). Positive cells in TUNEL staining decreased after PD150606 treating. However, Z-VAD-FMK had no influence on AIF, calpain expression or cell apoptosis. CONCLUSION: The AIF-related apoptosis pathway is involved in the process of Glu-induced SGN injury. Furthermore, the inhibition of calpain can prevent AIF from releasing the nuclear or inducing SGN apoptosis.

The role of RIP3 mediated necroptosis in ouabain-induced spiral ganglion neurons injuries.

Spiral ganglion neuron (SGN) injury is a generally accepted precursor of auditory neuropathy. Receptor-interacting protein 3 (RIP3) has been reported as an important necroptosis pathway mediator that can be blocked by necrostatin-1 (Nec-1). In our study, we sought to identify whether necroptosis participated in SGN injury. Ouabain was applied to establish an SGN injury model. We measured the auditory brain-stem response (ABR) threshold shift as an indicator of the auditory conditions. Positive ß3-tubulin immunofluorescence staining indicated the surviving SGNs. RIP3 expression was evaluated using immunofluorescence, quantitative real-time polymerase chain reaction and western blot. SGN injury promoted an increase in RIP3 expression that could be suppressed by application of the necroptosis inhibitor Nec-1. A decreased ABR threshold shift and increased SGN density were observed when Nec-1 was administered with apoptosis inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD). These results demonstrated that necroptosis is an indispensable pathway separately from apoptosis leading to SGN death pathway, in which RIP3 plays an important role.

Acoustical stimulus changes the expression of stromal cell-derived factor-1 in the spiral ganglion neurons of the rat cochlea.

Neural stem cell (NSC) transplantation into the cochlea has been tested as a treatment for spiral ganglion neuron (SGN) degenerative disease and injury in various animal models. A recent study has shown evidence of functional recovery after transplantation of the stem cells into a degenerated-SGN model. Chemokine stromal cell-derived factor-1 (SDF-1, or known as CXC chemokine ligand-12, CXCL-12) signaling through CXCR4 has previously been identified as a key step in the homing of the stem cells within the injury areas; meanwhile, studies have revealed that the SDF-1/CXCR4 axis is also involved in axon guidance and pathfinding. A study found that transplanted neural precursor cells can migrate to the root of the auditory nerve when animals are subjected to an augmented acoustic environment (AAE). In accordance with these studies, we hypothesize that AAE will up-regulate the expression of SDF-1 in acoustic nerves. We tested our hypothesis by examining the expression of SDF-1 in different acoustic environments, and the results were confirmed by the auditory brainstem response (ABR), immunohistochemical and RT-PCR analyses. The results showed that SDF-1 was expressed at a relatively low level in the SGNs under normal animal unit acoustic conditions (40-50 dB). Moreover, it was significantly up-regulated in the SGNs under the 75 dB (augmented physiological process without hearing loss) and 90 dB AAE (pathological process with light hearing loss) conditions; however, under the 115 dB AAE (pathological process with severe hearing loss) condition, the expression of SDF-1 was not up-regulated. The results confirmed that appropriately augmented acoustical stimuli lead to the up-regulation of SDF-1, which may assist in the migration of the transplanted cells and the subsequent establishment of essential synaptic contacts between the exogenous cells and the host auditory pathway.

Wnt1 from cochlear schwann cells enhances neuronal differentiation of transplanted neural stem cells in a rat spiral ganglion neuron degeneration model.

Although neural stem cell (NSC) transplantation is widely expected to become a therapy for nervous system degenerative diseases and injuries, the low neuronal differentiation rate of NSCs transplanted into the inner ear is a major obstacle for the successful treatment of spiral ganglion neuron (SGN) degeneration. In this study, we validated whether the local microenvironment influences the neuronal differentiation of transplanted NSCs in the inner ear. Using a rat SGN degeneration model, we demonstrated that transplanted NSCs were more likely to differentiate into microtubule-associated protein 2 (MAP2)-positive neurons in SGN-degenerated cochleae than in control cochleae. Using real-time quantitative PCR and an immunofluorescence assay, we also proved that the expression of Wnt1 (a ligand of Wnt signaling) increases significantly in Schwann cells in the SGN-degenerated cochlea. We further verified that NSC cultures express receptors and signaling components for Wnts. Based on these expression patterns, we hypothesized that Schwann cell-derived Wnt1 and Wnt signaling might be involved in the regulation of the neuronal differentiation of transplanted NSCs. We verified our hypothesis in vitro using a coculture system. We transduced a lentiviral vector expressing Wnt1 into cochlear Schwann cell cultures and cocultured them with NSC cultures. The coculture with Wnt1-expressing Schwann cells resulted in a significant increase in the percentage of NSCs that differentiated into MAP2-positive neurons, whereas this differentiation-enhancing effect was prevented by Dkk1 (an inhibitor of the Wnt signaling pathway). These results suggested that Wnt1 derived from cochlear Schwann cells enhanced the neuronal differentiation of transplanted NSCs through Wnt signaling pathway activation. Alterations of the microenvironment deserve detailed investigation because they may help us to conceive effective strategies to overcome the barrier of the low differentiation rate of transplanted NSCs.

G-CSF attenuates noise-induced hearing loss.

In this study, we investigated the effects of granulocyte colony-stimulating factor (G-CSF) for the treatment of noise-induced hearing loss (NIHL) in a guinea pig model. Forty guinea pigs were randomly divided into four groups: control, noise (white noise, 3 h/d for 2 days at 115 dB), noise+G-CSF (350 µg/kg/d for 5 days), and noise+saline. Auditory brainstem response (ABR) and distortion product otoacoustic emission (DPOAE) were used to determine the hearing threshold and outer hair cell function, respectively, in each group. Cochlear morphology was examined to evaluate hair cell injury induced by intense noise exposure. Fourteen days after noise exposure, the noise+G-CSF group had a lower ABR value than the noise group (P<0.05) or the noise+saline group (P<0.01). At most frequencies, the DPOAE value of the noise+G-CSF group showed a significant rise (P<0.05) compared to the noise group or the noise+saline group. Neither the ABR value nor the DPOAE value differed between the noise group and the noise+saline group. The morphology of the phalloidin-stained organ of Corti was consistent with the functional measurements. In conclusion, G-CSF can preserve hearing in an experimental model of NIHL in guinea pigs, by preserving hair cells after intense noise exposure.

Stem cell transplantation via the cochlear lateral wall for replacement of degenerated spiral ganglion neurons.

Spiral ganglion neurons (SGNs) are poorly regenerated in the mammalian inner ear. Because of this, stem cell transplantation has been used to replace injured SGNs, and several studies have addressed this approach. However, the difficulty of delivering stem cells into the cochlea and encouraging their migration to Rosenthal's canal (RC), where the SGNs are located, severely restricts this therapeutic strategy. In this study, we attempted to establish a new stem cell transplantation route into the cochlea via the cochlear lateral wall (CLW). First, we tested the precision of this route by injecting Fluorogold into the CLW and next assessed its safety by mock surgeries. Then, using a degenerated SGN animal model, we transplanted neural stem cells (NSCs), derived from the olfactory bulb of C57BL/6-green fluorescent protein (GFP) mice, via the CLW route and examined the cells' distribution in the cochlea. We found the CLW transplantation route is precise and safe. In addition, NSCs migrated into RC with a high efficiency and differentiated into neurons in a degenerated SGN rat model after the CLW transplantation. This result revealed that the basilar membrane (BM) may have crevices permitting the migration of NSCs. The result of this study demonstrates a novel route for cell transplantation to the inner ear, which is important for the replacement of degenerated SGNs and may contribute to the treatment of sensorineural hearing loss.

Up-regulation of stromal cell-derived factor-1 enhances migration of transplanted neural stem cells to injury region following degeneration of spiral ganglion neurons in the adult rat inner ear.

Neural stem cell (NSC) transplantation into the cochlea is widely used for the treatment of spiral ganglion neuron (SGN) degenerative disease and injury in the animal models, but the migration of the transplanted NSCs to the injury region is difficult and the mechanism is still unclear. In this study, we aimed to validate whether the SGN-degenerated cochlear microenvironment plays a role in the NSC migration and investigated whether stromal cell-derived factor-1 (SDF-1) was involved in the NSCs migration. Using a rat SGN degeneration model, we demonstrated that the transplanted NSCs are more likely to migrate to the injury region during the early post-injury (EPI) than the late post-injury (LPI) stage and the control cochlea. We found that the expressions of SDF-1 increased transiently after SGN degeneration. Additionally, we showed that the NSCs express CXCR4, a receptor for SDF-1. We observed that the region to which the transplanted NSC localized coincides with the region where the SDF-1 is highly expressed following the degeneration of SGNs. Finally, we observed that the increased SDF-1 is derived from the Schwann cells in the SGN-degenerated model. These results suggest that SDF-1, which is derived from cochlear Schwann cells and up-regulated in the early injury microenvironment, plays a beneficial role in the NSC migration to the injury region. Optimizing SDF-1 expression in the host microenvironment or increasing the CXCR4 expression of the donor stem cells may improve the migration efficiency of transplanted cells toward the injury region in the cochlea.

Inhalation of hydrogen gas attenuates ouabain-induced auditory neuropathy in gerbils.

AIM: Auditory neuropathy (AN) is a hearing disorder characterized by abnormal auditory nerve function with preservation of normal cochlear hair cells. This study was designed to investigate whether treatment with molecular hydrogen (H(2)), which can remedy damage in various organs via reducing oxidative stress, inflammation and apoptosis, is beneficial to ouabain-induced AN in gerbils. METHODS: AN model was made by local application of ouabain (1 mmol/L, 20 mL) to the round window membrane in male Mongolian gerbils. H(2) treatment was given twice by exposing the animals to H(2) (1%, 2%, and 4%) for 60 min at 1 h and 6 h after ouabain application. Before and 7 d after ouabain application, the hearing status of the animals was evaluated using the auditory brainstem response (ABR) approach, the hear cell function was evaluated with distortion product otoacoustic emissions (DPOAE). Seven days after ouabain application, the changes in the cochleae, especially the spiral ganglion neurons (SGNs), were morphologically studied. TUNEL staining and immunofluorescent staining for activated caspase-3 were used to assess the apoptosis of SGNs. RESULTS: Treatment with H(2) (2% and 4%) markedly attenuated the click and tone burst-evoked ABR threshold shift at 4, 8, and 16 kHz in ouabain-exposed animals. Neither local ouabain application, nor H(2) treatment changed the amplitude of DPOAE at 4, 8, and 16 kHz. Morphological study showed that treatment with H(2) (2%) significantly alleviated SGN damage and attenuated the loss of SGN density for each turn of cochlea in ouabain-exposed animals. Furthermore, ouabain caused significantly higher numbers of apoptotic SGNs in the cochlea, which was significantly attenuated by the H(2) treatment. However, ouabain did not change the morphology of cochlear hair cells. CONCLUSION: The results demonstrate that H(2) treatment is beneficial to ouabain-induced AN via reducing apoptosis. Thus, H(2) might be a potential agent for treating hearing impairment in AN patients.

[Detrimental effects of ouabain on cochlear spiral ganglion cells in rats].

OBJECTIVE: To investigate the detrimental effects of ouabain on cochlear spiral ganglion neurons (SGCs) in vivo and in vitro. METHODS: Seventy-five male SD rats were randomly divided into 5 groups. In addition to the normal control group, rats in other 4 groups received 0.01, 0.02, 0.05 mmol/L of Ouabain or saline through cochlear scala tympani drilling. Seven days after surgery, the hearing threshold was measured by distortion product otoacoustic emissions (DPOAE) and auditory brainstem response (ABR) in rats. In the in vitro study, SGNs were isolated from SD rats (E14) and treated with 1 × 10(-8) mmol/L of Ouabain. The damaged of SGCs were detected after ouabain treatment using immunohistochemistry, transmission electron microscope and scanning electron microscope in vitro. RESULTS: After administration of Ouabain, DPOAE did not change significantly. No significant difference in the amplitude of DPOAE could be observed among all the groups (P > 0.05). Compared with saline and normal control, ABR threshold was significantly increased in the Ouabain treated groups (P < 0.05), which correlated with the concentration of Ouabain. Electron microscopy showed that after treated with Ouabain, SGCs presented degenerative changes, including collapse of organelle structures, the karyotheca dissolved, myelin sheath disintegrating. Ouabain could damage type I SGCs but not type II SGNs. CONCLUSIONS: Ouabain can damage SGCs, either in the in vivo or in vitro conditions.

Combined use of decellularized allogeneic artery conduits with autologous transdifferentiated adipose-derived stem cells for facial nerve regeneration in rats.

Natural biological conduits containing seed cells have been widely used as an alternative strategy for nerve gap reconstruction to replace traditional nerve autograft techniques. The purpose of this study was to investigate the effects of a decellularized allogeneic artery conduit containing autologous transdifferentiated adipose-derived stem cells (dADSCs) on an 8-mm facial nerve branch lesion in a rat model. After 8 weeks, functional evaluation of vibrissae movements and electrophysiological assessment, retrograde labeling of facial motoneurons and morphological analysis of regenerated nerves were performed to assess nerve regeneration. The transected nerves reconstructed with dADSC-seeded artery conduits achieved satisfying regenerative outcomes associated with morphological and functional improvements which approached those achieved with Schwann cell (SC)-seeded artery conduits, and superior to those achieved with artery conduits alone or ADSC-seeded artery conduits, but inferior to those achieved with nerve autografts. Besides, numerous transplanted PKH26-labeled dADSCs maintained their acquired SC-phenotype and myelin sheath-forming capacity inside decellularized artery conduits and were involved in the process of axonal regeneration and remyelination. Collectively, our combined use of decellularized allogeneic artery conduits with autologous dADSCs certainly showed beneficial effects on nerve regeneration and functional restoration, and thus represents an alternative approach for the reconstruction of peripheral facial nerve defects.

Repair of facial nerve defects with decellularized artery allografts containing autologous adipose-derived stem cells in a rat model.

The purpose of this study was to investigate the effects of a decellularized artery allograft containing autologous adipose-derived stem cells (ADSCs) on an 8-mm facial nerve branch lesion in a rat model. At 8 weeks postoperatively, functional evaluation of unilateral vibrissae movements, morphological analysis of regenerated nerve segments and retrograde labeling of facial motoneurons were all analyzed. Better regenerative outcomes associated with functional improvement, great axonal growth, and improved target reinnervation were achieved in the artery-ADSCs group (2), whereas the cut nerves sutured with artery conduits alone (group 1) achieved inferior restoration. Furthermore, transected nerves repaired with nerve autografts (group 3) resulted in significant recovery of whisking, maturation of myelinated fibers and increased number of labeled facial neurons, and the latter two parameters were significantly different from those of group 2. Collectively, though our combined use of a decellularized artery allograft with autologous ADSCs achieved regenerative outcomes inferior to a nerve autograft, it certainly showed a beneficial effect on promoting nerve regeneration and thus represents an alternative approach for the reconstruction of peripheral facial nerve defects.

[Culture of neural stem cells from rat olfactory bulb].

OBJECTIVE: To establish the method of in vitro culture of neural stem cells (NSC) from rat olfactory bulb and investigate the characteristics of its proliferation and differentiation. METHODS: NSC from postnatal one-day (P1) and adult rat olfactory bulb were isolated and cultured in serum free media with epidermal growth factor (EGF) and fibroblast growth factor-basic (bFGF). Antibodies against NSC (nestin), neuronal (neuronal specific enolase, NSE), astrocytic (glial fibrillary acidic protein, GFAP) and oligodendrocytic (galactocerebroside, GalC) markers were used to identify NSC and specific neural cells differentiated from NSC with immunocytochemical staining. Growth curve of olfactory bulb NSC was measured using MTT method. RESULTS: Nestin immuno-positive NSC were isolated and cultured from P1 and adult rat olfactory bulb which could differentiate into neurons, astrocytes and oligodendrocytes. The forming rates of neurosphere from P1 and adult rat olfactory bulb were 20% approximately 30% and 0.1% respectively. The proliferation of olfactory bulb NSC depended on EGF and bFGF, in which EGF increased proliferation of cells stronger than bFGF. CONCLUSIONS: NSC with self-renewal capacity and potential multi-differentiation were cultured from P1 and adult rat olfactory bulb.