Lianjun Lu

Proliferation, multipotency and neuronal differentiation of cryopreserved neural progenitor cells derived from the olfactory neuroepithelium of the adult rat

The use of olfactory neuroepithelium neural progenitor cells for transplantation has attracted attention in the treatment of many neurological disorders, which require efficient recovery methods and cryopreservation procedures. The purpose of this study was to evaluate different cryopreservation techniques for neural progenitor cells derived from the olfactory neuroepithelium (ONe NPCs) in adult rats. Initially, we compared the survival rates of cryopreserved ONe NPCs treated with six different cryoprotectants: dimethylsulfoxide (DMSO), ethylene glycol (EG) and glycerol, each with or without 10% FBS and with two different storage periods in liquid nitrogen (−196 °C), specifically 3 days short‐term storage and 3 months long‐term storage. We assessed the recovery efficiency of ONe NPCs after freezing and thawing by viability testing and colony‐forming assay as well as immunocytochemistry under different conditions. No significant difference in the survival rate was observed among these different cryoprotectants. With these protocols, ONe NPCs retained their multipotency and differentiated into glial (GFAP‐positive), neuronal (NeuN‐positive) and oligodendroglia (Galc‐positive) cells. Collectively, our results imply that, under optimal conditions, ONe NPCs might be cryopreserved for periods of >3 months without losing their proliferative and multipotency activities.

Effects of noradrenaline on the GABA response in rat isolated spiral ganglion neurons in culture.

In the present study, the modulatory effects of noradrenaline (NA) on the GABA response were investigated in the isolated cultured spiral ganglion neurons of rat by using nystatin perforated patch recording configuration under voltage‐clamp conditions. NA reversibly depressed GABA response in a concentration‐dependent manner and neither changed the reversal potential of the GABA response nor affected the apparent affinity of GABA to its receptor. α2‐adrenoceptor agonist and antagonist, clonidine and yohimbine mimicked and blocked the NA action on the GABA response, respectively. N‐[2(methylamino)ethyl]‐5‐isoquinoline sulfonamide dihydrochloride (H‐89), a protein kinase A inhibitor, mimicked the effect of NA on the GABA response. NA failed to affect the GABA response in the presence of both cAMP and protein kinase A modulator. However, NA still depressed the GABA response even in the presence of both phorbol‐12‐myristate‐13‐acetate, a protein kinase C activator and chelerythrine, a protein kinase C inhibitor. These results suggest that the NA suppression of the GABA response is mediated by α2‐adrenoceptor which reduces intracellular cAMP formation through the inhibition of adenylyl cyclase. Therefore, NA input to the spiral ganglion neurons may modulate the auditory transmission by affecting the GABA response.

A comparison of the proliferative capacity and ultrastructure of proliferative cells from the cochleae of newborn rats of different ages

OBJECTIVE Recent reports have shown that multipotent stem cells/progenitor cells that are capable of proliferation and regeneration are present in mammalian cochleae. However, progenitor cells have not been isolated from the adult cochlea. We examined the proliferative potential of cells derived from neonatal rats of various ages. The determination of the differences between the proliferative cells from rats of different ages may provide clues to the mechanisms controlling the destiny of these cells. METHODS Proliferative cells were isolated from the cochleae of 1-, 7-, and 14-day-old rats, and the proliferative capacity and ultrastructure of the cells from each age group were assessed using flow cytometry and transmission electron microscopy, respectively. RESULTS During the first two postnatal weeks, the number of proliferative cells gradually fell to zero. This decrease occurred in parallel with the impairment of the proliferative capacity of the cells and the accumulation of proliferative cells in G0/G1. In addition, some of the cells exited the cell cycle by means of gradual maturity and apoptosis. CONCLUSIONS Our study suggests that cochlear proliferative cells are remnants of the progenitor cells that originally gave rise to the sensory epithelium. The disappearance of the cochlear proliferative cells in adult mammalian cochleae may result from their differentiation and/or apoptosis.

Primary cholesteatoma of petrous bone presenting as cervical fistula

A unique case of primary cholesteatoma of the petrous bone is described. The patient presented with a cutaneous fistula of the anterior cervical triangle and was initially diagnosed and treated as sebaceous cyst or imflammatory lympha node for 3 months. Later examination showed white mass in the right external auditory canal and a severe conductive hearing loss. Radiological findings revealed a massive soft tissue consistent with cholesteatoma in the right petrous bone. The lesion was successfully managed with an endoscopy-assisted technique leaving the neurovascular structures in situ without operative complications. A review of the literature shows that only a few cases of cervical abscess associated with mastoid cholesteatoma have been reported. We also discussed the diagnosis, clinical features and surgical strategy for petrous bone cholesteatoma.

Phenotypic characterization of GPR120-expressing cells in the interstitial tissue of pancreas

GPR120 functions as a plasma membrane receptor for unsaturated long-chain free fatty acids and involves in GLP-1 secretion, adipogenesis and the control of energy balance. Pancreas is the key organ in fuel and energy metabolism. Here GPR120 expression in human and rat pancreas was observed by RT-PCR, and the distribution and phenotypes of GPR120-positive cells in human and rat pancreas were shown by immunohistochemical staining. GPR120 mRNA expression was found in human and rat pancreas. GPR120-positive cells were scattered mainly in the interstitial tissues of human and rat pancreas, and they were not co-localized with nestin, vimentin, alpha-SMA and glucagon, respectively. However, GPR120 was distributed on the cells positively stained by CD68, the specific marker of macrophages, and on the cells positive stained by CD34 and CD117, the markers of interstitial cells. In conclusion, this study demonstrates the expression of GPR120 in pancreas and shows the distribution of GPR120 in human and rat pancreas.

GW9508 inhibits insulin secretion by activating ATP-sensitive potassium channels in rat pancreatic β-cells

GW9508 is an agonist of G protein-coupled receptor 40 (GPR40) that is expressed in pancreatic β-cells and is reported to regulate insulin secretion. However, the effects of GW9508 on pancreatic β-cells in primary culture have not been well investigated. This study measured the acute effects of GW9508 on insulin secretion from rat pancreatic islets in primary culture, and the insulin secretion-related events such as the changes in membrane potential, ATP-sensitive potassium currents (KATP currents), and intracellular Ca(2+) concentrations ([Ca(2+)]i) of rat islet β-cells were also recorded. GW9508 (10-40 μM) did not influence basal insulin levels at 2 mM glucose, but it (above 20 μM) significantly inhibited 5 and 15 mM glucose-stimulated insulin secretion (GSIS). GW9508 did not inhibit insulin secretion stimulated by tolbutamide, the closer of KATP channels. GW9508 activated KATP channels and blocked the membrane depolarization and the increase in [Ca(2+)]i that were stimulated by glucose. GW9508 itself stimulated a transient increase in [Ca(2+)]i, which was fully blocked by depletion of intracellular Ca(2+) stores with thapsigargin or by inhibition of phospholipase C (PLC) activity with U73122. GW9508-induced activation of KATP channels was only partly inhibited by U73122 treatment. In conclusion, although it stimulates a transient release of Ca(2+) from intracellular Ca(2+) stores via activation of PLC, GW9508 inhibits GSIS by activating KATP channels probably in a distal step to GPR40 activation in rat β-cells.

Exposure to acoustic stimuli promotes the development and differentiation of neural stem cells from the cochlear nuclei through the clusterin pathway.

Stem cell therapy has attracted widespread attention for a number of diseases. Recently, neural stem cells (NSCs) from the cochlear nuclei have been identified, indicating a potential direction for the treatment of sensorineural hearing loss. Acoustic stimuli play an important role in the development of the auditory system. In this study, we aimed to determine whether acoustic stimuli induce NSC development and differentiation through the upregulation of clusterin (CLU) in NSCs isolated from the cochlear nuclei. To further clarify the underlying mechanisms involved in the development and differentiation of NSCs exposed to acoustic stimuli, we successfully constructed animal models in which was CLU silenced by an intraperitoneal injection of shRNA targeting CLI. As expected, the NSCs from rats treated with LV-CLU shRNA exhibited a lower proliferation ratio when exposed to an augmented acoustic environment (AAE). Furthermore, the inhibition of cell apoptosis induced by exposure to AAE was abrogated after silencing the expression of the CLU gene. During the differentiation of acoustic stimuli-exposed stem cells into neurons, the number of astrocytes was significantly reduced, as evidenced by the expression of the cell markers, microtubule associated protein-2 (MAP-2) and glial fibrillary acidic protein (GFAP), which was markedly inhibited when the CLU gene was silenced. Our results indicate that acoustic stimuli may induce the development and differentiation of NSCs from the cochlear nucleus mainly through the CLU pathway. Our study suggests that CLU may be a novel target for the treatment of sensorineural hearing loss.

Polyclonal antibodies to LIM proteins CRP2 and CRIP2 reveal their subcellular localizations in olfactory precursor cells.

In this study, we describe the presence of CRP2 (cysteine- and glycine-rich protein 2) and CRIP2 (cysteine-rich intestinal protein 2), which are members of group 2 LIM proteins, in rat olfactory precursor cells by reverse transcription polymerase chain reaction. We have developed polyclonal antibodies against CRP2 and CRIP2 individually. Specificity of the antibodies was demonstrated by Western blot analysis, using CRP2 and CRIP2 transfected cells. No cross-reactivity was observed between the antibodies. Furthermore, we used the antibodies to determine the expression and localization of CRP2 and CRIP2 in olfactory precursor cells by Western blot analysis and immunofluorescence staining. Our results demonstrated that in undifferentiated olfactory precursor cells CRP2 was distributed both in the nucleus and the cytoplasm, whereas CRIP2 was predominantly localized in the cytoplasm. While the olfactory precursor cells differentiated into end cells, only the expression of CRIP2 would be detected. The function of these LIM proteins in olfactory precursor cells warrants further study.

Infrared video patch-clamp technique for spiral ganglion neurons in rat cochlear slices

Conclusion: Cochlear slice and infrared video patch-clamp techniques can be used in real-time observation. They provide a good method and platform for further study of the electrophysiological properties and auditory transduction mechanism of spiral ganglion neuron (SGN). Objective: To establish the isolated rat cochlear slice technique combined with the infrared video patch-clamp technique to explore the electrophysiological properties of the SGN. Materials and methods: SD rats were divided into three groups according to postnatal days (0–2 days, 3–6 days and 7–14 days). After quickly making SD rat cochlear slices, the electrophysiological properties of the SGN were observed using the infrared differential interference contrast technique and patch-clamp recording, and the factors that affect the cochlear slice quality and patch-clamp recording were analysed. Results: The successful slice rate was highest in 3–6-day-old SD rats and two to four slices could be prepared using each cochlea. It is crucial to maintain the connection of partial skull with the cochlea and the volute integrity when producing slices. The position of the cochlear axis and blade, and the slice preparation time were also important factors affecting slice quality and cell activity. SGN cells in good condition could easily be found using the infrared video patch-clamp technique to help the assessment of the seal test process. SGN resting membrane potential of whole-cell recording was −45.6±5.3 mV (n=52) and the currents of Na+ and K+ could be recorded.