Culture of Primary Neurons from Dissociated and Cryopreserved Mouse Trigeminal Ganglion.

Corneal nerves originate from the ophthalmic branch of the trigeminal nerve, which enters the cornea at the limbus radially from all directions toward the central cornea. The cell bodies of the sensory neurons of trigeminal nerve are located in the trigeminal ganglion (TG), while the axons are extended into the three divisions, including ophthalmic branch that supplies corneal nerves. Study of primary neuronal cultures established from the TG fibers can therefore provide a knowledge basis for corneal nerve biology and potentially be developed as an in vitro platform for drug testing. However, setting up primary neuron cultures from animal TG has been dubious with inconsistency among laboratories due to a lack of efficient isolation protocol, resulting in low yield and heterogenous cultures. In this study, we used a combined enzymatic digestion with collagenase and TrypLE to dissociate mouse TG while preserving nerve cell viability. A subsequent discontinuous Percoll density gradient followed by mitotic inhibitor treatment effectively diminished the contamination of non-neuronal cells. Using this method, we reproducibly generated high yield and homogenous primary TG neuron cultures. Similar efficiency of nerve cell isolation and culture was further obtained for TG tissue cryopreserved for short (1 week) and long duration (3 months), compared to freshly isolated tissues. In conclusion, this optimized protocol shows a promising potential to standardize TG nerve culture and generate a high-quality corneal nerve model for drug testing and neurotoxicity studies.

An epidemiological analysis of Nocardia seriolae isolated from a wide range of aquatic animals in Taiwan, based on their genotype and enzymatic activity.

Chronic disease following Nocardia seriolae infection in a wide range of aquatic animals has been reported in many Asian countries and recently in America and Mexico. This study aimed to investigate the epidemiological relationship among N. seriolae isolates in Taiwan by investigating their genotype and enzymatic activities. A total of 66 strains isolated from 14 known and four unknown host fish from five sites in Taiwan were characterized using five combined methods. High genotypic diversity was recognized among the isolates with 10 pulsotypes being identified from the pulsed-field gel electrophoresis method and 21 reptypes from the repetitive extragenic palindromic amplification method; however, no natural plasmids were detected in this bacterial population. Pulsotypes A8 and RI analysed by PFGE and repPCR, respectively, were found to be predominant within five sites in Taiwan over 17 years of isolation. Enzymatically, the majority of isolates displayed high leucine arylamidase, ß-glucosidase and α-glucosidase activities but were negative for lipase, α-galactosidase, ß-glucuronidase, N-acetyl-glucosaminidase, α-mannosidase and α-fucosidase activities. We identified a strong association between genotype and enzymatic activity since the majority of pulsotypes displayed the same type of enzymatic profile. This study provides comprehensive and potential epidemiological data, which will aid the fish farming activities and prevention method development.

Correction: Exploration of biomimetic poly(γ-benzyl-L-glutamate) fibrous scaffolds for corneal nerve regeneration.

Correction for 'Exploration of biomimetic poly(γ-benzyl-L-glutamate) fibrous scaffolds for corneal nerve regeneration' by Tien-Li Ma et al., J. Mater. Chem. B, 2022, 10, 6372-6379, https://doi.org/10.1039/D2TB01250B.

Exploration of biomimetic poly(γ-benzyl-L-glutamate) fibrous scaffolds for corneal nerve regeneration.

Poly(γ-benzyl-L-glutamate) (PBG) made biomimetic scaffolds are explored as candidate materials for corneal nerve regeneration and neurotrophic keratopathy treatment. The PBG with built-in neurotransmitter glutamate was synthesized and fabricated into 3D fibrous scaffolds containing aligned fibers using electrospinning. In in vitro experiments, primary mouse trigeminal ganglia (TG) cells were used. Immunohistochemistry (IHC) analysis shows that TG cells cultured on PBG have no cytotoxic response for 21 days. Without any nerve growth factor, TG cells have the longest neurite length of 225.3 µm in the PBG group and 1.3 times the average length as compared with the polycaprolactone and no scaffold groups. Also, aligned fibers guide the neurite growth and extension unidirectionally. In vivo assays were carried out by intracorneal implantation of PBG on clinical New Zealand rabbits. The external eye photos and in vivo confocal microscopy (IVCM) show a low immune response. The corneal neural markers (ßIII tubulin and SMI312) in the IHC analysis are consistent with the position stained by glutamate of implanted scaffolds, indicating that PBG induces neurogenesis. PBG exhibits mechanical stiffness to resist material deformation possibly caused by surgical operations. The results of this study demonstrate that PBG is suitable for corneal nerve regeneration and the treatment of neurotrophic keratopathy.

Phenotypic and genotypic analysis of Edwardsiella isolates from Taiwan indicates wide variation with a particular reference to Edwardsiella tarda and Edwardsiella anguillarum.

Edwardsiella spp. is a gram-negative, facultatively anaerobic, intracellular bacteria threatening the aquaculture industry worldwide. Noticeably, E. tarda is now genotypically classified into three distinct groups (E. tarda, E. piscicida and E. anguillarum), but morphologically, it is unclear due to varying degrees of virulence in different fish hosts. Hence, to reclassify E. tarda, we investigated differences in genotypes, phenotypes and pathogenicity. We collected Edwardsiella isolates from five different counties of Taiwan between 2017 and 2021. At first, gyrB gene was amplified for a phylogenetic tree from 40 isolates from different fish and one reference isolate, BCRC10670, from the human. Thirty-nine strains clustered into E. anguillarum, 1 strain into E. piscicida and 1 strain into E. tarda from human strain. Second, all isolates were characterized using various phenotypic (API 20E biochemical profiles) and genotypic (pulsed-field gel electrophoresis [PFGE], and virulence-related gene detection). SpeI digestion revealed 10 pulsotypes and I-CeuI into 7 pulsotypes. Virulent genes (citC, gadB, katB, mukF and fimA) confirmed in 35, 31, 28, 37 and 38 isolates, respectively. Finally, in vivo challenge test in milkfish (Chanos chanos) indicated the highest mortality from E. anguillarum. Overall, results revealed unique features with Edwardsiella spp. genotypes and pathogenicity, which are relevant to the host and provide useful insights for future vaccine development.

Effect of storage temperature and time on erythrocyte sedimentation rate.

OBJECTIVE: This paper explores the effect of blood sample storage temperature and time on the erythrocyte sedimentation rate (ESR) by using the Weiss method. METHODS: Whole blood samples were collected from 80 patients and diluted 1:9 with sodium citrate solution. Each sample was split into two tubes. Using the Weiss method, ESR was tested within 1 h of collection, and one sample was placed at 4 °C and the other at room temperature (23 ± 2 °C). ESR was then measured at 2, 4, 6, 8, 12, and 24 h. The data were statistically analyzed with consideration for temperature and time. RESULTS: ESR decreased gradually over 6 h at room temperature, but the results were not statistically significant. Similarly, there was no significant difference in the decline of ESR within 8 h at 4 °C. However, ESR results decreased significantly after the samples were stored at room temperature for more than 6 h or at 4 °C for more than 8 h. ESR reduction was lower in the samples stored at 4 °C than in those stored at room temperature over the same time period. CONCLUSION: Blood sample storage temperature and duration can affect the measurement of ESR using the Weiss method. ESR testing should be completed within 4 h of sample collection in clinical work.

[Effect of acupuncture for dysphagia after stroke based on fiberoptic endoscopic swallowing function evaluation].

OBJECTIVE: To observe the effect of acupuncture combined with regular treatment and swallowing function training on pharyngeal motor, sensory function and penetration-aspiration function in patients with dysphagia after stroke. METHODS: A total of 60 patients with dysphagia after stroke were randomly divided into a control group and an observation group, 30 patients in each group. Both groups were treated with conventional treatment and swallowing function training; in addition, the observation group was treated with acupuncture at Lianquan (CV 23), Fengfu (GV 16), Yifeng (TE 17). All the treatments were given once a day, 5 days a week, for totally 4 weeks. In the two groups, the pharyngeal motor and sensory function, penetration-aspiration scores were evaluated by fiberoptic endoscopic evaluation of swallowing (FEES), and the Kubota water swallowing test scores were assessed before and after treatment, and the clinical effects were compared. RESULTS: After treatment, the pharyngeal motor and sensory function in the two groups were all higher than those before treatment (P<0.05), and those in the observation group were better than the control group (P<0.05). After treatment, the penetration-aspiration scores and Kubota water swallowing test scores in the two groups were all lower than those before treatment (P<0.05), and those in the observation group were lower than the control group (P<0.05). The total effective rate was 93.3% (28/30) in the observation group, which was better than 73.3% (22/30) in the control group (P<0.05). CONCLUSION: Acupuncture combined with regular treatment and swallowing training could improve the pharyngeal motor and sensory function, and penetration-aspiration scores in patients with dysphagia after stroke.

Genotyping and phenotyping of Lactococcus garvieae isolates from fish by pulse-field gel electrophoresis (PFGE) and electron microscopy indicate geographical and capsular variations.

Lactococcus garvieae is the etiological agent of Lactococcosis, an evolving disease affecting many fish species and causing significant economic losses worldwide. Assessing pathogen relatedness and bacterial population structure is critical for determining the epidemiology of L. garvieae infections and in establishing effective pathogen management methods. The previously published morphological and genetic studies point to a clonal population structure, as seen in other fish bacteria. In the present study, the pulsed-field gel electrophoresis (PFGE) method was utilized to define a population of 41 Taiwanese isolates from outbreaks with comparisons to four well-characterized non-Taiwanese isolates previously published. Two restriction enzymes (ApaI and SmaI) were utilized individually for PFGE analysis (cut-off value = 90.0%), revealing genetic heterogeneity across L. garvieae isolates, with ApaI and SmaI yielding 12 and seven distinct PFGE band patterns, respectively. The phylogenic analysis using internal transcribed spacer region clustered all L. garvieae isolates in the same clad. Furthermore, the electron microscopic results confirmed the absence of capsular gene cluster (CGC) in previously characterized Taiwanese vaccine strain (S3) from grey mullet. Overall, our findings emphasize the importance of analysing the morphological and genetic diversity in L. garvieae being correlated for proper taxonomic classification in vaccine strain selection and epidemiological studies.

Melatonin ameliorates bupivacaine-induced spinal neurotoxicity in rats by suppressing neuronal NLRP3 inflammasome activation.

Bupivacaine is a common local anesthetic that causes neurotoxicity when used at clinical concentrations. Melatonin (MT), is a potent neuroprotective molecule. The study aimed to characterize the neuroprotective effects of MT on spinal neurotoxicity induced by bupivacaine in rats. It showed that bupivacaine, by intrathecal injection, induced spinal injury, and that the protein levels of Nod-like receptor protein 3 (NLRP3), cleaved caspase-1, and the N-terminal region of gasdermin D (GSDMD-N) were significantly increased. NLRP3 was expressed mainly in neurons and microglia. MT treatment ameliorated bupivacaine-induced spinal cord injury in rats by suppressing activation of neuronal NLRP3 inflammasomes.

The effect of human platelet lysate on corneal nerve regeneration.

AIM: This study aimed to test whether human platelet lysate (HPL) has neurotrophic ability for corneal nerve regeneration. METHODS: We measured the neurotrophic factors in human peripheral serum (HPS) and two commercially available HPLs, UltraGRO and PLTMax. In vitro, we compared the growth rates, neuronal differentiation and immunostaining of neuron markers in mouse neuroblastoma cell line (Neuro-2a) and primary culture of mouse trigeminal ganglion cells that were cultivated in different concentrations of fetal bovine serum, HPS and HPL. In vivo, we created corneal wounds on Sprague Dawley rats with a rotating burr and evaluated the effects of topical HPL on wound healing and corneal nerve regeneration by in vivo confocal microscopy and corneal aesthesiometry. RESULTS: HPLs had significantly higher concentrations of various neurotrophic factors compared with HPS (p<0.05). In Neuro-2a cells, 3% HPL was better at promoting neuronal growth and differentiation compared with HPS at the same concentration. HPL was also found to have superior neurotrophic effects compared with HPS in primary cultures of mouse trigeminal ganglion cells. In vivo, HPL-treated eyes had better corneal epithelial wound healing rate, nerve regeneration length and corneal touch threshold compared with eyes treated with artificial tears (p<0.05). CONCLUSION: HPL has significantly higher concentrations of neurotrophic factors compared with HPS. It showed not only in vitro but also in vivo corneal neurotrophic abilities. Our results suggest that HPL may have a potential role in the treatment of diseases related to corneal nerve damage or degeneration.

Effect of air-lifting on the stemness, junctional protein formation, and cytokeratin expression of in vitro cultivated limbal epithelial cell sheets.

PURPOSE: The aim of this study is to evaluate the effects of air-lifting on the stemness, junctional protein formation, and cytokeratin expression of rabbit limbal stem cells cultivated in vitro, and to find out the proper timing of air-lifting before transplantation as limbal epithelial cell sheets for the treatment of limbal insufficiency. MATERIALS AND METHODS: Limbal epithelial cells were isolated from the limbus of New Zealand white rabbits and cultivated in vitro. After the cells became confluent, different durations of air-lifting (0, 1, 2, 4, and 7 days) were performed. At the end of cultivation, immunohistochemistry on cryosections was performed and observed by fluorescein microscopy and in vitro confocal microscopy for cytokeratins (K3, K10, K12, K13, and K14), junctional and structural proteins (ZO-1, p120, and actin) and stem cell markers (ABCG2 and P63). Scanning electron microscopy was used for observing the microstructure of superficial cells. Transepithelial electrical resistance (TEER) was used to measure the transepithelial permeability. RESULTS: The expression of K3, K10, K12, K13, K14, and ABCG2 showed no differences in pattern and location among different groups of airlifting. A time-dependent increase in corneal epithelial thickness was found after air-lifting. In vitro confocal microscopy demonstrated that K3, p120, and ZO-1 were expressed on the apical cell layer, whereas P63 and ABCG2 were expressed more on the basal epithelial layer. Scanning electron microscopy of the superficial layer demonstrated that airlifting induced time-dependent increase in the size of surface epithelial cells and triggered cellular differentiation. TEER results demonstrated a time-dependent increase of transepithelial electric resistance. CONCLUSIONS: During limbal epithelial cell expansion in vitro, air-lifting can increase cellular stratification, enlarge surface cells, trigger cellular differentiation, and increase the transepithelial barrier. However, the expression of cellular junctional, stem cell and cytokeratin markers seems to have no obvious differences in pattern and localization.

A new isolation method of human limbal progenitor cells by maintaining close association with their niche cells.

In human corneal epithelium, self-renewal and fate decision of stem cells are highly regulated in a niche microenvironment called palisades of Vogt in the limbus. Herein, we discovered that digestion with dispase, which cleaves off the basement membrane, did not remove the entire basal epithelial progenitor cells. In contrast, digestion with collagenase isolated on cluster consisting of not only entire epithelial progenitor cells but also their closely associated mesenchymal cells because of better preservation of some basement membrane matrix. Collagenase isolated more basal epithelial progenitor cells, which were p63α+ and small in the size (8 µm in diameter), and generated significantly more holoclones and meroclones on 3T3 fibroblast feeder layers than dispase. Further, collagenase isolated more small pan-cytokeratin-/p63α-/vimentin+ cells with the size as small as 5 µm in diameter and heterogeneously expressing vimentin, Oct4, Sox2, Nanog, Rex1, Nestin, N-cadherin, SSEA4, and CD34. Maintenance of close association between them led to clonal growth in a serum-free, low-calcium medium, whereas disruption of such association by trypsin/EDTA resulted in no clonal growth unless cocultured with 3T3 fibroblast feeder layers. Similarly, on epithelially denuded amniotic membrane, maintenance of such association led to consistent and robust epithelial outgrowth, which was also abolished by trypsin/EDTA. Epithelial outgrowth generated by collagenase-isolated clusters was significantly larger in diameter and its single cells yielded more holoclones on 3T3 fibroblast feeder layers than that from dispase-isolated sheets. This new isolation method can be used for exploring how limbal epithelial stem cells are regulated by their native niche cells.

Cholesterol deficiency perturbs actin signaling and glutamate homeostasis in hippocampal astrocytes.

This study was undertaken to explore the possibility that cholesterol deficiency may perturb the physiological functions of astrocytes, thus rendering cells vulnerable to the cytotoxicity induced by glutamate (Glu). Cholesterol deprivation induces astrocyte stellation, which is accompanied by disruption of cortical actin, and phosphorylation of extracellular signal-regulated kinase (ERK) in an astrocyte-specific manner. Moreover, cholesterol reduction decreases the activity of glutamine synthetase (GS) while enhancing the capacity of Glu transporter. Using [(3)H]d-aspartate as a tracer, we found a marked efflux of [(3)H]d-aspartate from cholesterol-deficient astrocytes after Glu stimulation. Changes in the actin cytoskeleton, cell morphology, ERK phosphorylation and GS level gradually recovered in astrocytes after the withdrawal of cholesterol depletion. Moreover, withdrawal of cholesterol deprivation attenuated cell loss in cholesterol-deficient astrocytes during Glu exposure. Taken together, our data suggest that, upon Glu exposure, there would be an increase in intracellular Glu as a consequence of enhanced Glu uptake and reduced degradation of Glu by GS in cholesterol-deficient astrocytes. This in turn leads to a concentration gradient favoring Glu release, thereby causing the accumulation of cytotoxic levels of Glu extracellularly. It is thus concluded that the detrimental effect of cholesterol deprivation may, in part, arise from the impairment in Glu homeostasis.