Protective effects of Slit2 on corneal epithelium and nerves in diabetic mice and its mechanism / 中华实验眼科杂志

Objective:To explore the promoting effects of slit guidance ligand 2 (Slit2) on the repair of corneal epithelium and nerve damage in diabetic mice and possible molecular mechanism.Methods:Sixty SPF C57BL/6 mice aged 5-6 weeks were divided into normal control group, diabetes model group and Slit2 injection group according to the random number table method, 20 for each group.Diabectic model was prepared by intraperitoneal injection of streptozotocin in the diabetes model group and Slit2 injection group.A mouse corneal epithelial injury repair model was established using electric epithelial scraper, and Slit2 recombinant protein was subconjunctivally injected immediately following modeling in the Slit2 injection group.The equal volume of phosphate buffer saline (PBS) was used in a same way in the diabetes model group.No intervention was performed in the normal control group.Corneal epithelial healing were examined at 24, 48 and 72 hours after corneal epithelial defect by corneal fluorescin staining.Real-time fluorescent quantitative PCR was used to detect the expression of Slit2 and its related receptors in the corneal epithelium of normal and diabetic model mice.Fluorescence staining of corneal wholemount with β-tubulin Ⅲ was used to observe the changes in corneal nerve morphology.Immunofluorescence staining was performed to detect the expression and distribution of Slit2 in mouse corneal epithelium in normal control group and diabetes model group, as well as the expression and distribution of Slit2, epidermal growth factor receptor (EGFR), extracellular-signal-regulated kinase (ERK), threonine protein kinase (AKT), β-catenin and Ki67 in the healing corneal epithelium of mice after corneal epithelium damage in different groups.The mouse corneal epithelial stem/progenitor cell line (TKE2) was divided into normal control group, high-glucose group and Slit2 treatment group.Western blot was performed to detect the expression of p-EGFR/EGFR and p-AKT/AKT in the TKE2 of the three groups.The expression of p-EGFR/EGFR and p-AKT/AKT in high glucose-cultured TKE2 with 0.01, 0.1 and 0.5 μg/ml Slit2 treatment for 10 minutes, and before and 10, 20, 30, 60, 120 minutes after 0.5 μg/ml Slit2 treatment was detected by Western blot.The effects of Slit2 on the axon regeneration of mouse trigeminal ganglion cells (TGs) were observed by immunofluorescence staining.The use and care of animals complied with the ARVO statement.This study protocol was approved by an Ethics Committee of Qingdao Eye Hospital of Shandong First Medical University (No.[2020]57).Results:At 48 and 72 hours after corneal epithelial scraping, the speed of corneal epithelial repair was significantly slowed down in diabetes model group in comparison with the normal control group and Slit2 injection group.The relative expression levels of Slit2 and its receptors Robo1, Robo2 and Robo4 mRNA in the normal corneal epithelium in the diabetes model group were significantly higher than those of the normal control group (all at P<0.05). The fluorescence intensity of Slit2 in normal corneal epithelium in diabetes model group was similar to the normal control group, and the fluorescence intensity of Slit2 in damaged corneal epithelium in diabetic mice was significantly weaker than that in normal control group.Corneal nerve plexus was denser at 7 days after corneal epithelial injury and the nerve fibers were increased with more branches in Slit2 injection group compared with diabetic group.The fluorescence intensity of p-EGFR, p-ERK, β-catenin and Ki67 in damaged corneal epithelium in normal control group and Slit2 injection group was stronger than that of the diabetes model group.The relative expression levels of p-EGFR/EGFR, p-AKT/AKT, and β-catenin in TKE2 in high-glucose group were significantly lower than those in normal control group and Slit2 treatment group (all at P<0.05). The relative expression levels of p-EGFR/EGFR and p-AKT/AKT in high glucose-cultured TKE2 after Slit2 treatment were significantly increased in comparison with before Slit2 treatment (both at P<0.05), and the relative expression levels of p-EGFR/EGFR and p-AKT/AKT in TKE2 were elevated as the increase of Slit2 concentration.The activation effect of 0.5 μg/ml Slit2 on EGFR and AKT pathways was most obvious.The synapse length of TGs cultured by high glucose was (40.52±5.44) μm, which was significantly shortened than (72.14±9.48) μm in normal control group and (73.04±4.66) μm in Slit2 injection group (both at P<0.05). Conclusions:Slit2 can protect the corneal epithelium by activating EGFR signaling pathway and play a protective role to neurons by increasing the density of corneal subepithelial plexus and promoting the growth of TGs axons in diabetic mice.

Mitochondrial Reactive Oxygen Species Elicit Acute and Chronic Itch via Transient Receptor Potential Canonical 3 Activation in Mice / 神经科学通报·英文版

Mitochondrial reactive oxygen species (mROS) that are overproduced by mitochondrial dysfunction are linked to pathological conditions including sensory abnormalities. Here, we explored whether mROS overproduction induces itch through transient receptor potential canonical 3 (TRPC3), which is sensitive to ROS. Intradermal injection of antimycin A (AA), a selective inhibitor of mitochondrial electron transport chain complex III for mROS overproduction, produced robust scratching behavior in naïve mice, which was suppressed by MitoTEMPO, a mitochondria-selective ROS scavenger, and Pyr10, a TRPC3-specific blocker, but not by blockers of TRPA1 or TRPV1. AA activated subsets of trigeminal ganglion neurons and also induced inward currents, which were blocked by MitoTEMPO and Pyr10. Besides, dry skin-induced chronic scratching was relieved by MitoTEMPO and Pyr10, and also by resveratrol, an antioxidant. Taken together, our results suggest that mROS elicit itch through TRPC3, which may underlie chronic itch, representing a potential therapeutic target for chronic itch.

Infecção latente pelo herpesvírus bovino tipo 1 em búfalos (Bubalus bubalis) no Rio Grande do Sul / Bovine herpesvirus type 1 latent infection in buffalo (Bubalus bubalis) in Rio Grande do Sul

Apesar dos bovinos serem considerados os hospedeiros naturais do BoHV-1, estudos sorológicos têm sugerido que búfalos podem ser suscetíveis ao BoHV-1 e a outros alfa-herpesvírus geneticamente relacionados. O objetivo deste estudo foi detectar a presença de DNA viral de BoHV-1 em 202 amostras de gânglios trigêmeos de búfalos, pela técnica de semi-nested PCR, para detecção de um segmento do gene codificante da glicoproteína D (gD) do BoHV-1. Além disso, 242 amostras de soro foram analisadas pela técnica de soroneutralização (SN) para a detecção de anticorpos neutralizantes contra BoHV-1, BoHV-5 e BuHV. Todas as amostras clínicas foram coletadas em um matadouro na cidade de Pelotas, RS, Brasil. O DNA de BoHV-1 foi detectado em 61 (30,1%) gânglios, e os resultados da SN demonstraram que 27,6% dos animais apresentaram anticorpos contra, pelo menos, um dos vírus testados. O sequenciamento genômico e a análise de 14 amplicons confirmaram a presença do DNA do BoHV-1 nos tecidos analisados. Em resumo, os resultados indicam que o BoHV-1 está distribuído em rebanhos bubalinos provenientes da região Sul do Brasil. Entretanto, são necessárias investigações adicionais, no sentido de elucidar o papel exato dos búfalos na epidemiologia das infecções pelo BoHV-1.(AU)

Although bovines are natural hosts for BoHV-1, serologic studies in several countries have suggested that buffaloes (Bubalus bubalis) may be susceptible to BoHV-1 and other genetically related alphaherpesvirus. This study aimed to investigate the presence of BoHV-1 DNA in trigeminal ganglia from 202 buffaloes by a semi-nested PCR to amplify partially the glycoprotein D (gD) gene of BoHV-1. Additionally, 242 serum samples were tested by serum neutralization (SN) for the detection of antibodies against BoHV-1, BoHV-5 and BuHV. All clinical samples were collected in a slaughterhouse located in Pelotas, RS, Brazil. BoHV-1 DNA was detected in 61 (30.1%) of the samples and SN revealed 27.6% of the animals with neutralizing antibodies against at least one of the tested viruses. Nucleotide sequencing of 15 amplicons followed by BLAST analysis confirmed the presence of BoHV-1 DNA in the analyzed tissues. Taken together, these data indicate that BoHV-1 infection is distributed in buffaloes in southern Brazil. However, the role of buffaloes in the BoHV-1 epidemiology needs further investigation.(AU)

Nimodipine down-regulates CGRP expression in the rat trigeminal nucleus caudalis.

L-type calcium channel blockers like verapamil are used in the prophylaxis of migraine. However, their effect on the expression of CGRP in the trigeminal nucleus caudalis (TNC) is unknown. It is important because an earlier study had shown that olcegepant, a CGRP receptor antagonist, acts at the level of the trigeminal spinal nucleus rather than the trigeminal ganglia. Nimodipine was used in the present study as it crosses the blood-brain barrier. The objective of the study was to determine the pattern of expression of calcitonin gene-related peptide (CGRP) in the TNC after administration of nimodipine and/or morphine. Wistar rats were injected with saline, morphine, nimodipine or morphine + nimodipine for 14 days. Subsequently, the lowest part of the medulla oblongata containing the spinal nucleus was removed and processed for immunohistochemical localization of CGRP. The density of expression was quantified using Image J software. The results were statistically analyzed. CGRP expression was noted over the superficial part of the TNC, which decreased significantly after nimodipine administration. Conversely, morphine produced an up-regulation. The expression was unchanged with reference to saline in the morphine + nimodipine treated group. Decreased expression of CGRP in the trigeminal nucleus caudalis after nimodipine is being reported for the first time. Also, whether CGRP expression can be used as a marker for predicting the therapeutic efficacy of an anti-migraine drug is currently being investigated.

Detección de la presencia de antígeno y ADN de virus herpes simple tipo 1 en ganglios trigeminales humanos / Viral DNA and antigen detection of type 1 herpes simplex virus in human trigeminal ganglia

Antecedentes: la infección por virus herpes simple tipo 1 (VHS-1) es una de las más frecuentesen la población humana; produce infecciones en mucosa oral, piel, ojos e inclusoen el sistema nervioso, que causa encefalitis. Después de la infección en la región orofacial,este virus puede permanecer en estado de latencia en el ganglio trigémino y eventualmentereactivarse. Objetivo: determinar la presencia de VHS-1 en ganglios trigeminales humanosmediante pruebas paralelas de PCR, RT-PCR e inmunohistoquímica. Métodos: previa aprobacióndel Comité de Ética de la Facultad de Odontología de la Universidad Nacional deColombia y del Instituto de Medicina Legal y Ciencias Forenses, se recolectaron dieciséispares de ganglios trigeminales humanos, que se procesaron tanto para extracción de ácidosnucleicos como para inmunohistoquímica. Resultados: en seis de los ocho donantesanalizados por inmunohistoquímica se encontró marcaje positivo para antígeno de VHS-1.Se halló que nueve de los donantes evaluados por PCR para VHS-1 y cinco de los diezexaminados para transcritos asociados a latencia (LAT) fueron positivos. Conclusión: seencontraron ganglios trigeminales en los que no se detectó virus y otros con distintosestados de infección (activa y latente). En casi todos los ganglios fue evidente el infiltradoinflamatorio asociado. El presente es el primer trabajo en el que se busca sistemáticamentetanto genoma viral como proteínas y transcritos LAT en ganglios trigeminales humanos, locual abre puertas para la investigación tanto de la epidemiología como de los fenómenosasociados a la LAT y reactivación del VHS-1...

Background: Infection by type 1 Herpes Simplex Virus (HSV-1) is the most frequent viralinfection in human population being able to cause injuries in oral mucosa, skin, cornea,and even the central nervous system causing encephalitis. After mucosal infection, HSV-1establishes a lifespan latent infection in trigeminal ganglia where it occasionally reactivatesinfecting primary sites again. It is little known about cell and molecular events responsiblefor infection reactivation and immune response in human ganglia. Objective: To standardizethe obtaining and processing of human trigeminal ganglia to detect specific HSVantigen, DNA and RNA. Methods: After approval of the study by the Universidad NacionalIRB, 32 trigeminal ganglia were obtained from 16 cadavers from the Colombian ForensicMedicine Institute. Results: Using PCR technique to detect viral DNA, it was found that 56.3 %of ganglia (9/16) amplified specific fragment and five out of ten with suitable quality RNAwere positive for latency associated transcript. Conclusion: Some trigeminal ganglia did notshow evidence of infection and some had different HSV-1 infection status (active or latent)with inflammatory cells infiltrate in almost all samples. This is the first work that detectssimultaneously genome, proteins and LAT of HSV-1 in human trigeminal ganglia, leading toexplore findings about the latency and r eactivation pr ocess...

Gabapentin Attenuates the Activation of Transient Receptor Potential A1 by Cinnamaldehyde

Gabapentin is used as an effective drug for relieving pain, but the main mechanism is still unclear. Recently, voltage-gated Ca2+ channel subunits are suggested for the main target for the analgesic action of gabapentin. We wonder whether gabapentin directly modulates other specific ion channels peripherally expressed in the sensory neurons. To test this, we used a heterologous expression system in which the cell lines transiently expressed thermosensitive transient receptor potential ion channels (thermoTRPs) as well as the primary cultured mouse trigeminal neurons. The application of gabapentin reduced the increases in the intracellular Ca2+ level caused by TRPA1 activation in the heterologous expression system whereas the responses via actions of other thermoTRPs were not dramatically affected by the gabapentin treatment. Gabapentin also attenuated the TRPA1-mediated intracellular Ca2+ increases in the cultured trigeminal neurons. These findings suggest TRPA1 in the peripheral sensory neurons as a novel target for the analgesic of gabapentin.