Evaluation of effects of riboflavin and/or ultraviolet-A on survival of rat limbal epithelial stem cells in ex-vivo

Purpose: To investigate the effects of riboflavin and/or ultraviolet?A (UV?A) irradiation on the cell viability of ex?vivo?cultured rat limbal stem cells (LSCs). Methods: LSCs of male Wistar rats (N = 12 eyes) were cultured, and immunofluorescence staining was performed to evaluate them. After characterization, these cells were assigned to four groups of control (C), a group that was exposed to UV?A radiation (UV), a group that was treated with riboflavin (R), and a group that cotreated with both UV?A and riboflavin (UV+R). To determine the cell viability of LSCs, these cells were subjected to MTT assay on days 1, 3, and 7 after exposure to UV?A and/or riboflavin. The duration of exposure to UV?A and riboflavin was similar to levels used during the conventional corneal collagen cross?linking procedure. Results: Compared with the viable cells in the control group, there was a significant decrease (P < 0.0001) in the number of LSCs in the UV group during all study days. In the R group, the level of viable LSCs was as same as the level of viable LSCs in the C group. Combined treatment with UV?A plus riboflavin significantly decreased the survival of LSCs on days 1 and 3 (P < 0.0001, P < 0.001, respectively) compared with the control group. Interestingly, in the UV+R group, the photosensitizing effect of riboflavin significantly decreased the cytotoxic effect of UV irradiation 7 days after exposure. Conclusion: These results suggest that the administered UV energy in the presence or absence of riboflavin can damage LSCs. Likewise, riboflavin could decrease the toxic effect of UVA on LSCs

Conjunctival collagen crosslinking for the management of bleb leak

The purpose of this article is to evaluate the effectiveness of collagen crosslinking of filtering blebs with 0.1% riboflavin stimulated with ultraviolet A radiation in a case of a thin-walled filtering bleb with leakage following an episode of late blebitis. The time to cessation of the bleb leakage was studied, as well as the intraocular pressure, the visual acuity, and the presence of adverse effects during 4 years of follow-up after treatment. A single session of crosslinking was effective in resolving the leakage of the filtering bleb. There were no adverse effects. The intraocular pressure and the visual acuity remained stable after 4 years of follow-up. Collagen crosslinking with 0.1% riboflavin stimulated with ultraviolet A radiation may prove to be a simple, painless, non-invasive, and repeatable treatment of the leaking bleb with the aim of strengthening the collagen fibers of the conjunctiva and avoiding more invasive surgical treatments.

Characteristics of corneal stromal demarcation line after different cross-linking methods in keratoconus and its influence on the cross-linking effect / 中华实验眼科杂志

Objective:To compare the characteristics of corneal stromal demarcation line after different surgical methods of riboflavin/ultraviolet A corneal collagen cross-linking (CXL) in early keratoconus, and analyze the influence of the demarcation line on the cross-linking effect.Methods:A non-randomized controlled clinical study was conducted.Sixty-nine eyes of 69 patients treated with riboflavin/ultraviolet A CXL in the Eye Hospital of Shandong First Medical University from May 2019 to February 2021 were included.According to the cross-linking methods, the patients were divided into epithelium-on treatment group (21 eyes) and epithelium-off treatment group (48 eyes). There were 25 eyes in 5.4 J energy group and 44 eyes in 7.2 J energy group.The morphology and changes of corneal stromal cross-linking reaction (corneal stromal demarcation line) were observed at 2 weeks, 1, 3 and 4 months after operation.Changes in the thinnest corneal thickness (TCT), uncorrected visual acuity (UCVA, LogMAR), best corrected visual acuity (BCVA, LogMAR) and corneal maximum curvature (Kmax) were recorded.This study adhered to the Declaration of Helsinki.The study protocol was approved by the Ethics Committee of Eye Hospital of Shandong First Medical University (No.2019.05). Written informed consent was obtained from each subject.Results:Of the 69 eyes after operation, 44 eyes (63.77%) had demarcation lines, and 25 eyes (36.23%) had no demarcation lines.The occurrence rate of demarcation lines in the epithelium-on treatment group was 79.17%(38/48), which was significantly higher than 28.57%(6/21) in the epithelium-off treatment group ( χ2=16.186, P<0.01). The occurrence rate of demarcation line in 5.4 J energy group was 72.00%(18/25), and the 7.2 J energy group was 56.80%(25/44), with no significant difference ( χ2=1.565, P=0.302). Slit lamp microscopy and anterior segment-optical coherence tomography showed that the demarcation line appeared at 1-2 weeks after operation, gradually converged and strengthened after 1 month, turned diffuse, blurred and faded by degrees after 2-3 months, and basically disappeared after 4 months.The depth of the demarcation line reached 141-423 μm, with an average depth of (263.44±84.22)μm.Scanning laser confocal microscopy showed that corneal stromal cells were activated and light reflection was enhanced after CXL.Collagen fibers extended vertically and horizontally, crisscrossed, and were in a reticular arrangement.The TCT decreased from preoperative (458.69±38.28)μm to (443.86±36.54)μm at 4 months after operation, showing a statistically significant difference ( t=6.705, P<0.001). There was no significant difference in the TCT reduction between groups with and without demarcation lines ( t=1.684, P=0.100). At 4 months postoperatively, the UCVA of all eyes increased from preoperative 0.74±0.37 to 0.69±0.38, and the difference was statistically significant ( t=2.109, P=0.039). There was no significant difference in BCVA between before and after operation ( t=1.006, P=0.319). There was no significant difference in change of UCVA and BCVA between groups with and without demarcation lines ( t=0.065, P=0.949; t=0.346, P=0.730). There was no significant difference in Kmax in all patients between before and after operation ( t=0.050, P=0.950). There was no significant difference in the Kmax change between groups with and without demarcation lines ( t=-0.739, P=0.464). The change in TCT in the epithelium-off treatment group was significantly greater than that in the epithelium-on treatment group ( t=2.815, P=0.008). There was no significant difference in UCVA, BCVA and Kmax changes between epithelium-on and epithelium-off treatment groups (all at P>0.05). There was no obvious corneal scarring, infectious keratitis, corneal endothelial decompensation or other complications. Conclusions:The demarcation line after CXL may be a sign of the depth of cross-linking reaction, which is more prone to occur after the epithelium-off operation method.Both the epithelium-on and epithelium-off operation methods have similar therapeutic effects.Demarcation line after different cross-linking methods has no significant influence on the cross-linking effect in keratoconus.

B2 or Not B2: A Systematic Literature Review on Migraine Headaches and Riboflavin Deficiency / B2 ou não B2: uma revisão sistemática da literatura sobre enxaquecas e deficiência de riboflavina

Objective: To assess the prevalence of riboflavin deficiency in patients with migraines or headaches.Methods: Systematic review of the literature following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. Results: Two articles met the inclusion and exclusion criteria, and neither of these discussed riboflavin deficiency prevalence in those patients who suffer from migraines. Conclusion: The prevalence of riboflavin deficiency in migraineurs and in headache patients is unknown.

Objetivo: Avaliar a prevalência de deficiência de riboflavina em pacientes com enxaquecas ou dores de cabeça. Métodos :Revisão sistemática da literatura seguindo as diretrizes Preferred Reporting Items for Systematic Reviews and Meta-Analyses. Resultados: Dois artigos preencheram os critérios de inclusão e exclusão, e nenhum deles discutiu a prevalência de deficiência de riboflavina em pacientes que sofrem de enxaqueca. Conclusão: A prevalência da deficiência de riboflavina em pacientes com enxaqueca e com dor de cabeça é desconhecida.

Reassessment of the Clinical Significance of the Demarcation Line in the Corneal Stroma in Crosslinking

To consider the clinical significance and features of the formation of a demarcation line in the corneal stroma with various methods of corneal crosslinking.Materials and Methods: Literature data on the evaluation of the effectiveness of various methods of crosslinking by the demarcation line in the corneal stroma were analyzed. The formation of a demarcation line during prophylactic and therapeutic excimer laser crosslinking was studied after various photorefractive operations, keratoconus and other pathologies of the cornea (168 operations).Results: With various methods of crosslinking, including prophylactic and therapeutic excimer laser corneal crosslinking, the depth of the demarcation line in the stroma varied from 1/3 to 2/3 of the corneal thickness. It was noted that the saturation of the corneal stroma with a 0.25% solution of riboflavin is accompanied by a large increasing effect of the optical density in the stroma above the demarcation line at a shallower depth of its occurrence. The severity of the aseptic inflammatory reaction after corneal crosslinking affected the optical density, shape, intensity, and depth of the demarcation line in the stroma. In some cases, the formation of a demarcation line in the stroma was noted when the stroma was saturated with riboflavin immediately after refractive keratoablation without additional UV irradiation. The demarcation line in the stroma was revealed during inflammatory processes in the corneal stroma without the participation of riboflavin and its activation by UV radiation. The study showed that the assessment of corneal crosslinking by the depth of the demarcation line is not an indicator of the photochemical process and the number of crosslinks formed in the corneal stroma.Conclusion: Based on the severity, shape and depth of the demarcation line, it is not possible to judge the density of crosslinks in the corneal stroma, which predetermine its strength properties after one or another method of corneal crosslinking.

Bio prospecting of Riboflavin producing bacteria from different riboflavin enriched food sources

Riboflavin is an essential, water-soluble vitamin (B2) and a component of basic cellular metabolism. The aim of the present study is to isolate and characterize riboflavin producing bacteria from different food sources. Ten different riboflavin enriched food sources were collected from Vellore district. Totally 72 bacterial strains were isolated and cultured on nutrient agar plates. Out of these, 43 strains were identified as riboflavin producers. Isolated bacterial strains HDS27, HDS07, HDS14, HDS18, HDS38 and HDS54 isolated from milk, mushroom, spinach, lamb kidney, beef liver and mackerel fish were found to be potent riboflavin producers. Based on morphological, biochemical and molecular characterization, the potent strains were identified as Lactobacillus plantarum (HDS27), Bacillus cereus (HDS07), Delftia tsuruhatensis (HDS14), Citrobacter freundii (HDS18), Enterobacter cloacae (HDS38) and Bacillus cereus (HDS54). The selected potent isolates HDS27 from milk and HDS07 from mushroom showed a maximum riboflavin production of 3.69 mg/L and 2.9mg/L respectively. The present study explores the riboflavin producing novel bacteria from different food sources. This is the first report that the Enterobacter cloacae isolated from beef liver, Delftia tsuruhatensis from spinach and Citrobacter freundii from lamb kidney has the ability to produce riboflavin. These potent strains could be a better starter for substituting the conventional bacteria for large scale production of riboflavin in industry.

Riboflavin did not provide anti-inflammatory or antioxidant effects in an experimental model of sepsis

We aimed to evaluate whether the administration of riboflavin to septic animals reduces inflammation, oxidative stress, organ dysfunction, and mortality. C57BL/6 mice, 6-8 weeks old, were allocated to the study group (polymicrobial sepsis induced by cecal ligation and puncture (CLP) + antibiotic + iv riboflavin), control (CLP + antibiotic + iv saline), or naïve (non-operated controls). Serum concentrations of alanine aminotransferase (ALT), creatine kinase-MB (CK-MB), urea, and creatinine, and markers of inflammation [interleukin (IL)-6, tumor necrosis factor (TNF)-α, keratinocyte-derived chemokine (KC), and macrophage inflammatory protein (MIP)-2)], and oxidative stress (malondialdehyde (MDA) were measured 12 h after the experiment. Animal survival rates were calculated after 7 days. Means between groups were compared using linear regression models adjusted under the Bayesian approach. No significant difference was observed between control and study groups in serum concentrations of IL-6 (95% credible interval) (-0.35 to 0.44), TNF-α (-15.7 to 99.1), KC (-0.13 to 0.05), MIP-2 (-0.84 to 0.06), MDA (-1.25 to 2.53), or ALT (-6.6 to 11.5). Serum concentrations of CK-MB (-145.1 to -30.1), urea (-114.7 to -15.1), and creatinine (-1.14 to -0.01) were higher in the study group. Survival was similar in both groups (P=0.8). Therefore, the use of riboflavin in mice undergoing sepsis induced by CLP did not reduce inflammation, oxidative stress, organ dysfunction, or mortality compared with placebo.

Trans-epithelial accelerated corneal collagen cross-linking for progressive keratoconus / 国际眼科杂志(Guoji Yanke Zazhi)

@#AIM:To investigate the clinical efficacy and safety of trans-epithelial accelerated corneal collagen cross-linking in the treatment of progressive keratoconus.METHODS: A prospective before-after self-control study. A total of 47 eyes of 37 patients with progressive keratoconus who underwent trans-epithelial accelerated corneal collagen cross-linking in our hospital from August 2016 to November 2019 were collected. Uncorrected visual acuity(UCVA), best corrected visual acuity(BCVA), refractive status, corneal transparency, maximum keratometry value of the anterior corneal surface, thinnest corneal thickness, corneal endothelial cell counts and intraocular pressure(IOP)were analysed preoperatively and at 1, 3, 6 and 12mo postoperative.RESULTS: UCVA of patients 1, 3, 6 and 12mo after surgery was higher than that before surgery, but there was no difference(<i>F</i>=1.372, <i>P</i>=0.261). BCVA at 1, 3, 6 and 12mo after surgery was higher than that before surgery, the difference was statistically significant(<i>F</i>=3.308, <i>P</i>=0.019). There were no differences in the spherical and cylindrical power, Kmax and thickness of the thinnest point of cornea at 1, 3, 6 and 12mo postoperatively compared with those before surgery(<i>F</i>=0.293, 1.378, 2.448, 1.970; <i>P</i>=0.881, 0.258, 0.061, 0.116). There was no difference in corneal endothelial cell counts between 1mo after surgery and before surgery(<i>t</i>=1.156, <i>P</i>=0.25). There was no difference in IOP at all postoperative time points compared with that before surgery(<i>F</i>=1.221, <i>P</i>=0.321). The corneal Haze(grade 1-2)appeared in 7 eyes after surgery, and subsided in 5 eyes from 3-6mo after surgery, and the corneal transparency recovered. The corneal nebula remained in 1 eye, and the corneal central stroma linear opacity existed in 1 eye, but the visual acuity of both eyes was not affected.CONCLUSION: Trans-epithelial accelerated corneal collagen cross-linking can significantly improve BCVA, stabilize refractive status, corneal morphology and thickness, prevent or delay the progression of keratoconus, and enable patients to obtain better visual function. At the same time, the operation time is short, postoperative complications are less, and the operation has good safety.

Progress in application and detection of riboflavin singlet oxygen photochemistry / 中国输血杂志

Photodynamic therapy (PDT), which uses photosensitizers to produce singlet oxygen to kill bacteria, was first discovered by scientists over 100 years ago and has been widely used in clinical treatment. Riboflavin is an endogenous and the most commonly used photosensitizer.A large number of studies have shown that riboflavin can produce singlet oxygen through photodynamic reaction(typeⅡ) after illumination.This method is currently widely used to kill pathogens such as bacteria and viruses in blood, and singlet oxygen is considered to be the key factor in the photodynamic effect.Singlet oxygen can induce cell apoptosis, necrosis and autophagy.At present, there are direct and indirect methods to detect singlet oxygen, but both with some limitations.This paper reviews production mechanism, action mechanism and detection of singlet oxygen produced by riboflavin during photodynamic therapy, which provides a basis for further application of photodynamic therapy in clinical practice and searching for a more suitable detection method for singlet oxygen.

Corneal crosslinking efficacy in patients with keratoconus under 18 years of age / Eficácia do crosslinking corneano em portadores de ceratocone com idade inferior a 18 anos

ABSTRACT Purpose: Keratoconus presents certain specificities in pediatric patients compared with adults. The greatest challenge is because the disease is typically more severe and progresses faster in children. This retrospective study aimed to report crosslinking procedure in patients under 18 years of age and their follow-up for at least 24 months after the procedure. Methods: Overall, 12 eyes from 10 patients were studied and data, such as visual acuity with and without correction, maximum keratometry, corneal thickness, foveal thickness, and endothelial microscopy, were assessed at both preoperative and postoperative visits. Corneal crosslinking was performed in all patients. Results: A tendency toward reduced Kmax and improved Corrected Distance Visual Acuity at all postoperative moments. Only one of the 12 eyes exhibited increased Kmax of more than 1 D during a time frame longer than 12 months. Regarding pachymetry, a tendency for corneal thinning was observed in the first four months after surgery. Conclusion: Encouraging results were obtained regarding the stabilization of the disease, progression, and procedural safety, corroborating to other authors' findings. The significance of early diagnosis and short-term follow-up were highlighted.

RESUMO Objetivo: O ceratocone na população pediátrica apresenta algumas particularidades em relação à população adulta. O maior desafio é devido à doença ser geralmente mais severa e rapidamente progressiva em crianças. Métodos: Este artigo utiliza uma análise retrospectiva para relatar o uso do crosslinking em jovens menores de 18 anos e sua evolução pelo menos 24 meses após o procedimento. Foram estudados 12 olhos de 10 pacientes, e dados como acuidade visual com e sem correção, ceratometria máxima, espessura corneana, espessura foveal e microscopia endotelial avaliados no pré e pós-operatórios. O crosslinking corneano foi realizado em todos os pacientes pelo mesmo cirurgião. Resultados: Observou-se uma tendência de redução do valor do Kmax e melhora da acuidade visual corrigida em todos os momentos de pós operatório. Com relação à paquimetria, observou-se afinamento corneano do ponto mais fino, nos primeiros quatro meses de pós-operatório. Conclusão: Resultados encorajadores foram obtidos com relação à estabilização da doença, progressão e segurança do procedimento, corroborando com as conclusões de outros autores. A importância do diagnóstico precoce e do acompanhamento a curto prazo do paciente deve ser destacada.

Relationship between irradiation time of riboflavin-ultraviolet A scleral collagen cross-linking and retinal injury / 中华实验眼科杂志

Objective:To investigate the effects of riboflavin-ultraviolet A scleral collagen cross-linking on the retina under different irradiation time, and to determine the safe irradiation time.Methods:Sixty healthy New Zealand white rabbits were randomly divided into control group (0 minute group), 10 minutes group, 20 minutes group, 30 minutes group and 40 minutes irradiation group according to the irradiation time, with 12 rabbits in each group.The left eye was irradiated with riboflavin-ultraviolet A scleral collagen (370 nm, 10 mW/cm 2). The histopathological change of retina was observed by light microscope and transmission electron microscope and compared among different groups.The concentration of MDA and the activities of SOD, CAT and GSH-Px in retinal tissue were detected by corresponding kits.The expression levels of SOD and CAT proteins in retinal tissue were detected by Western blot method.The study protocol was approved by the Binzhou Medical University Laboratory Animal Ethical Committee (No.2017-80). The use and care of animals complied with the statement of ARVO and the Regulation on the Management of Laboratory Animal Quality of China. Results:Under the light microscope, the structure of the retinas in the control group was orderly arranged.Under the transmission electron microscope, the lamellar structure in the inner segment and the mitochondrial structure in the outer segment of the photoreceptor cells were intact, and the mitochondrial ridge was continuous in the control group.There was no obvious difference in retinal morphology between the 10 minutes irradiation group and the control group under both the light microscope and the transmission electron microscope, and the retinal damage became more severe with the prolongation of irradiation time.The concentration of MDA in the retina of each group was elevated gradually with the increase of irradiation time, and the difference was statistically significant ( F=65.25, P<0.05). The concentration of MDA was (11.31±1.84), (14.94±1.04)and (18.25±1.42)nmol/mgprot in the 20 minutes, 30 minutes and 40 minutes irradiation groups respectively, which were significantly higher than (1.13±0.02)nmol/mgprot in the control group (all at P<0.05). The MDA concentration in 20 minutes, 30 minutes and 40 minutes irradiation groups was increased successively, showing statistically significant differences (all at P<0.05). With the prolongation of irradiation time, the activities of SOD, CAT and GSH-Px as well as the expression levels of SOD and CAT proteins were significantly decreased gradually ( F=44.09, 34.18, 35.60, 115.75, 78.86; all at P<0.05). The differences between the control group and 20 minutes, 30 minutes, 40 minutes irradiation groups, and the differences among 20 minutes, 30 minutes, 40 minutes irradiation groups were statistically significant (all at P<0.05). Conclusions:Riboflavin-ultraviolet A 10 mW/cm 2 scleral collagen cross-linking irradiation for 10 minutes is safe.Excessive irradiation time can cause damage to the retina of rabits.

Clinical observation on transepithelial rapid cross-linking combined with RGPCL in the treatment of keratoconus / 国际眼科杂志(Guoji Yanke Zazhi)

@#AIM: To investigate the clinical effect of transepithelial rapid cross-linking combined with wearing rigid gas permeable contact lens(RGPCL)in keratoconus patients.<p>METHODS: This was a retrospective study of patients with progressive keratoconus diagnosed in the Second Affiliated Hospital of Fujian University of Traditional Chinese Medicine in October 2014, and underwent transepithelial rapid keratoplasty. After surgery 16 patients(25 eyes)used RGPCL. Average age was 25.31±5.92 years old, maximum preoperative corneal curvature was 58.96±6.76D, thinnest corneal thickness was 439.52±53.24μm. Rapid cross-linking was performed using riboflavin and 45mW/cm² ultraviolet light(UV)irradiation with a total energy of 7.2J/cm². RGPCL was used after surgery. Follow up of 3-6a, average follow-up time was 52.44±11.39mo. The best corrected visual acuity \〖(BCVA), in RGPCL\〗, corneal curvature of flat meridian(K₁), corneal curvature of steep meridian(K₂), maximum corneal curvature(Kmax), corneal refractive power(MPP), Baiocchi Calossi Versaci index of anterior and posterior surface(BCVf and BCVb)asymmetry index of anterior surface curvature(SIf), asymmetry index of posterior surface curvature(SIb), the highest point of anterior cone cornea(KVf), the highest point of posterior cone cornea(KVb), the change of corneal thickness(ThkMin), lens opacity, and corneal endothelial cell count were observed.<p>RESULTS: All patients had a successful operation, and no intraoperative complications. On the first day after surgery, all cases felt slight tingling and slight photophobia tears. After 1d of operation, the corneal epithelium was removed in two eyes. While 3d after re-wearing the bandage, the epithelium healed. None of lens in the patients was opacified during follow-up, and there was no significant difference in corneal endothelial cell count compared with preoperative(<i>P</i>>0.05). The BCVA(wearing RGPCL)after operation is obviously improved compared with preoperative. All postoperative Kmax, SIf, KVf, BCVf, BCVb were lower than preoperative(<i>P</i><0.05). <p>CONCLUSION: Transepithelial rapid corneal cross-linking combined with RGPCL is safe and effective in controlling the progress of keratoconus. And the long-term effect is stable.

Application of limbal protection technique in epithelium-on corneal collagen cross-linking / 国际眼科杂志(Guoji Yanke Zazhi)

@#AIM: To evaluate the clinical effect of a limbus protection technique during epithelium-on corneal collagen cross-linking(Epi-on CXL)for keratoconus treatment.<p>METHODS: Prospectively clinical study. A total of 15 patients(30 eyes)with diagnosed binocular progressive keratoconus were selected and treated with Epi-on CXL in my hospital from Jan. to Dec. 2019, and divided into 2 groups, Limbus-protecting Group and Control Group. The differences in corrected distance visual acuity, astigmatism, Km, thickness at the thinnest point of cornea, postoperative discomfort score, corneal epithelium healing time, and tear breakup time were compared between the two groups pre- and postoperatively.<p>RESULTS: The corrected distance visual acuity and astigmatism were improved at 3mo postoperatively(<i>P</i><0.05). The thickness at the thinnest point of cornea and the tear breakup time decreased after Epi-on CXL surgeries(<i>P</i><0.05). No difference was found in parameters of corrected distance visual acuity, astigmatism, Km, thickness at the thinnest point of cornea, tear break up time, and postoperative discomfort sore between the two groups(<i>P</i>>0.05). However, the postoperative corneal epithelium healing time in Limbus-protecting Group was shorter than in Control Group(3.20±0.56d <i>vs</i> 3.73±0.96d, <i>P</i>=0.041).<p>CONCLUSION:This limbal protection technique can reduce the corneal epithelium healing time after Epi-on CXL surgery, which preliminarily shows the feasibility of this technique.

The effect of parasitic ions on riboflavin permeability and cross-linking effectiveness in iontophoresis-assisted scleral cross-linking / 生物医学工程学杂志

The effect of parasitic ions on the results of ultraviolet A (UVA) cross-linking in iontophoresis was still not clear. In this work, the porcine sclera was cross-linked by riboflavin lactate Ringer's solution (group A) and riboflavin normal saline (group B)

Comparative analysis of miRNA profiles of platelets treated and untreated with riboflavin and ultraviolet-B light / 中国输血杂志

【Objective】 To analyze the changes of microRNA (miRNA) expression profiles on day 1 and day 5 after storage with or without riboflavin and ultraviolet-B (UVB) light (VB2-PRT) treatment, and to explore the molecular mechanism of miRNAs involved in the regulation of platelet storage lesion (PSL) under VB2-PRT treatment. 【Methods】 20 apheresis platelet concentrates (5mL / sample), collected from voluntary donors, were split into two group after mixing and agitation. One was treated with riboflavin (final concentration 50 μmol/L) plus 6.24 J/mL UVB light(E group), and the other worked as a control group (C group) without any treatment. Both groups were subjected to agitated storage at (22±2) ℃ horizontally. The platelet concentrates were sampled on d1 and d5 (5mL) during storage, named as E1, E5, C1 and C5 groups, respectively, and sequenced by DNA nanoball (DNB) sequencing technology. The differentially expressed miRNAs between E and C groups were screened by using DEGseq and MA-plot analysis software, and GO function enrichment analysis and KEGG pathway enrichment analysis were further performed when the different expression between groups reached twofold and above. 【Results】 Compared with C1 group, 487 miRNAs with significantly different expression (P<0.01) were screened in E1 group, including 220 up-regulated miRNAs, such as miR-146a and let-7b, and 267 down regulated miRNAs, such as miR-7 and miR-1260. Compared with C5 group, 229 miRNAs with significantly different expression (P<0.01) were screened in E5, including 80 miRNAs with up-regulated expression, such as miR-423 and miR-378, and 149 down regulated miRNAs, such as miR-451 and miR-30.The target genes with differentially expressed miRNAs in E1 vs C1 groups and E5 vs C5 groups were similar in the numbers of enriched GO terms, including cell components, organelles, cell membrane and other cell structures, molecular functions such as adhesion, catalysis, molecular transformation, transportation, transcription factors and receptor activity, cell processing, metabolism, biological regulation, stress and other biological processes etc. Compared with E1 and C1 groups, E5 and C5 groups lacked of signal pathways related to environmental adaptation, translation and mucin synthesis, however, it increased inositol phosphate metabolism, phosphatidylinositol signaling system and chemokine signaling pathway. 【Conclusion】 The expression profiles of platelets miRNAs treated with VB2-PRT has changed significantly after storage for a period of time. Functional prediction suggests that these miRNAs might be involved in the regulation of platelet PSL induced by VB2-PRT.

Expression analysis of miRNA profiles in apheresis platelets at the end of storage under riboflavin and ultraviolet-Blight treatment / 中国输血杂志

【Objective】 To investigate the changes of platelet microRNA (miRNA) expression profiles on d1 and d5 during storage with riboflavin and ultraviolet-B (UVB) light (VB2-PRT) treatment, and to explore the molecular mechanism of miRNAs involved in the regulation of platelet storage lesion (PSL) under VB2-PRT treatment. 【Methods】 20 apheresis platelet concentrates (5mL / sample) were collected from voluntary blood donors. After mixing and shaking, the samples was treated with riboflavin (final concentration 50 μmol/L) and 6.24J/mL UVB light for 8min, then split into two aliquots and agitated stored at (22±2) ℃. The concentrates were sampled (5mL) on d1 and d5, respectively, and sequenced by DNA nanoball (DNB) sequencing technology. The differentially expressed miRNAs between the two groups (at different storage periods) were screened by DEGseq and MA-plot analysis software. The miRNAs, reached more than 2 times different expression between groups, were considered significant different(P<0.01). The miRanda and TargetScan softwares were used to predict the target genes. Gene Ontology (GO) function enrichment analysis and Kyoto Encyclopedia of genes and genomes (KEGG) pathway enrichment analysis were performed on the target genes of significant differentially expressed miRNAs. The expression of miRNAs was verified by real-time fluorescence quantitative PCR (qRT-PCR). 【Results】 miRNA expression profile: compared with d1 platelets, there were 590 miRNAs with significantly different expression (P< 0.01) in d5 group, including 255 up-regulated miRNAs (such as miR-99b, miR-7) and 335 down regulated miRNAs (such as miR-451a, miR-19b). Among the 272 known miRNAs, 112 were up-regulated and 160 were down regulated. There were 318 new miRNAs sequences. The enriched GO terms of target genes of differentially expressed miRNAs in d5 and d1 groups included cell components, organelles, cell membranes and other cellular structures, molecular functions such as adhesion, catalysis, molecular conversion, transportation, transcription factor and receptor activity, and biological processes such as cell processing, metabolism, biological regulation and stress. The corresponding pathways in the top 10 of KEGG enrichment were mainly secretion, glucose metabolism, signal transduction, membrane transport, translation, environmental adaptation and other signal pathways. The six randomly selected differentially expressed miRNAs verified by qRT-PCR was consistent with those of DNB sequencing. 【Conclusion】 The expression profiles of platelets miRNAs has changed significantly between d1- and d5-storage under VB2-PRT treatment. Functional prediction suggests that these miRNAs might be involved in the regulation of platelet PSL underVB2-PRT treatment.

Accelerated corneal cross-linking with hypo-osmolar riboflavin in thin keratoconic corneas: 2-year follow-up / Cross-linking corneano acelerado com riboflavina hiposmolar em córneas ceratocônicas finas: 2 anos de acompanhamento

ABSTRACT Purpose: This study was performed to evaluate the outcomes of accelerated corneal cross-linking in keratoconic corneas with thinnest pachymetry values of <400 µm. Methods: The study included 28 eyes of 24 patients. The uncorrected and best-corrected visual acuities (logMAR), flattest and steepest keratometric readings, central corneal thickness at the thinnest point, corneal higher-order aberrations, and contrast sensitivity were assessed before and at 1, 3, 6, 12, and 24 months after corneal cross-linking. Result: The mean best-corrected visual acuity and contrast sensitivity increased (p=0.02, p=0.03, respectively), whereas the mean uncorrected visual acuity did not significantly differ (p>0.05) at 24 months after corneal cross-linking, compared with measurements before corneal cross-linking. Although the mean flattest keratometric reading showed no significant change (p=0.58), the mean steepest keratometric reading was reduced when compared with its value before corneal cross-linking (p=0.001). No change was observed in the mean central corneal thickness at the thinnest point at 24 months after corneal cross-linking, compared with its value before corneal cross-linking (p=0.12). Conclusion: Accelerated corneal cross-linking in keratoconic eyes with thin corneas could halt the progression of keratoconus in corneas thinner than 400 µm at 24 months after treatment.

RESUMO Objetivo: Este estudo foi realizado para avaliar os resultados do cross-linking corneano acelerado em córneas ceratocônicas com os valores mais baixos de paquimetria <400 µm. Métodos: O estudo incluiu 28 olhos de 24 pacientes. As acuidades visuais não corrigidas e melhor corrigidas (logMAR), leituras ceratométricas mais planas e íngremes, espessura corneana central no ponto mais fino, aberrações corneanas de mais alta ordem e a sensibilidade ao contraste foram avaliadas antes e em 1, 3, 6, 12 e 24 meses após a realização do do cross-linking. Resultados: A média da acuidade visual melhor corrigida e a sensibilidade ao contraste aumentaram (p=0,02, p=0,03, respectivamente), enquanto a média da acuidade visual não corrigida não diferiu significativamente (p>0,05) aos 24 meses após o cross-linking, comparada com medidas antes do procedimento. Embora a leitura da média da ceratometria mais plana não tenha apresentado alteração significativa (p=0,58), a leitura ceratométrica mais íngreme diminuiu quando comparada ao seu valor antes do cross-linking (p=0,001). Não foi observada alteração na média da espessura corneana central no ponto mais fino aos 24 meses após o cross-linking em comparação com seu valor antes do procedimento (p=0,12). Conclusão: O cross-linking corneano acelerado nos olhos ceratocônicos com córneas finas pode interromper a progressão do ceratocone nas córneas mais finas que 400 µm 24 meses após o tratamento.

Research progress of corneal collagen cross-linking in the treatment of keratoconus / 国际眼科杂志(Guoji Yanke Zazhi)

@#Corneal collagen cross-linking(CXL)is a new treatment for corneal diseases such as primary or secondary keratoconus, infectious keratitis and bullous keratopathy. CXL is used to increase the biomechanical stability of the stromal tissue and prevent the progression of keratopathy based on Laws of Photochemistry, which is widely used in clinical practice at present. Currently, commonly used method in clinical practice is the traditional “epithelium-off” corneal cross-linking method(dresden protocol), but the classical method takes a long time, and there may be postoperative complications such as poor healing of the corneal epithelium and infection. In recent years, a number of studies have improved the classical method, such as the choice of riboflavin solution immersed in the cornea, increasing the energy of ultraviolet light to shorten the accelerated cross-linking time of irradiation and cross-epithelial CXL. Thus, this paper reviews clinical and basic researches of the current use of non-classical CXL in the treatment of keratoconus.

Application of hypotonic riboflavin in corneal crosslinking in patients with keratoconus / 国际眼科杂志(Guoji Yanke Zazhi)

@#AIM:To compare the results of corneal cross-linking(CXL)with isotonic(IR)and hypotonic riboflavin(HR)in patients with keratoconus.<p>METHODS: Retrospective study of 29 eyes/keratoconus patients submitted to CXL, 15 eyes with application of IR(IR group)and 14 eyes with application of HR(HR group). The parameters analysed included(1-year follow-up): best corrected visual acuity(BCVA), sphere and cylinder, central and finer pachymetry, mean and maximum keratometry(Km and Kmax respectively), complications and progression.<p>RESULTS: An increase on the BCVA scale(LogMAR, logarithm of the minimal angle of resolution)was observed in the two groups: 0.26±0.57(IR)and 0.47±0.72(HR)before treatment, and 0.13±0.79(IR)and 0.29±1.52(HR)at the 1y. Only at 1y, the difference was statistically significant(<i>P</i>=0.018, group IR with higher BCVA). The central pachymetry(μm)decreased at 1mo in both groups, and increased in the following months: 497±28 μm(IR)and 432±14 μm(HR)before treatment, and 480±31 μm(IR)and 424±15 μm(HR)to the 1y. The thinner pachymetry(μm)presented the same evolution: 487±29 μm(IR)and 410±20 μm(HR)before treatment, and 468±33 μm(IR)and 413±13μm(HR)at 1y. Km and Kmax decreased in both groups(<i>P</i>>0.05). 6 eyes from each group presented transitory haze. No eye progressed to the 1y.<p>CONCLUSION:The use of hypotonic riboflavin seems to be a valid alternative for performing the traditional corneal cross-linking technique in eyes with a central corneal thickness of <400 μm.

Brown Vialetto Van Laere syndrome, a fatal disease with a simple solution: a case series

Brown Vialetto Van Laere (BVVL) syndrome is an extremely rare neuro metabolic disorder postulated to be caused by a defect in riboflavin transporter. The disease is characterized by progressive hearing loss with ataxia and difficulty in swallowing and breathing. The diagnosis of the disease requires great deal of suspicion on the part of treating physician. Here authors present 2 cases of BVVL who presented to us with dysphagia and hearing loss and responded to therapy. Brown Vialetto Van Laere (BVVL) syndrome is an extremely rare neuro metabolic disorder postulated to be caused by a defect in Riboflavin transporter. The disease is characterised by progressive hearing loss with ataxia and difficulty in swallowing and breathing. It is a subset in multiple acyl CoA dehydrogenase deficiency (MADD disorder). Age of onset is generally first to third decade of life. Lower cranial nerve involvement with LMN and UMN signs concomitantly is the striking feature. There is no specific treatment for BVVL except supportive care. Response to high dose riboflavin (20mg/kg/day) has produced promising results but the results may take anywhere from 1 week to 12 months to appear.