Use of a tissue clearing technique combined with retrograde trans-synaptic viral tracing to evaluate changes in mouse retinorecipient brain regions following optic nerve crush.

Successful establishment of reconnection between retinal ganglion cells and retinorecipient regions in the brain is critical to optic nerve regeneration. However, morphological assessments of retinorecipient regions are limited by the opacity of brain tissue. In this study, we used an innovative tissue cleaning technique combined with retrograde trans-synaptic viral tracing to observe changes in retinorecipient regions connected to retinal ganglion cells in mice after optic nerve injury. Specifically, we performed light-sheet imaging of whole brain tissue after a clearing process. We found that pseudorabies virus 724 (PRV724) mostly infected retinal ganglion cells, and that we could use it to retrogradely trace the retinorecipient regions in whole tissue-cleared brains. Unexpectedly, PRV724-traced neurons were more widely distributed compared with data from previous studies. We found that optic nerve injury could selectively modify projections from retinal ganglion cells in the hypothalamic paraventricular nucleus, intergeniculate leaflet, ventral lateral geniculate nucleus, central amygdala, basolateral amygdala, Edinger-Westphal nucleus, and oculomotor nucleus, but not the superior vestibular nucleus, red nucleus, locus coeruleus, gigantocellular reticular nucleus, or facial nerve nucleus. Our findings demonstrate that the tissue clearing technique, combined with retrograde trans-synaptic viral tracing, can be used to objectively and comprehensively evaluate changes in mouse retinorecipient regions that receive projections from retinal ganglion cells after optic nerve injury. Thus, our approach may be useful for future estimations of optic nerve injury and regeneration.

Comprehensive Management of Infantile Hemangiomas Involving the Periorbital Region.

ABSTRACT: Periocular infantile hemangioma (pIH) is associated with a risk of vision loss and requires urgent medical intervention. We evaluated the outcomes of the comprehensive management of 44 pIH patients (aged 2-6 months) with superficial (n = 11), deep (n = 15), and mixed (n = 18) subtypes, treated with a standard propranolol dose (2 mg kg -1 d -1 ) without any obvious side effects. Obvious and slight improvements were observed in 70.5% and 29.5% of patients, respectively. Obvious improvement was found in 96.3% of patients 3 months or younger but only 29.4% of patients older than 3 months ( P = 0.036) after 4 weeks of treatment. Thirteen patients demonstrated slight improvement and were treated with an increased propranolol dose (3 mg kg -1 d -1 ) in combination with corticosteroid injections or plastic surgery, which led to good outcomes. The patients were followed up for 12 to 18 months, with no cases of amblyopia or ametropia. Oral propranolol was effective in the treatment of pIH. Earlier propranolol use was associated with better outcomes. An increased propranolol dose combined with corticosteroid injections or plastic surgery is appropriate for patients with propranolol resistance.

Effects of curcumin nanoparticles on proliferation and VEGF expression of human retinal pigment epithelial cells.

AIM: To investigate the effects of curcumin (Cur) nanoparticles loaded with chitosan derivatives grafted by deoxycholic acid (Chit-DC) on human retinal pigment epithelial (hRPE) cell proliferation and vascular endothelial growth factor (VEGF) mRNA expression. METHODS: Cur nanoparticles were synthesized with Chit-DC as the carrier and Cur as the supported drug. Cell counting kit-8 (CCK-8) method was used to detect the effects of different concentrations of Cur/Chit-DC, Chit-DC, and Cur on the proliferation of hRPE cells for different times. The changes of Cur/Chit-DC and Cur on hRPE cell cycle were determined by flow cytometry. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the mRNA expression levels of VEGF in hRPE cells treated with Cur, Chit-DC and Cur/Chit-DC at 10 µg/mL for 24h. RESULTS: Different concentrations of Chit-DC nanoparticle treated hRPE cells had no significant difference in terms of optical density (OD) values compared with the control group at 24h and 48h. Moreover, there was no change in the cell morphology under a light microscope. After 24h treatment with Cur/Chit-DC and Cur, the percentage of G0-G1 phase cells increased and the percentage of S phase cells decreased in all concentration groups. Cur/Chit-DC and Cur in all concentration groups inhibited the proliferation of hRPE cells in a time and dose dependent manner, and reduced the expression level of VEGF mRNA. CONCLUSION: The Cur/Chit-DC nanoparticles can release Cur continuously and have sustained release function. Both Cur/Chit-DC nanoparticles and Cur could inhibit hRPE cells cultured in vitro, and could reduce the expression level of VEGF mRNA in hRPE cells.

Ultrasonography predicts the results of labial salivary gland biopsy in patients with suspected Sjögren's syndrome: a matrix risk model.

OBJECTIVE: Although a positive result of labial salivary gland biopsy (LSGB) is critical for the diagnosis of Sjögren's syndrome, rheumatologists prefer assessing the non-invasive objective items and hope to learn the predicted probability of positive LSGB before referring patients with suspected Sjögren's syndrome to receive biopsy. This study aimed to explore the predictive value of combined B-mode ultrasonography (US) and shear-wave elastography (SWE) examination on LSGB results. METHODS: A derivation cohort and later a validation cohort of patients with suspected Sjögren's syndrome were recruited. All participants received clinical assessments, B-mode US and SWE examination on bilateral parotid and submandibular glands before LSGB. Positive LSGB was defined by a focus score ⩾1 per 4 mm2 of glandular tissue. RESULTS: In the derivation cohort of 91 participants, either the total US scores or the total SWE values of four glands significantly distinguished patients with positive LSGB from those with negative results (area under the curve (AUC) = 0.956, 0.825, both p < 0.001). The positive predictive value (PPV) was 100% in patients with total US scores ⩾9 or with total SWE values ⩾33 kPa. The negative predictive value (NPV) was 100% in patients with total US scores <5, but 68% in patients with total SWE values <27 kPa. A matrix risk model was derived based on the combination of total US scores and total SWE values. Patients can be stratified into high, moderate, and low risk of positive LSGB. In the validation cohort of 52 participants, the PPV was 94% in the high-risk subpopulation and the NPV was 93% in the low-risk subpopulation. CONCLUSION: A novel matrix risk model based on the combined B-mode US and SWE examination can help rheumatologists to make a shared decision with suspected Sjögren's syndrome patients on whether the invasive procedure of LSGB should be performed.

Optical coherence tomography angiography assessment of 577 nm laser effect on severe non-proliferative diabetic retinopathy with diabetic macular edema.

AIM: To quantitatively evaluate the effect of the combined use of 577-nm subthreshold micropulse macular laser (SML) and multi-point mode pan retinal laser photocoagulation (PRP) on severe non-proliferative diabetic retinopathy (NPDR) with central-involved diabetic macular edema (CIDME) using optical coherence tomography angiography (OCTA). METHODS: In this observational clinical study, 86 eyes of 86 NPDR patients with CIDME who underwent SML and PRP treatment were included. Images were obtained 1d before laser and post-laser (1d, 1wk, 1, 3, and 6mo) using AngioVue software 2.0. Best corrected visual acuity (BCVA, LogMAR), foveal avascular zone area (FAZ), choriocapillary flow area (ChF), parafoveal vessel density (PVD), capillary density inside disc (CDD), peripapillary capillary density (PCD), macular ganglion cell complex thickness (mGCCT), central macular thickness (CMT), and subfoveal choroidal thickness (ChT) were compared between pre- and post-laser treatment. RESULTS: BCVA remained stable during 6mo post-laser therapy (pre-laser vs 6mo post-laser: 0.53±0.21 vs 0.5±0.15, P>0.05). PVD, ChF, ChT, CMT, and mGCCT significantly increased 1d post-laser therapy [pre-laser vs 1d post-laser: superficial PVD (%), 40.51±3.42 vs 42.43±4.68; deep PVD (%), 42.66±3.67 vs 44.78±4.52; ChF, 1.72±0.21 vs 1.9±0.12 mm2; ChT, 302.45±69.74 vs 319.38±70.93 µm; CMT, 301.65±110.78 vs 320.86±105.62 µm; mGCCT, 105.71±10.72 vs 115.46±9.64 µm; P<0.05]. However, PVD, ChF and ChT decreased to less than baseline level at 6mo post-laser therapy (pre-laser vs 6mo post-laser: superficial PVD (%), 40.51±3.42 vs 36.32±4.19; deep PVD (%), 42.66±3.67 vs 38.76±3.74; ChF, 1.72±0.21 vs 1.62±0.09 mm2; ChT, 302.45±69.74 vs 289.61±67.55 µm; P<0.05), whereas CMT and mGCCT decreased to baseline level at 6mo post-laser therapy (CMT, 301.65±110.78 vs 297.77±90.23 µm; mGCCT, 105.71±10.72 vs 107.05±11.81 µm; P>0.05). Moreover, FAZ continuously increased while CDD and PCD continuously decreased in 6mo after laser therapy. CMT and ChT had a significant positive correlation with ChF and PVD in most post-laser stages. CONCLUSION: During a 6-month follow-up period after combined use of SML and PRP therapy, BCVA remained stable and there was a decreased trend in macular edema. Blood flow increased at 1d post-laser therapy and reduced at 6mo post-laser therapy.

TLR7 deficiency contributes to attenuated diabetic retinopathy via inhibition of inflammatory response.

Diabetic retinopathy (DR) is a major microvascular complication of diabetes, resulting in neuronal dysfunction, retinal vascular leakage, and apoptosis within the retina. Innate immunity plays an important role in the pathogenesis of type 2 diabetes (T2D) and related complications. The toll-like receptors (TLRs), central to innate immunity, are essential participants in the progression and pathogenesis of the disease and its complications. In the study, streptozotocin (STZ) was combined with whole-body hypoxia for quicker induction of early-stage diabetic retinopathy (DR) in the wild type (WT) and TLR7-knockout (KO) C57BL/6 mice. The effects of TLR7 were also investigated in fructose-treated retinal pigment epithelial (RPE) cells. In the retinas of WT/DR mice, abnormal a-wave and b-wave activity, hyperfluorescence, and reduced retinal thickness were observed. DR development was associated with enhanced TLR7 expression, whose deletion dramatically reduced VEGF expression levels. And the secretion of pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, IL-6, IL-18 and IL-12, was highly reduced by TLR7-deficiency in DR mice. Consistently, WT/DR mice exhibited higher phosphorylation of IκB kinase α (IKKα), inhibitor of NF-κB α (IκBα) and nuclear factor κB (NF-κB), which were found to be down-regulated in KO/DR mice. Similarly, DR-induced mitogen-activated protein kinases (MAPKs) activation was blocked by TLR7-knockout. In vitro, fructose incubation-triggered inflammation was reversed by TLR7 knockdown, accompanied with inactivated NF-κB and MAPKs pathways. And reduced reactive oxygen species (ROS) generation was observed in TLR7-knockdown cells with fructose treatment. Together, inhibiting TLR7 suppressed diabetic retinopathy by reducing inflammation and suggested a potential application in clinics.

A simple classification of cranial-nasal-orbital communicating tumors that facilitate choice of surgical approaches: analysis of a series of 32 cases.

Cranial-nasal-orbital communicating tumors involving the anterior and middle skull base are among the most challenging to treat surgically, with high rates of incomplete resection and surgical complications. Currently, there is no recognized classification of tumors with regard to the choice of surgical approaches. From January 2004 to January 2014, we classified 32 cranial-nasal-orbital communicating tumors treated in our center into three types according to the tumor body location, scope of extension and direction of invasion: lateral (type I), central (type II) and extensive (type III). This classification considerably facilitated the choice of surgical routes and significantly influenced the surgical time and amount of hemorrhage during operation. In addition, we emphasized the use of transnasal endoscopy for large and extensive tumors, individualized treatment strategies drafted by a group of multidisciplinary collaborators, and careful reconstruction of the skull base defects. Our treatment strategies achieved good surgical outcomes, with a high ratio of total resection (87.5 %, 28/32, including 16 cases of benign tumors and 12 cases of malignant tumors) and a low percentage of surgical complications (18.8 %, 6/32). Original symptoms were alleviated in 29 patients. The average KPS score improved from 81.25 % preoperatively to 91.25 % at 3 months after surgery. No serious perioperative complications occurred. During the follow-up of 3 years on average, four patients with malignant tumors died, including three who had subtotal resections. The 3-year survival rate of patients with malignant tumors was 78.6 %, and the overall 3-year survival rate was 87.5 %. Our data indicate that the simple classification method has practical significance in guiding the choice of surgical approaches for cranial-nasal-orbital communicating tumors and may be extended to other types of skull base tumors.

[Study on the integrated monitoring program regarding mouse and main mouse-borne disease in Zhejiang province].

OBJECTIVE: To study the integrated monitoring program regarding mouse and plague, hemorrhagic fever of renal syndrome (HFRS) and leptospirosis. METHODS: Integrated monitoring plan was used. A designated office coordinated 5 departments' actions within the Zhejiang Provincial Center for Disease Control and Prevention (CDC). Cage-trapping method was conducted to monitor the density of mice from June to October, respectively. RESULTS: Lishui municipal CDC had finished the integrated monitoring program on mouse and mouse-borne disease while the Longyou CDC had finished the field investigation, using the integrated monitoring program. Specimens were sent to provincial CDC. The integrated monitoring program needed more number of personnel and better coordination.Lishui reported 3 leptospirosis cases and 58 HFRS cases in 2009, with the incidence rates as 0.13 and 2.44 per 100 000, respectively. Longyou reported 2 leptospirosis case and 1 HFRS cases in 2009, with the incidence rates as 0.49 and 0.25 per 100 000, respectively. Lishui and Longyou had no plague case. Lishui caught 91 mice in 2009 and the density was 4.17%. Longyou caught 37 mice in 2009, with the density as 1.18 percent. Most mice caught from Lishui were Apodemus agrarius and the next was Mus musculus. In Longyou the Rattus tanezumi ranked the first, followed by Apodemus agrarius. The positive rate of HFRS antigen in Lishui and Longyou were 10.42% and 4.59% respectively. The positive rate of HFRS antibody in Longyou was 3.70%. The culture positive rate of leptospirosis in mouse renal of Lishui and Longyou were 0 and 0.98% respectively. The culture positive rate of leptospirosis in pig renal, duck renal, frog renal and cattle urine of Longyou was 0. The culture positive rate of leptospirosis in duck blood of Longyou was 80%. CONCLUSION: The integrated monitoring program on mouse and mouse-borne disease seemed to be feasible and could promote the integrated surveillance and control program on mouse and mouse-borne diseases in China.

Ophthalmic drug-loaded N,O-carboxymethyl chitosan hydrogels: synthesis, in vitro and in vivo evaluation.

AIM: to investigate the ability of drug-loaded N,O-carboxymethyl chitosan (CMCS) hydrogels to modulate wound healing after glaucoma filtration surgery. METHODS: the drug-loaded CMCS hydrogels were in situ synthesized using genipin as the crosslinker in the presence of 5-fluorouracil (5FU) or bevacizumab. Their structures were characterized by FTIR, ultraviolet-visible (UV-vis) spectroscopy and scanning electron microscopy (SEM). In-vitro drug release experiments and in vivo evaluation in rabbits were performed. RESULTS: the results of FTIR, UV-vis spectroscopy and SEM analyses indicated that 5FU was encapsulated into the CMCS hydrogels that were crosslinked by genipin. The in vitro drug release experiments showed that nearly 100% of 5FU was released from the drug-loaded hydrogels within 8 h, but less than 20% bevacizumab was released after 53 h. The in vivo evaluation in rabbits indicated that the drug-loaded CMCS hydrogels were nontoxic to the cornea and were gradually biodegraded in the eyes. Furthermore, the drug-loaded CMCS hydrogels effectively inhibited conjunctival scarring after glaucoma filtration surgery and controlled postoperative intraocular pressure (IOP). CONCLUSION: the drug-loaded CMCS hydrogels provide a great opportunity to increase the therapeutic efficacy of glaucoma filtration surgery.

Suppression of IkappaBalpha increases the expression of matrix metalloproteinase-2 in human ciliary muscle cells.

PURPOSE: An increase of matrix metalloproteinase-2 (MMP-2) has been found to improve outflow through the uveoscleral pathway. This experiment was designed to test whether reduction of inhibitor of nuclear factor kappa B alpha (IkappaBalpha) levels could enhance MMP-2 expression in human ciliary muscle (HCM) cells in vitro. METHODS: The small interfering RNA (siRNA) targeting inhibitor of nuclear factor kappa B (IkappaBalpha) was transfected into HCM cells. The mRNA and protein levels of IkappaBalpha, nuclear factor-kappa B (NF-kappaB)p65, MMP-2, tissue inhibitor of metalloproteinase-2 (TIMP-2), and membrane-type 1 matrix metalloproteinase (MT1-MMP) in HCM cells were examined 24 h, 48 h, and 72 h after IkappaBalpha siRNA transfection by real-time reverse transcription polymerase chain reaction (RT-PCR) and western blot. The activation of NF-kappaBp65 was determined through the translocation of NF-kappaBp65 by fluorescence microscope. Gelatin zymography was used to detect the secretion and activity of MMP-2. RESULTS: Real-time RT-PCR and western blot showed that transfection of IkappaBalpha siRNA led to gradual downregulation of IkappaBalpha and TIMP-2 both at the mRNA and protein level after 24 h, 48 h and 72 h. The IkappaBalpha and TIMP-2 mRNA levels decreased 92.7%+/-1.6% and 70.3%+/-13.1%, respectively, and the protein levels were reduced 87.3%+/-2.0% and 62.9%+/-0.8% (p<0.01), respectively, when compared to the control 72 h after siRNA transfection. Conversely, the MMP-2 and MT1-MMP mRNA and protein levels increased in the time-dependent manner after IkappaBalpha siRNA transfection. The MMP-2 and MT1-MMP mRNA levels increased 178%+/-4.6% and 165%+/-8.2%, respectively, while protein levels were raised to 162%+/-3.7% and 157.6%+/-5.7% (p<0.01), respectively, when compared to the control 72 h after IkappaBalpha siRNA transfection. Although no obvious changes were seen in either mRNA or protein levels of total NF-kappaBp65 (p>0.05), the protein level of NF-kappaBp65 increased dramatically in the nucleus as revealed by western blot and fluorescence staining 24 h, 48 h, and 72 h after IkappaBalpha siRNA transfection. Moreover, gelatin zymography indicated that the secretion and activity of MMP-2 in treated cells were higher than those in the control cells. The maximum increases of pro-MMP-2 and active-MMP-2 were 172%+/-15% and 151%+/-14% (p<0.01), respectively, when compared to the control at the experiment's conclusion 72 h after siRNA transfection. CONCLUSIONS: Expression and activity of MMP-2 was enhanced by the IkappaBalpha siRNA in HCM cells through the activation of the NF-kappaB signaling pathway. Our results suggested that IkappaBalpha may therefore be a potential target for controlling the uveoscleral outflow pathway in glaucoma.

[The inhibitive effects of 5-fluorouracil-loaded polylactic acid nanoparticles on human Tenon's capsule fibroblast in vitro].

OBJECTIVE: To investigate the inhibitive effects of 5-fluorouracil-loaded polylactic acid nanoparticles (5-FU-PLA-NPs) on human Tenon's capsule fibroblasts in vitro. METHODS: This paper was experimental study. MTT assay was performed to estimate the effects of 0.1 mg/L, 1.0 mg/L, 10.0 mg/L, 100.0 mg/L, 1000.0 mg/L unloaded polylactic acid nanoparticles (PLA-NPs) on fibroblasts proliferation on 48 h, 72 h. MTT assay was performed to estimate the effects of 0.1 mg/L, 1.0 mg/L, 10.0 mg/L, 100.0 mg/L, 1000.0 mg/L original 5-FU and 5-FU-PLA-NPs on fibroblasts' proliferation on 7 consecutive days respectively. The inhibitory rate of the two drugs against the fibroblasts was calculated. Cells were exposed to 100 mg/L original 5-FU and 5-FU-PLA-NPs respectively for 7 days. Semi-quantitative RT-PCR was used to observe the mRNA expression of type III procollagen at the 1st day, 5th day and 7th day. RESULTS: PLA-NPs had no cytotoxicity on the fibroblasts. Both 5-FU-PLA-NPs and original 5-FU could inhibit the proliferation of the fibroblasts and make the expression of type III procollagen mRNA lower in dose-dependent and time-dependent manner. The inhibitive effect of original 5-FU was more effective than 5-FU-PLA-NPs in the initial period, but the inhibitive effect of 5-FU-PLA-NPs was more effective than original 5-FU in long time. CONCLUSION: PLA has good biocompatibility and safety, and can be used as a carrier of ophthalmic drugs. 5-FU-PLA-NPs shows slow-releasing function. 5-FU-PLA-NPs can be proposed as a potential controlled and targeted ophthalmic delivery system for the treatment of antifibrosis after glaucoma filtering surgery.

[Experimental study on the effects of interferon-gamma gene on Tenon's capsule fibroblasts in vitro].

OBJECTIVE: To investigate the effects of interferon-gamma (IFN-gamma) gene on Tenon's capsule fibroblasts in vitro. METHODS: Using lipofectAMINE, IFN-gamma gene was transferred to human Tenon's capsule fibroblasts with plasmid pcDNA3IFN-gamma in vitro. Cells treated with plasmid pcDNA3 without IFN-gamma gene were used as the controls. The transfected fibroblasts were screened by G418. The expression of IFN-gamma was determined by RT-PCR, immunohistochemistry and flow cytometry assay. The effects of IFN-gamma on the proliferation of Tenon's capsule fibroblasts were evaluated by flow cytometry and MTT. RESULTS: The Tenon's capsule fibroblasts transferred the IFN-gamma gene could express the IFN-gamma RNA and protein transiently. The proliferation of the fibroblasts transferred the IFN-gamma gene was inhibited. CONCLUSION: The proliferation of the Tenon's capsule fibroblasts in vitro can be inhibited by transferred IFN-gamma gene.

[The inhibition effect of photodynamic on human Tenon capsule fibroblast cells].

OBJECTIVE: To investigate the inhibition of photodynamic effect on human Tenon capsule fibroblast cells in vitro. METHODS: The human Tenon capsule fibroblast cells were divided into nine groups named as group A to I, each group had four wells. Group A to G, each group was added photosensitizer benzoporphyrin derivative monoacid ring A (BPD) with end concentration of 2.50 x 10(3) g/L, 1.25 x 10(3) g/L, 0.62 x 10(3) g/L, 0.31 x 10(3) g/L, 0.16 x 10(3) g/L, 0.08 x 10(3) g/L, 0.04 x 10(3) g/L respectively. Then the culture cells were irradiated by 689 nm diode laser with dosage of 2.4 J/cm(2) after BPD treatment for 15 minutes. Group H was treated with Mitomycin C (MMC) at concentration of 0.2 g/L. Group I was control without any treatment. All the cells were kept cultured for another 24 hours and then MTT colorimetric assay was applied to measure the relative inhibitory rate of photodynamic effect on the cells. RESULTS: There is statistical significance between group A to H and group I with the method of one way analysis of variance. When compared to group I, the relative inhibitory rates of group A to G are 93.3%, 91.0%, 90.3%, 87.1%, 66.0%, 41.6%, 12.5% respectively, and the inhibitory rate of group H (MMC) is 93.0%. CONCLUSION: Photodynamic effect can inhibit the proliferation of human Tenon capsule fibroblast cells in vitro. The inhibitory rate appears to be dependent on the concentration of the photo sensitizer.