Vibrational Therapies for Vocal Fatigue.

BACKGROUND: Vibration is commonly used to relax tension in the limb and truck muscle. Vibration used directly on the muscle concerned and vertical vibration used on the whole-body through a foot platform have been reported in the literature to be useful to release muscle tension. AIM: The present study investigated the effect of indirect whole-body vibration (WBV) and direct localized perilaryngeal vibration (LPV) on the phonatory functions of nondysphonic individuals with vocal fatigue. METHODS: Forty-four subjects (mean age = 21.67 years) with normal voice, were randomly assigned to either the WBV group, the LPV group, or the Control (sham hand-held vibratory device) group. They performed karaoke singing for at least 95 minutes. They then received either WBV through a Turbosonic vibratory machine, LPV with a Novofan vibrator, or a sham vibrator for 10 minutes. The highest pitch produced, and self-reported vocal fatigue score were taken before singing, after singing, and after the intervention. Data were analyzed separately for the gender subgroups. RESULTS: All subject groups showed significant reduction of vocal function (highest pitch production, and vocal fatigue score) after singing. Following the vibrational interventions, both the WBV and LPV groups showed significantly recovery in the highest pitch production and the perception of vocal fatigue (P < 0.002) than the Control groups. CONCLUSION: Vibrational therapy, whether it is localized vibration on the peri-laryngeal muscles, or whole-body vibration, is more effective than voice rest per se in relieving vocal fatigue. Vibrational methods are recommended for treating vocal fatigue.

Nasopharyngeal carcinoma MHC region deep sequencing identifies HLA and novel non-HLA TRIM31 and TRIM39 loci.

Despite pronounced associations of major histocompatibility complex (MHC) regions with nasopharyngeal carcinoma (NPC), causal variants underlying NPC pathogenesis remain elusive. Our large-scale comprehensive MHC region deep sequencing study of 5689 Hong Kong Chinese identifies eight independent NPC-associated signals and provides mechanistic insight for disrupted transcription factor binding, altering target gene transcription. Two novel protective variants, rs2517664 (Trs2517664 = 4.6%, P = 6.38 × 10-21) and rs117495548 (Grs117495548 = 3.0%, P = 4.53 × 10-13), map near TRIM31 and TRIM39/TRIM39-RPP21; multiple independent protective signals map near HLA-B including a previously unreported variant, rs2523589 (P = 1.77 × 10-36). The rare HLA-B*07:05 allele (OR < 0.015, P = 5.83 × 10-21) is absent in NPC, but present in controls. The most prevalent haplotype lacks seven independent protective alleles (OR = 1.56) and the one with additional Asian-specific susceptibility rs9391681 allele (OR = 2.66) significantly increased NPC risk. Importantly, this study provides new evidence implicating two non-human leukocyte antigen (HLA) genes, E3 ubiquitin ligases, TRIM31 and TRIM39, impacting innate immune responses, with NPC risk reduction, independent of classical HLA class I/II alleles.

Clinical Outcome-Related Mutational Signatures Identified by Integrative Genomic Analysis in Nasopharyngeal Carcinoma.

PURPOSE: Investigation of biological mechanisms underlying genetic alterations in cancer can assist the understanding of etiology and identify the potential prognostic biomarkers. EXPERIMENTAL DESIGN: We performed an integrative genomic analysis for a total of 731 nasopharyngeal carcinoma cases from five independent nasopharyngeal carcinoma cohorts to identify the genetic events associated with clinical outcomes. RESULTS: In addition to the known mutational signatures associated with aging, APOBEC and mismatch repair (MMR), a new signature for homologous recombination deficiency (BRCAness) was discovered in 64 of 216 (29.6%) cases in the discovery set including three cohorts. This signature appeared more frequently in the recurrent and metastatic tumors and significantly correlated with shorter overall survival (OS) in the primary tumors. Independent prognostic value of MMR and BRCAness signatures was revealed by multivariable Cox analysis after adjustment for clinical parameters and stratification by studies. The cases with both signatures had much worse clinical outcome than those without these signatures [hazard ratio (HR), 12.4; P = 0.002]. This correlation was confirmed in the validation set (HR, 8.9; P = 0.003). The BRCAness signature is highly associated with BRCA2 pathogenic germline or somatic alterations (7.8% vs. 0%; P = 0.002). Targeted sequencing results from a prospective nasopharyngeal carcinoma cohort (N = 402) showed that the cases carrying BRCA2 germline rare variants are more likely to have poor OS and progression-free survival. CONCLUSIONS: Our study highlights importance of defects of DNA repair machinery in nasopharyngeal carcinoma pathogenesis and their prognostic values for clinical implications. These signatures will be useful for patient stratification to evaluate conventional and new treatment for precision medicine in nasopharyngeal carcinoma.

Clinical utility of serial analysis of circulating tumour cells for detection of minimal residual disease of metastatic nasopharyngeal carcinoma.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is an important cancer in Hong Kong. We aim to utilise liquid biopsies for serial monitoring of disseminated NPC in patients to compare with PET-CT imaging in detection of minimal residual disease. METHOD: Prospective serial monitoring of liquid biopsies was performed for 21 metastatic patients. Circulating tumour cell (CTC) enrichment and characterisation was performed using a sized-based microfluidics CTC chip, enumerating by immunofluorescence staining, and using target-capture sequencing to determine blood mutation load. PET-CT scans were used to monitor NPC patients throughout their treatment according to EORTC guidelines. RESULTS: The longitudinal molecular analysis of CTCs by enumeration or NGS mutational profiling findings provide supplementary information to the plasma EBV assay for disease progression for good responders. Strikingly, post-treatment CTC findings detected positive findings in 75% (6/8) of metastatic NPC patients showing complete response by imaging, thereby demonstrating more sensitive CTC detection of minimal residual disease. Positive baseline, post-treatment CTC, and longitudinal change of CTCs significantly associated with poorer progression-free survival by the Kaplan-Meier analysis. CONCLUSIONS: We show the potential usefulness of application of serial analysis in metastatic NPC of liquid biopsy CTCs, as a novel more sensitive biomarker for minimal residual disease, when compared with imaging.

Leukocyte telomere length associates with nasopharyngeal carcinoma risk and survival in Hong Kong Chinese.

Telomere shortening occurs as an early event in tumorigenesis. The TERT-CLPTM1L locus associates with nasopharyngeal carcinoma (NPC) risk. It remains unknown if leukocyte telomere length (LTL) associates with NPC risk and survival. The relative LTL (rLTL) was measured by quantitative-PCR in 2,996 individuals comprised of 1,284 NPC cases and 1712 matched controls. The odds ratio (OR) and 95% confidence intervals (CI) were calculated by logistic regression. The hazard ratio (HR) and 95% CI were calculated by Cox regression for survival analysis with rLTL and other clinical parameters in 1,243 NPC with a minimum follow-up period of 25 months. NPC patients had significantly shorter telomere length than controls. Shorter rLTL significantly associated with increased NPC risk, when the individuals were dichotomized into long and short telomeres based on median-split rLTL in the control group (OR = 2.317; 95% CI = 1.989-2.700, p = 4.10 × 10-27 ). We observed a significant dose-response association (ptrend  = 3.26 × 10-34 ) between rLTL and NPC risk with OR being 3.555 (95% CI = 2.853-4.429) for the individuals in the first quartile (shortest) compared with normal individuals in the fourth quartile (longest). A multivariate Cox regression analysis adjusted by age demonstrated an independent effect of rLTL on NPC survival for late-stage NPC patients, when the individuals were categorized into suboptimal rLTL versus the medium rLTL based on a threshold set from normal (HR = 1.471, 95% CI = 1.056-2.048, p = 0.022). Shorter blood telomeres may be markers for higher susceptibility for NPC risk. Suboptimal rLTL may be a poor prognostic factor for advanced NPC patients, as it associates independently with poor survival.

Differential transcriptomic response in the spleen and head kidney following vaccination and infection of Asian seabass with Streptococcus iniae.

Vaccination is an important strategy in the protection of aquaculture species from major diseases. However, we still do not have a good understanding of the mechanisms underlying vaccine-induced disease resistance. This is further complicated by the presence of several lymphoid organs that play different roles when mounting an immune response. In this study, we attempt to elucidate some of these mechanisms using a microarray-based approach. Asian seabass (Lates calcarifer) were vaccinated against Streptococcus iniae and the transcriptomic changes within the spleen and head kidney at one and seven days post-vaccination were profiled. We subsequently challenged the seabass at three weeks post-vaccination with live S. iniae and similarly profiled the transcriptomes of the two organs after the challenge. We found that vaccination induced an early, but transient transcriptomic change in the spleens and a delayed response in the head kidneys, which became more similar to one another compared to un-vaccinated ones. When challenged with the pathogen, the spleen, but not the head kidneys, responded transcriptomically at 25-29 hours post-challenge. A unique set of genes, in particular those involved in the activation of NF-κB signaling, was up-regulated in the vaccinated spleens upon pathogen challenge but not in the un-vaccinated spleens. A semi-quantitative PCR detection of S. iniae using metagenomic DNA extracted from the water containing the seabass also revealed that vaccination resulted in reduction of pathogen shedding. This result indicated that vaccination not only led to a successful immune defense against the infection, but also reduced the chances for horizontal transmission of the pathogen. In conclusion, we have provided a transcriptomic analysis of how the teleost spleen and head kidneys responded to vaccination and subsequent infection. The different responses from the two organs are suggestive of their unique roles in establishing a vaccine-induced disease resistance.

Onset of an outbreak of Bipolaris hawaiiensis fungal endophthalmitis after intravitreal injections of triamcinolone.

PURPOSE: To report a series of cases with fungal endophthalmitis occurring after intravitreal injection of triamcinolone derived from a single lot prepared by a compounding pharmacy. DESIGN: Retrospective, observational case series. PARTICIPANTS: Seventeen eyes treated with triamcinolone obtained from a single lot subsequently found to be contaminated with Bipolaris hawaiiensis. METHODS: A retrospective chart review in a single retina practice was performed for 15 patients (n = 17 eyes) who received intravitreal injections of triamcinolone obtained from a single compounding pharmacy. Medical records and cytologic and microbiologic results were reviewed from December 2011 through January 2013. MAIN OUTCOME MEASURES: Visual acuity; presence of vitreous cell, anterior chamber cell, or both; and fungal detection in samples obtained by vitreous needle aspiration or vitreous biopsy. RESULTS: Fungal endophthalmitis developed in 82% (14/17) of eyes after intravitreal triamcinolone obtained from the same lot. Median onset was 83 days (range, 6-322 days). Preinjection visual acuity ranged from 20/20 to counting fingers (median, 20/50). Median visual acuity at last follow-up was 20/400 (range, 20/30-no light perception). The most common signs and symptoms included decreased vision (57% [8/14]), vitreous cell (64% [9/14]), and anterior chamber cell (50% [7/14]). Fungus was detected by cytologic or culture examination in 7% (1/14) from initial vitreous tap. By comparison, vitreous samples obtained by pars plana vitrectomy (PPV) resulted in fungus-positive cytologic results in 43% (6/14) of eyes and positive culture results in 36% (5/14) of eyes. All culture-positive specimens (100% [5/5]) were identified as B. hawaiiensis. Overall, fungal infection was confirmed in 57% (8/14) of eyes by either cytologic or microbiologic analysis. CONCLUSIONS: Fungal endophthalmitis resulting from B. hawaiiensis developed in a series of eyes after intravitreal injections of triamcinolone obtained from a single compounding pharmacy. Clinical presentation of infection can be delayed up to 10 months. Vitreous tap may be inadequate, and direct vitreous biopsy by PPV may be preferred to identify fungal endophthalmitis and facilitate prompt diagnosis and treatment.

Characterization of microaneurysm closure after focal laser photocoagulation in diabetic macular edema.

PURPOSE: To characterize microaneurysm closure following focal laser photocoagulation in diabetic macular edema (DME) using simultaneous fluorescein angiography (FA) and spectral-domain optical coherence tomography (SD-OCT). DESIGN: Retrospective observational case series. METHODS: Leaking microaneurysms (n = 123) were analyzed in eyes (n = 29) with nonproliferative diabetic retinopathy (NPDR) that underwent navigated focal laser photocoagulation in DME and were followed at 3, 6, and 12 months. Closure of diabetic microaneurysms was characterized in detail following focal laser using SD-OCT. RESULTS: Closure rate of microaneurysms by both FA and SD-OCT was 69.9% (84/123), 79.7% (98/123), and 82.9% (102/123) at 3, 6, and 12 months, respectively. Microaneurysm closure rate increased at 6 and 12 months compared to 3 months (P < .003, P < .001). Over half of closed microaneurysms (45/86, 52.3%) left hyperreflective spots while the remaining half (41/86, 47.7%) disappeared without any hyperreflectivity by SD-OCT at 3 months. Hyperreflective spots decreased at 6 (36/99, 36.4%) and 12 months (17/102, 16.7%) with a concomitant increase in complete loss of reflectivity at 6 (63/99, 63.6%) and 12 months (85/102, 83.3%). Smaller outer and inner diameters and heterogeneous lumen reflectivity were positively associated with microaneurysm closure at 12 months (P < .0001, P < .001, P < .03). CONCLUSIONS: Characterization of microaneurysms following focal laser photocoagulation resulted in hyperreflective spots and complete resolution of all reflectivity using SD-OCT. Smaller microaneurysms and those with heterogeneous lumen were positively associated with microaneurysm closure. These findings provide greater understanding of localized retinal changes following focal laser photocoagulation in DME treatment.

Evolution of the capsular operon of Streptococcus iniae in response to vaccination.

Streptococcus iniae causes severe septicemia and meningitis in farmed fish and is also occasionally zoonotic. Vaccination against S. iniae is problematic, with frequent breakdown of protection in vaccinated fish. The major protective antigens in S. iniae are the polysaccharides of the capsule, which are essential for virulence. Capsular biosynthesis is driven and regulated by a 21-kb operon comprising up to 20 genes. In a long-term study, we have sequenced the capsular operon of strains that have been used in autogenous vaccines across Australia and compared it with the capsular operon sequences of strains subsequently isolated from infected vaccinated fish. Intriguingly, strains isolated from vaccinated fish that subsequently become infected have coding mutations that are confined to a limited number of genes in the cps operon, with the remainder of the genes in the operon remaining stable. Mutations in strains in diseased vaccinated fish occur in key genes in the capsular operon that are associated with polysaccharide configuration (cpsG) and with regulation of biosynthesis (cpsD and cpsE). This, along with high ratios of nonsynonymous to synonymous mutations within the cps genes, suggests that immune response directed predominantly against capsular polysaccharide may be driving evolution in a very specific set of genes in the operon. From these data, it may be possible to design a simple polyvalent vaccine with a greater operational life span than the current monovalent killed bacterins.

Characterization of diabetic microaneurysms by simultaneous fluorescein angiography and spectral-domain optical coherence tomography.

PURPOSE: To correlate spectral-domain optical coherence tomography (SD-OCT) findings of perfused diabetic microaneurysms with leakage status on fluorescein angiography (FA) using simultaneous FA and SD-OCT. DESIGN: Retrospective, observational case series. METHODS: A total of 173 microaneurysms were analyzed in 50 eyes (14 mild nonproliferative diabetic retinopathy [NPDR]; 22 moderate NPDR; 9 severe NPDR; 5 proliferative diabetic retinopathy) of 40 diabetic patients using simultaneous FA and SD-OCT. The characteristics of microaneurysms were evaluated by 2 masked observers using SD-OCT and correlated with leakage status on FA. RESULTS: External diameter of microaneurysms averaged 104 µm (range 43-266 µm). Some microaneurysm centers (15/173; 9%) and the outermost extent of microaneurysms (113/173; 68%) were localized to the outer half of the retina. Almost all microaneurysms spanned more than 1 retinal layer (157/173; 91%). Most microaneurysms had an internal lumen with homogeneous reflectivity (109/173; 63%) and moderate reflectivity (87/173; 50%). Retinal thickness through microaneurysms as well as the presence of adjacent hyporeflectivity on SD-OCT correlated with increasing leakage status seen on FA (P < .001). Microaneurysm dimensions, percent depth within the retina, retinal layer location, and internal reflectivity by SD-OCT did not correlate significantly with FA leakage status. CONCLUSIONS: Simultaneous FA and SD-OCT allows detailed characterization of perfused diabetic microaneurysms. Increased FA leakage of diabetic microaneurysms positively correlated with perianeurysm fluid and retinal thickness. Perfused microaneurysms seen by SD-OCT were localized deeper than the inner nuclear layer.

Retinal adherence and fibrillary surface changes correlate with surgical difficulty of epiretinal membrane removal.

PURPOSE: To correlate surgical difficulty of epiretinal membrane (ERM) removal with characteristics of ERM adherence seen by spectral-domain optical coherence tomography (SD-OCT). DESIGN: Prospective observational case series. METHODS: Surgical difficulty was correlated with extent of ERM adherence by SD-OCT using masked observers in consecutive eyes undergoing ERM removal (N=31). Surgical videos were analyzed and difficulty of ERM removal (grade 1-3) was determined in 4 quadrants as well as the fovea by consensus of observers masked to SD-OCT findings. Extent of ERM adhesion was categorized (focal, broad, or complete) by masked observers using SD-OCT. The presence of fibrillary changes between the ERM and retinal nerve fiber layer (RNFL) was also evaluated. Surgical difficulty of ERM removal for each quadrant and fovea was compared to extent of ERM adherence and presence of fibrillary changes. RESULTS: Assessment of ERM adherence using SD-OCT between masked observers was highly concordant (kappa=0.9178). Surgical difficulty of ERM removal was strongly associated with more extensive ERM adherence to the retina observed by SD-OCT. Complete ERM adherence correlated with an 8.6-fold increased surgical difficulty of ERM removal compared to focal adherence (P<.0001). The presence of fibrillary changes between the ERM and RNFL also correlated with a 25.5-fold increased difficulty of surgical removal compared to the absence of fibrillary changes (P<.0001). CONCLUSION: Extent of ERM-retinal adhesion and presence of fibrillary changes determined by SD-OCT provide reliable preoperative assessment of surgical difficulty. Furthermore, SD-OCT analysis may help localize surgically advantageous coordinates to initiate ERM removal.

Primary trabeculectomy surgery performed by residents at a county hospital.

PURPOSE: To evaluate the safety and efficacy of trabeculectomies performed by ophthalmology residents at a metropolitan county hospital, under the supervision of attending physicians. METHODS: A retrospective analysis of resident-performed trabeculectomies at the San Francisco General Hospital from the period of 1994 to 2004 was performed. The preoperative and postoperative ocular data of 50 eyes in 35 patients were evaluated. Of the 50 cases, 47 procedures were performed as primary trabeculectomies and 3 were revisions of trabeculectomies. RESULTS: The average follow-up period was 28.9+/-17.6 months with an intraocular pressure (IOP) decrease from 23.2+/-9.4 mm Hg preoperatively to 11.3+/-4.4 mm Hg at last follow-up, for a mean reduction of 11.9+/-10.5 mm Hg (51.6%) (P<0.0001). Follow-up periods ranged from 3 months to over 6 years. The number of medications required decreased from 3.2+/-1.1 to 0.6+/-0.1 (P<0.0001). Success, defined by a postoperative IOP < or =21 mm Hg or a decreased postoperative IOP of at least 25% from preoperative pressure if the preoperative IOP was already < or =21 mm Hg, was observed in 42 eyes (84%) at last follow-up. Best-corrected visual acuity was stable or improved in 22 eyes (44%) and was noted to decrease 2 or more Snellen lines in 28 eyes (56%). Notable complications included 3 cases (6%) of persistent hypotony (IOP<5), 1 case (2%) of late endophthalmitis, and 1 case (2%) of phthisis. Seven eyes (14%) required subsequent penetrating glaucoma procedures due to bleb failure. CONCLUSIONS: Results of this study suggest that the outcomes of trabeculectomies performed by residents at a county hospital can have a high success rate, comparable with previous studies in the literature. Rates of complications are overall similar to those found in the published literature.

Contribution of imidazoline receptors and alpha2-adrenoceptors in the rostral ventrolateral medulla to sympathetic baroreflex inhibition by systemic rilmenidine.

OBJECTIVES: To determine whether the hypotensive and sympathetic baroreflex inhibition by rilmenidine administered systemically are mediated via imidazoline receptors in the rostral ventrolateral medulla (RVLM). METHODS: Initial dose-response curves to rilmenidine were determined in urethane anaesthetized rabbits. Effects of a single intravenous dose of rilmenidine (445 microg/kg) on the renal sympathetic nerve activity (RSNA) baroreflex were examined before and after microinjection into the RVLM of the mixed imidazoline/alpha2-adrenoceptor antagonist idazoxan and the alpha2-adrenoceptor antagonist 2-methoxyidazoxan (2-MI). RESULTS: Intravenous administration of rilmenidine lowered mean arterial pressure and RSNA, inhibited the RSNA baroreflex range by 33% and shifted the baroreflex curve to the left. Idazoxan injected into the RVLM reversed the hypotension and completely restored the baroreflex curve at doses that did not affect the hypotension produced by the selective alpha2-adrenoceptor agonist alpha-methylnoradrenaline. The alpha2-adrenoceptor antagonist, 2-MI also reversed the rilmenidine sympatho-inhibition suggesting that alpha2-adrenoceptors are activated as well. CONCLUSIONS: The results of the present study show that the hypotensive and sympatho-inhibitory actions of systemic rilmenidine are primarily mediated via imidazoline receptors in the RVLM. However, alpha2-adrenoceptors are also involved, probably as a direct result of the imidazoline receptor action.

Proceedings of the Third International Symposium on Retinopathy of Prematurity: an update on ROP from the lab to the nursery (November 2003, Anaheim, California).

The Third International Symposium on Retinopathy of Prematurity (ROP) was convened with the aim of cross fertilizing the horizons of basic and clinical scientists with an interest in the pathogenesis and management of infants with ROP. Ten speakers in the clinical sciences and ten speakers in the basic sciences were recruited on the basis of their research to provide state of the art talks. The meeting was held November 9, 2003 immediately prior to the American Academy of Ophthalmology meeting; scholarships were provided for outreach to developing countries and young investigators. This review contain the summaries of the 20 platform presentations prepared by the authors and the abstracts of presented posters. Each author was asked to encapsulate the current state of understanding, identify areas of controversy, and make recommendations for future research. The basic science presentations included insights into the development of the human retinal vasculature, animal models for ROP, growth factors that affect normal development and ROP, and promising new therapeutic approaches to treating ROP like VEGF targeting, inhibition of proteases, stem cells, ribozymes to silence genes, and gene therapy to deliver antiangiogenic agents. The clinical presentations included new insights into oxygen management, updates on the CRYO-ROP and ETROP studies, visual function in childhood following ROP, the neural retina in ROP, screening for ROP, management of stage 3 and 4 ROP, ROP in the third world, and the complications of ROP in adult life. The meeting resulted in a penetrating exchange between clinicians and basic scientists, which provided great insights for conference attendees. The effect of preterm delivery on the normal cross-talk of neuroretinal and retinal vascular development is a fertile ground for discovering new understanding of the processes involved both in normal development and in retinal neovascular disorders. The meeting also suggested promising potential therapeutic interventions on the horizon for ROP.

Expression of protein kinase CK2 in astroglial cells of normal and neovascularized retina.

We previously documented protein kinase CK2 involvement in retinal neovascularization. Here we describe retinal CK2 expression and combined effects of CK2 inhibitors with the somatostatin analog octreotide in a mouse model of oxygen-induced retinopathy (OIR). CK2 expression in human and rodent retinas with and without retinopathy and in astrocytic and endothelial cultures was examined by immunohistochemistry, Western blotting, and reverse transcriptase-polymerase chain reaction. A combination of CK2 inhibitors, emodin or 4,5,6,7-tetrabromobenzotriazole, with octreotide was injected intraperitoneally from postnatal (P) day P11 to P17 to block mouse OIR. All CK2 subunits (alpha, alpha', beta) were expressed in retina, and a novel CK2alpha splice variant was detected by reverse transcriptase-polymerase chain reaction. CK2 antibodies primarily reacted with retinal astrocytes, and staining was increased around new intraretinal vessels in mouse OIR and rat retinopathy of prematurity, whereas preretinal vessels were negative. Cultured astrocytes showed increased perinuclear CK2 staining compared to endothelial cells. In the OIR model, CK2 mRNA expression increased modestly on P13 but not on P17. Octreotide combined with emodin or 4,5,6,7-tetrabromobenzotriazole blocked mouse retinal neovascularization more efficiently than either compound alone. Based on its retinal localization, CK2 may be considered a new immunohistochemical astrocytic marker, and combination of CK2 inhibitors and octreotide may be a promising future treatment for proliferative retinopathies.

Differential expression of pro- and antiangiogenic factors in mouse strain-dependent hypoxia-induced retinal neovascularization.

Clinical observations suggest that genetic factors may influence heterogeneity of angiogenic responses in cardiovascular disease, proliferative diabetic retinopathy, and neoplasia. Experiments among mouse strains using a corneal micropocket assay indicate that extent of angiogenesis may be genetically determined. Here, we established the strain-dependence of hypoxia-induced retinal angiogenesis in multiple mouse strains which paralleled the rank order found for bFGF-induced corneal angiogenesis. Using quantitative real-time RT-PCR, strain-related gene expression differences in retina/choroid between C57BL/6J and 129S3/SvIM, inbred strains with relatively low and high levels of angiogenesis, respectively, after 0, 6, 12, 24, 48, and 96 h hypoxia were determined for vascular endothelial growth factor (VEGF) and angiopoietin-2 (Ang-2), angiogenic ligands potently induced by hypoxia, and for pigment epithelium-derived factor (PEDF) and thrombospondin-1 (TSP-1), endogenous broad-spectrum antiangiogenic factors. Indirect ELISA was used to correlate VEGF and PEDF protein levels with mRNA expression. At the onset of hypoxia, both PEDF and TSP-1 levels were increased over 15-fold and VEGF was increased over 10-fold compared to Ang-2 in both strains. At the onset of neovascularization (48 h), both VEGF and Ang-2 mRNA levels were increased in the more angiogenic 129S3/SvIM strain (P < 0.02), which was not observed among developmental control animals. PEDF expression was higher in the less angiogenic C57BL/6J strain at 6, 12, 24, and 96 h hypoxia (P < 0.03), while TSP-1 expression was higher in C57BL/6J throughout the entire time course of hypoxia (4 days) compared to 129S3/SvIM (P < 0.02). Among developmental control animals, PEDF and TSP-1 expression was also increased at P14 and P16 in C57BL/6J strain compared to 129S3/SvIM (P < 0.02). Strain-dependent expression of both pro- and antiangiogenic growth factors may determine heterogeneity in the angiogenic response and potentially, susceptibility to angiogenesis-dependent diseases.

Mouse strain-dependent heterogeneity of resting limbal vasculature.

PURPOSE: Heterogeneity of the extent of angiogenesis induced by exogenous growth factors may be determined by genetic influences. Because angiogenesis is the formation of new vessels from preexisting ones, strain-related influences on naïve resting limbal vessel phenotype and gene expression were determined in mice having divergently low and high angiogenic responses. METHODS: Resting limbal vessel surface area and density and extent of bFGF-induced corneal angiogenesis were determined in C57BL/6J, BALB/cJ, F1 intercross identical with C57BL/6J X 129S3/SvIM, and 129S3/SvIM mouse strains by quantitative three-dimensional reconstruction confocal microscopy. Strain-related influences on pro- and antiangiogenic gene expression in naïve cornea were determined by quantitative real-time RT-PCR. RESULTS: The strain-dependent rank order of resting limbal vessel surface area and resting vessel density paralleled bFGF-induced neovascularization: 129S3/SvIM > BALB/cJ, F1 > C57BL/6J (P < 0.0006). Pigment epithelium-derived factor (PEDF) was increased more than 67-fold compared to Ang-2 in resting cornea of both C57BL/6J and 129S3/SvIM strains (P < 0.0001; P < 0.0001), suggesting a strongly antiangiogenic environment. The corneas of the C57BL/6J mice demonstrated 1.8-, 1.5-, and 1.7-fold increased mRNA levels for Flt-1, VEGF, and bFGF, respectively (P < 0.02; P < 0.04; P < 0.02); however, TSP-1 expression was increased 2.4-fold compared with 129S3/SvIM (P < 0.0004). CONCLUSIONS: Strain-dependent differences in the resting limbal vessel surface area and density correlated with heterogeneity in the extent of bFGF-induced angiogenesis. Differences in pro- and antiangiogenic gene expression levels in resting cornea may influence vascular limbal phenotype during quiescence and may predict susceptibility to angiogenesis-dependent diseases.

Interleukin-6 in retinal ischemia reperfusion injury in rats.

PURPOSE: To study the role of interleukin (IL)-6 after retinal ischemia-reperfusion (I/R) injury in rats. METHODS: Intraocular pressure of adult male Lewis albino rats was raised to create retinal ischemia for 1 hour. Retinal reperfusion was reestablished, and the animals were killed at various time points after the injury. Their eyes were enucleated and processed for immunohistochemistry to detect IL-6 and ED-1 (a marker of microglial/phagocytic cells), enzyme-linked immunosorbent assay (ELISA) of IL-6 protein, and semiquantitative real-time RT-PCR for IL-6 mRNA. The neuroprotective effect of IL-6 was evaluated by giving intravitreal injections of 150 or 300 ng rat recombinant IL-6 to eyes immediately after I/R injury and counting cresyl violet-stained retinal ganglion cell layer cells (RGCLCs) and fluorochrome-labeled retinal ganglion cells (RGCs) on flat preparations of retinas at 7 days. RESULTS: IL-6-positive cells appeared after I/R injury in the inner plexiform layer (IPL) and the inner nuclear layer (INL). Their numbers were significantly higher 18 hours after the injury, and most of these cells were also ED-1 positive. ELISA showed noticeable increases in endogenous retinal IL-6 protein levels 8 hours after I/R injury. Semiquantitative real-time RT-PCR showed significant increases in endogenous retinal IL-6 mRNA levels between 2 and 18 hours. Exogenously added IL-6 prevented between 50% and 70% of RGC loss after I/R injury. CONCLUSIONS: IL-6 is upregulated after retinal I/R injury, and its expression by microglia/phagocytic cells may protect RGC layer neurons from I/R injury. Exogenously added IL-6 protects the inner retina after I/R injury.

Matrix metalloproteinase-9-dependent exposure of a cryptic migratory control site in collagen is required before retinal angiogenesis.

Retinal neovascularization is a leading cause of human blindness. However, little is known concerning the molecular mechanisms controlling retinal neovascularization in vivo. Here we provide evidence that exposure of a collagen type IV cryptic epitope detected by monoclonal antibody (mAb) HUIV26, delineates sites of vascular bud formation and represents one of the earliest structural remodeling events required before vessel out-growth. Exposure of these cryptic sites was inhibited in matrix metalloproteinase (MMP)-9-deficient but not MMP-2-deficient mice implicating MMP-9 in their exposure. Retinal endothelial cell interactions with the HUIV26 epitopes induced endothelial cell migration, which was blocked by mAb HUIV26. Importantly, subcutaneous administration of mAb HUIV26 potently inhibited retinal angiogenesis in vivo. Taken together, these findings suggest a novel mechanism in which MMP-9 facilitates exposure of HUIV26 cryptic sites, thereby promoting retinal endothelial cell migration and neovascularization in vivo.