Wound Healing in a Porcine Model of Retinal Holes.

Purpose: To investigate retinal wound healing, we created a new porcine model of retinal hole and identified the cells involved in hole closure. Methods: Sixteen landrace pigs underwent vitrectomy, and a subretinal bleb was created before cutting a retinal hole using a 23G vitrector. No tamponade was used. Before surgery and one, two, and four weeks after surgery, the eyes were examined by optical coherence tomography and color fundus photos. At the end of follow-up, the eyes were enucleated for histology. Tissue sections of 5 µm were prepared for hematoxylin-eosin staining and immunohistochemical analysis with antibodies to retinal glial and epithelial cells. Results: Retinal holes below 1380 µm in diameter closed spontaneously within four weeks, whereas larger holes remained open. Hole closure was mediated by central movement of the edges of the hole and in most cases the formation of a gliotic plug. Fluorescence microscopy revealed that the plug consisted of cells positive for glial fibrillary acidic protein, indicating the presence of macroglial cell types. Specifically, the plug was positive for S100 calcium-binding protein B, mainly representing astrocytes, while it was negative for anti-glutamine syntethase, representing Müller glia. These findings suggest that astrocytes are the predominating cell type in the plug. Minimal glial reaction was seen in the retinal holes that did not close. Conclusions: We present a new porcine model for investigating large retinal holes. The retinal holes closed by approximation of hole edges, and the remnant retinal defect was closed with an astroglial plug.

Controlled Subretinal Injection Pressure Prevents Damage in Pigs.

INTRODUCTION: Administration of retinal gene and stem cell therapy in patients with retinal degenerative diseases is in many cases dependent on a subretinal approach. It has been indicated that manual subretinal injection is associated with outer retinal damage, which may be explained by a high flow rate in the injection cannula. In the present porcine study, we evaluated flow-related retinal damage after controlled subretinal injection at different flow rates. METHODS: The flow rate through a 41G cannula was estimated at different injection pressures (6-48 pounds per square inch [PSI]) in an in vitro setup. A linear correlation between the flow rate and injection pressure was found from 6 to 32 PSI. In full anesthesia, 12 pigs were vitrectomized and received a controlled subretinal injection of 300 µL balanced saline solution at injection pressures of 14, 24, and 32 PSI (four in each group). Prior to surgery and 2 and 4 weeks after surgery, the eyes were examined by multifocal electroretinogram (mfERG) and fundus photographs. At the end of follow-up, the eyes were enucleated for histology. RESULTS: The in vitro flow study determined that the flow in a 41G cannula shifts from laminar to turbulent at 32 PSI and that the manual injection flow is turbulent. In the porcine study, we showed a significant difference in retinal pigment epithelium (RPE) damage between the three pressure groups (p = 0.0096). There was no significant difference in damage to the outer retina (p = 0.1526), but the high-pressure group (32 PSI) had the most outer retinal damage. The middle-pressure group (24 PSI) showed minimum retinal damage. There was no significant change in the mfERG ratios during follow-up. DISCUSSION/CONCLUSION: This study indicates that an injection pressure at approximately 24 PSI might be safe for subretinal delivery. Retinal damage at low injection pressures may be explained by mechanical damage to the RPE due to prolonged needle time in the subretinal space, while retinal damage at high pressures can be related to high flow in the injection cannula. Controlled subretinal injection pressure of 24 PSI showed minimum mechanical- and flow-related damage to the porcine retina.

Systemic vascular air embolus following CT-guided transthoracic needle biopsy: a potentially fatal complication.

Following an uncomplicated CT-guided transthoracic biopsy, a patient becomes unconscious and subsequently dies despite immediate cardiac resuscitation. The patient felt well during the procedure but started complaining about dizziness and chest pain when he sat up. When he again was put in a supine position, cardiac arrest was noted. A CT scan performed when the symptoms initiated was afterwards rigorously reviewed by the team and revealed air located in the left ventricle, aorta and right coronary artery.We present a rare but potentially lethal complication following CT-guided transthoracic needle biopsy-systemic vascular air embolus. Knowledge and evidence about the complication are sparse because of low incidence and varying presentation. However, immediate initiation of treatment can save a life, and awareness of the complication is therefore crucial.

Patients Own Safety Incidents Reports to the Danish Patient Safety Database Possess a Unique but Underused Learning Potential in Patient Safety.

OBJECTIVE: The aim of the study was to analyze how patients' own reports of safety incidents to the Danish Patient Safety Database can contribute to patient safety. BACKGROUND: Patient involvement enhances patient safety; however, there is a shortage of tools capable of systematically capturing and usage of patients' own reports. Since 2012, the Danish Patient Safety Database (DPSD) has comprised such a tool. METHODS: A total of 209,263 incident reports were compared across the following four reporting groups: patients, relatives, doctors, and nurses. Using thematic comparison, 300 narratives from each group were compared with respect to differences and similarities in the wording of the safety incident. RESULTS: Only a tiny proportion of safety incidents were reported by patients' themselves (1.4%). Most of these (86%) were accepted for processing in the DPSD. Almost 90% of the accepted incidents were classified successfully. Patients' own reports were longer, more often "less severe incidents," and more often reported by female patients. Thematic content analyses revealed incident descriptions from health professionals as terse, unemotional, and extensively using medical terminology and abbreviations. In contrast patients' reports were lengthy, emotional, and focused on relations to health personal, health consequences, and communication errors. CONCLUSIONS: Despite the very low ratio of patients reporting an observed incident to DPSD, the finding that most patients own reports are accepted and classified makes the DPSD a promising, comprehensive tool to capture patients' own reports. However, the rich, contextualized descriptions seem insufficiently captured by established nomenclature.

Neurotrophic Effects of Vascular Endothelial Growth Factor B and Novel Mimetic Peptides on Neurons from the Central Nervous System.

Vascular endothelial growth factor B (VEGFB) is a pleiotropic trophic factor, which in contrast to the closely related VEGFA is known to have a limited effect on angiogenesis. VEGFB improves survival in various tissues including the nervous system, where the effect was observed mainly for peripheral neurons. The neurotrophic effect of VEGFB on central nervous system neurons has been less investigated. Here we demonstrated that VEGFB promotes neurite outgrowth from primary cerebellar granule, hippocampal, and retinal neurons in vitro. VEGFB protected hippocampal and retinal neurons from both oxidative stress and glutamate-induced neuronal death. The VEGF receptor 1 (VEGFR1) is required for VEGFB-induced neurotrophic and neuroprotective effects. Using a structure-based approach, we designed short peptides, termed Vefin1-7, mimicking the binding interface of VEGFB to VEGFR1. Vefins were analyzed for their secondary structure and binding to VEGF receptors and compared with previously described peptides derived from VEGFA, another ligand of VEGFR1. We show that Vefins have neurotrophic and neuroprotective effects on primary hippocampal, cerebellar granule, and retinal neurons in vitro with potencies comparable to VEGFB. Similar to VEGFB, Vefins were not mitogenic for MCF-7 cancer cells. Furthermore, one of the peptides, Vefin7, even dose-dependently inhibited the proliferation of MCF-7 cells in vitro. Unraveling the neurotrophic and neuroprotective potentials of VEGFB, the only nonangiogenic factor of the VEGF family, is promising for the development of neuroprotective peptide-based therapies.

Loss of retinal tension and permanent decrease in retinal function: a new porcine model of rhegmatogenous retinal detachment.

PURPOSE: Permanent loss of visual function after rhegmatogenous retinal detachment can occur despite successful surgical reattachment in humans. New treatment modalities could be explored in a detachment model with loss of retinal function. In previous porcine models, retinal function has returned after reattachment, regardless of height and duration of detachment. Difference in retinal tension between the models and the disease might explain these different outcomes. This study investigates, for the first time in an in vivo porcine model, another characteristic of rhegmatogenous retinal detachment - the loss of retinal tension. METHODS: Left eyes (n = 12) of 3-month-old domestic pigs were included. Baseline multifocal electroretinogram (mfERG) and a fundus photograph were obtained following anaesthesia (isoflurane). The pigs were vitrectomized, saline was injected subretinally, and the RPE was removed. The eyes were evaluated at 2, 4 and 6 weeks after surgery. Four eyes were enucleated at each evaluation for histologic examinations. RESULTS: A retinal detachment structurally resembling rhegmatogenous retinal detachment was induced in 11 out of 12 pigs. MfERG amplitudes were significantly decreased despite partial reattachment four and 6 weeks after detachment. The retinal thickness decreased with 27%, the inner nuclear layer degenerated, Müller cells hypertrophied, and outer segments were lost. In the ganglion cell layer, cellularity increased and there was cytoplasmic staining with Cyclin D1. Vimentin and GFAP staining for glial cells increased. After 2 weeks of detachment, the ganglion cells had lost their nucleus and nucleolus. CONCLUSIONS: Loss of retinal tension in the detached retina seems to induce permanent damage with loss of retinal function. Death of ganglion cells, observed as soon as 2 weeks after detachment, explains the permanent loss of retinal function. The new model enables investigations of time-relationship between retinal detachment and lasting damage in addition to exploration of novel treatment modalities.

Bruch's membrane allows unhindered passage of up to 2 µm latex beads in an in vivo porcine model.

PURPOSE: It has been proposed that changes in the permeability of Bruch's membrane play a role in the pathogenesis of age-related macular degeneration (AMD). This paper investigates, in an in vivo porcine model, the migration of fluorescent latex beads across the Bruch's membrane after subretinal injection. METHODS: Forty-one healthy eyes of 33 three-month-old domestic pigs received a subretinal injection of 0.5, 1.0, 2.0, or 4.0 µm fluorescent latex beads. Between three hours and five weeks after injection evaluations were performed with fundus photographs and histology. Fluorescent beads were identified in unstained histologic sections using the rhodamine filter with the light microscope. RESULTS: The fluorescent latex beads relocated from the subretinal space. Intact beads up to 2.0 µm were found in the choroid, sclera, and extrascleral space. The smaller beads were also found inside choroidal and extrascleral blood vessels. In contrast, the larger beads of 4.0 µm did not pass the Bruch's membrane. CONCLUSION: Subretinally implanted beads up to 2.0 µm pass the Bruch's membrane intact and cross the blood-ocular barrier. The intact beads are found in the choroid, sclera and inside blood vessels. The results give reason to consider the role of subretinal clearance and passage of Bruch's membrane in the development of AMD.

Retrospective analysis of radial EBUS outcome for the diagnosis of peripheral pulmonary lesion: sensitivity and complications.

BACKGROUND: The purpose of the current study was to clarify the sensitivity and complication rate of the radial (endobronchial ultrasound, EBUS) without the use of guide-sheath (GS) and fluoroscopy for lung cancer (LC), by measuring the distance from the orifice of the bronchus to the pulmonary lesion, as well as to analyze factors that can predict the diagnostic outcome. MATERIALS AND METHODS: A total of 147 patients with peripheral pulmonary lesions (PPL) underwent radial EBUS-guided transbronchial biopsy (TBB) in between August 1, 2013, and August 31, 2014. We analyzed retrospectively radiological data, diagnostic work-up in everyday clinical settings, final diagnosis and complication rates, as well as factors influencing the diagnostic outcome. RESULTS: Around 63.9% of PPLs were visualized by ultrasound. A definitive malignant diagnosis was established in 39 patients (26.5%) using radial EBUS. In the remaining 108 patients, additional procedures were performed. We missed LC diagnosis in 40 cases that results in a sensitivity of 49%. For malignant lesions visualized by radial EBUS, the sensitivity was 60%, compared with 24% for not visualized lesions. For malignant lesions, logistic regression was performed to identify the factors that had significant influence on visualization of the lesion and on diagnostic yield. Logistic regression analysis showed significant odds ratios (OR) for visualization depending on location of the lesion; upper lobe lesions were identified more frequent with OR of 3.85 (95% CI 1.42 - 10.98, p=0.009). Size above 30 mm had a non-significant OR of 2.11 (95% CI 0.80-5.73, p=0.134) for visualization.Diagnostic yield was only significantly influenced by visualization with the radial EBUS, OR 3.70 (95% CI 1.35-11.02, p=0.014). Location (p=0.745) and size above 30 mm (p=0.308) showed no significant increase in diagnostic yield.Other lesion characteristics defined on computed tomography, such as distance to carina and pleura, did not show any significant influence on the diagnostic yield. The complications rate was low with three cases of pneumothorax. CONCLUSION: Radial EBUS has definitely its place in the diagnostic work-up of PPL, especially for the lesions that can be visualized by radial ultrasound. However, prospective randomized controlled studies are necessary to raise the diagnostic yield and to define factors that can predict the outcome, which will consequently enable selection of the 'right' patients for this diagnostic procedure.

The functional role of the novel biomarker karyopherin α 2 (KPNA2) in cancer.

In recent years, Karyopherin α 2 (KPNA2) has emerged as a potential biomarker in multiple cancer forms. The aberrant high levels observed in cancer tissue have been associated with adverse patient characteristics, prompting the idea that KPNA2 plays a role in carcinogenesis. This notion is supported by studies in cancer cells, where KPNA2 deregulation has been demonstrated to affect malignant transformation. By virtue of its role in nucleocytoplasmic transport, KPNA2 is implicated in the translocation of several cancer-associated proteins. We provide an overview of the clinical studies that have established the biomarker potential of KPNA2 and describe its functional role with an emphasis on established associations with cancer.

Diagnosis of bladder cancer recurrence based on urinary levels of EOMES, HOXA9, POU4F2, TWIST1, VIM, and ZNF154 hypermethylation.

BACKGROUND: Non muscle invasive bladder cancer (NMIBC) has the highest recurrence rate of any malignancy and as many as 70% of patients experience relapse. Aberrant DNA methylation is present in all bladder tumors and can be detected in urine specimens. Previous studies have identified DNA methylation markers that showed significant diagnostic value. We evaluated the significance of the biomarkers for early detection of tumor recurrence in urine. METHODOLOGY/PRINCIPAL FINDINGS: The methylation levels of EOMES, HOXA9, POU4F2, TWIST1, VIM, and ZNF154 in urine specimens were measured by real-time PCR (MethyLight). We analyzed 390 urine sediments from 184 patients diagnosed with NMIBC. Urine from 35 age-matched control individuals was used to determine the methylation baseline levels. Recurrence was diagnosed by cystoscopy and verified by histology. Initially, we compared urine from bladder cancer patients and healthy individuals and detected significant hypermethylation of all six markers (P<0.0001) achieving sensitivity in the range 82%-89% and specificity in the range 94%-100%. Following, we validated the urinary hypermethylation for use in recurrence surveillance and found sensitivities of 88-94% and specificities of 43-67%. EOMES, POU4F2, VIM and ZNF154 were more frequently methylated in urine from patients with higher grade tumors (P ≤ 0.08). Univariate Cox regression analysis showed that five markers were significantly associated with disease recurrence; HOXA9 (HR=7.8, P=0.006), POU4F2 (HR=8.5, P=0.001), TWIST1 (HR=12.0, P=0.015), VIM (HR=8.0, P=0.001), and ZNF154 (HR=13.9, P<0.001). Interestingly, for one group of patients (n=15) we found that hypermethylation was consistently present in the urine samples despite the lack of tumor recurrences, indicating the presence of a field defect. CONCLUSION/SIGNIFICANCE: Methylation levels of EOMES, HOXA9, POU4F2, TWIST1, VIM, and ZNF154 in urine specimens are promising diagnostic biomarkers for bladder cancer recurrence surveillance.