Subretinal AAV delivery of RNAi-therapeutics targeting VEGFA reduces choroidal neovascularization in a large animal model.

Neovascular age-related macular degeneration (nAMD) is a frequent cause of vision loss among the elderly in the Western world. Current disease management with repeated injections of anti-VEGF agents accumulates the risk for adverse events and constitutes a burden for society and the individual patient. Sustained suppression of VEGF using gene therapy is an attractive alternative, which we explored using adeno-associated virus (AAV)-based delivery of novel RNA interference (RNAi) effectors in a porcine model of choroidal neovascularization (CNV). The potency of VEGFA-targeting, Ago2-dependent short hairpin RNAs placed in pri-microRNA scaffolds (miR-agshRNA) was established in vitro and in vivo in mice. Subsequently, AAV serotype 8 (AAV2.8) vectors encoding VEGFA-targeting or irrelevant miR-agshRNAs under the control of a tissue-specific promotor were delivered to the porcine retina via subretinal injection before CNV induction by laser. Notably, VEGFA-targeting miR-agshRNAs resulted in a significant and sizable reduction of CNV compared with the non-targeting control. We also demonstrated that single-stranded and self-complementary AAV2.8 vectors efficiently transduce porcine retinal pigment epithelium cells but differ in their transduction characteristics and retinal safety. Collectively, our data demonstrated a robust anti-angiogenic effect of VEGFA-targeting miR-aghsRNAs in a large translational animal model, thereby suggesting AAV-based delivery of anti-VEGFA RNAi therapeutics as a valuable tool for the management of nAMD.

Porcine models of choroidal neovascularization: A systematic review.

Animal models of choroidal neovascularization (CNV) are extensively used in translational studies of CNV formation and to evaluate angiostatic treatment strategies. However, the current paucity of large animal models compared with rodent models constitutes a knowledge gap regarding the clinical translation of findings. Ocular anatomical and physiological similarities to humans suggest the pig as a relevant model animal. Thus, a systematic survey of porcine CNV models was performed to identify pertinent model parameters and suggest avenues for model standardization and optimization. A systematic search was performed in PubMed and EMBASE on November 28, 2022 for porcine models of CNV. Following inclusion by two investigators, data from the articles were extracted according to a predefined protocol. A total of 14 articles, representing 19 independent porcine CNV models were included. The included models were almost equally divided between laser-induced (53%) and surgically-induced (47%) models. Different specified breeds of domestic pigs (71%) were most commonly used in the studies. All studies used normal animals. Female pigs were reported used in 43% of the studies, while 43% did not report on sex of the animals. Younger pigs were typically used. The surgical models reported consistent CNV induction following mechanical Bruch's membrane rupture. The laser models used variants of the infrared diode laser (40%) or the frequency-doubled Nd:YAG laser (50%). Both lasers enabled successful CNV induction with reported induction rates ranging from 60 to 100%. Collateral damage to the neuroretina was reported for the infrared diode laser. CNV evaluation varied across studies with fluorescein angiography (50%) as the most used in vivo method and retinal sections (71%) as the most used ex vivo method. In interventional studies, quantification of lesions was in general performed between 7 and 14 days. The field of porcine CNV models is relatively small and heterogeneous and almost equally divided between surgically-induced and laser-induced models. Both methods have allowed successful modeling of CNV formation with induction rates comparable to those of non-human primates. However, the field would benefit from standardization of model parameters and reporting. This includes laser parameters and validation of CNV formation as well as methods of CNV evaluation and statistical analysis.

Animal Models of Choroidal Neovascularization: A Systematic Review.

Purpose: Animal models of choroidal neovascularization (CNV) are extensively used to characterize the pathophysiology of chorioretinal diseases with CNV formation and to evaluate novel treatment strategies. This systematic review aims to give a detailed overview of contemporary animal models of CNV. Methods: A systematic search was performed in PubMed and EMBASE from November 20, 2015, to November 20, 2020, for mammalian animal models of CNV. Following inclusion by two investigators, data from the articles were extracted according to a predefined protocol. Results: A total of 380 full articles, representing 409 independent animal models, were included. Mice were by far the most utilized animal (76%) followed by rats and non-human primates. The median age of rodents was 8 weeks but with a wide range. Male animals were used in 44% of the studies, but 32% did not report the sex. CNV was laser induced in 89% of the studies, but only 44% of these reported sufficiently on standard laser parameters. Surprisingly, 28% of the studies did not report a sample size for quantitative CNV evaluation. Less than half of the studies performed quantitative in vivo evaluation, and 73% evaluated CNV quantitatively ex vivo. Both in vivo and ex vivo evaluations were conducted primarily at day 7 and/or day 14. Conclusions: The laser-induced mouse model is the predominant model for experimental CNV. The widespread use of young, healthy male animals may complicate clinical translation, and inadequate reporting challenges reproducibility. Definition and implementation of standardized methodologic and reporting guidelines are attractive.

In vivo quantification of rolling and adhered leukocytes in human sepsis.

BACKGROUND: The use of in vivo videomicroscopy at the bedside has demonstrated microcirculatory flow disturbances in sepsis. The ability of in vivo videomicroscopy to detect changes in the prevalence of rolling and adhered leukocytes that occur in sepsis is not well-described in humans. We sought to (1) develop methodology for accessing and quantifying sublingual leukocyte rolling and adherence with sidestream dark field (SDF) imaging; (2) compare the number of rolling and adhered leukocytes between patients with septic shock and non-infected controls; and (3) compare the number of rolling and adhered leukocytes between survivors and non-survivors of septic shock. METHODS: We included adult (age > 18 years) patients in the emergency department presenting with septic shock prospectively enrolled in the ProCESS trial. We recruited comparison non-infected patients as emergency department controls. Using a SDF videomicroscope, we obtained image sequences from the sublingual mucosa, quantifying rolling and adhered leukocytes per 1 mm × 1 mm visual field in a standardized 3-s clip. We report data as median and interquartile range and depicted as box plots. We compared groups using the Mann-Whitney U test, considering a p value < 0.05 significant. RESULTS: We included a total of 64 patients with septic shock and 32 non-infected controls. The median number of adhered leukocytes per field in the sepsis group was 1.0 (IQR 0-3.5) compared to 0 (0-0) in the non-infected group (p < 0.001). The median number of rolling leukocytes was 26 (10.3-42) in the sepsis group and 9.8 (4.8-17.3) in the non-infected group (p < 0.001) per field. Among the patients with sepsis (n = 64), there was an increased number of adhered leukocytes in non-survivors compared to survivors (3.0 (1-5.5) vs. 1.0 (0-3.0)) (p < 0.05); however, there was no difference in rolling leukocytes (35 (20-48) vs. 26 (10-41)) (p = 0.31). CONCLUSIONS: Our results demonstrated a higher number of rolling and adhered leukocytes in patients with septic shock when compared to non-infected controls, and an increased number of adhered leukocytes in non-survivors. TRIAL REGISTRATION: ClinicalTrials.gov , NCT00793442 ; Registered on 19 November 2008 PG0GM076659 (US NIH Grant/Contract). First submitted 18 July 2007. First posted 2 August 2007.

Ocular albinism with infertility and late-onset sensorineural hearing loss.

Ocular albinism type 1 (OA1) is caused by mutations in the GPR143 gene located at Xp22.2. The manifestations, which are due to hypopigmentation, are confined to the eyes and optic pathway. OA1 associated with late-onset sensorineural hearing loss was previously reported in a single family and hypothesized to be caused by a contiguous gene deletion syndrome involving GPR143 and the adjacent gene, TBL1X. Here, we report on a family with OA1, infertility, late-onset sensorineural hearing loss, and a small interstitial Xp microdeletion including the GPR143, TBL1X, and SHROOM2 genes. In addition, we re-examined a patient previously described with OA1, infertility and a similar Xp deletion with audiologic follow-up showing a late-onset sensorineural hearing loss. Our results raise an intriguing question about the possibility for TBL1X (absence) involvement in this type of hearing loss. However, our study cannot claim a causative relationship and more convincing evidence is needed before the hypothesis can be accepted that TBL1X could be involved in late-onset sensorineural hearing loss and that ocular albinism with late-onset sensorineural hearing loss can present itself as a contiguous gene deletion/microdeletion syndrome. The finding of infertility in all affected male patients demonstrates that this deletion, including the SHROOM2 gene, may be a potentially causative X-linked genetic factor of male infertility.

Genotype-phenotype correlations, dystonia and disease progression in spinocerebellar ataxia type 14.

BACKGROUND: Spinocerebellar ataxia type 14 is a rare form of autosomal dominant cerebellar ataxia caused by mutations in protein kinase Cγ gene. Clinically, it presents with a slowly progressive, mainly pure cerebellar ataxia. METHODS: Using next generation sequencing, we screened 194 families with autosomal dominant cerebellar ataxia and normal polyglutamine repeats. In-depth phenotyping was performed using validated clinical rating scales neuroimaging and electrophysiological investigations. RESULTS: We identified 25 individuals from 13 families carrying pathogenic mutations in protein kinase Cγ gene. A total of 10 unique protein kinase Cγ gene mutations have been confirmed of which 5 are novel and 5 were previously described. Our data suggest that the age at onset is highly variable; disease course is slowly progressive and rarely associated with severe disability. However, one third of patients presented with a complex ataxia comprising severe focal and/or task-induced dystonia, peripheral neuropathy, parkinsonism, myoclonus, and pyramidal syndrome. The most complex phenotype is related to a missense mutation in the catalytic domain in exon 11. CONCLUSION: We present one of the largest genetically confirmed spinocerebellar ataxia type 14 cohorts contributing novel variants and clinical characterisation. We show that although protein kinase Cγ gene mutations present mainly as slowly progressive pure ataxia, more than a third of cases had a complex phenotype. Overall, our case series extends the phenotype and suggests that protein kinase Cγ gene mutations should be considered in patients with slowly progressive autosomal dominant cerebellar ataxia, particularly when myoclonus, dystonia, or mild cognitive impairment are present in the absence of polyglutamine expansion. © 2018 The Authors. Movement Disorders published by Wiley Periodicals, Inc. on behalf of International Parkinson and Movement Disorder Society.

Animal models of cardiac arrest: A systematic review of bias and reporting.

AIM OF THE REVIEW: Animal models are essential in advancing resuscitation research but are susceptible to various biases compromising internal validity, which may explain unsuccessful transition to human clinical trials. This study aimed to assess risk of bias in animal studies of cardiac arrest. DATA SOURCES: This study was based on a previous systematic review of all animal cardiac arrest studies published between March 8, 2011 and March 8, 2016 in PubMed and EMBASE. For this study, we focused on interventional studies and selected a random sample of 50 pig and 50 rat studies. We used a modified version of the SYRCLE's risk of bias tool for animal studies. Bias assessment was performed by two independent reviewers. RESULTS: 92% of pig studies and 88% of rat studies used randomization to assign interventions, but the methodology was unknown or insufficiently reported in 60% and 68% of the studies, respectively. Correct timing of randomization was lacking or unclear in over half of the studies. 40% of pig studies and 28% of rat studies reported insufficient baseline characteristics. When possible, blinding was not performed/reported in 68% of rat studies and 31% of pig studies. Blinding of outcome assessors was missing or inadequately reported in 65% of pig studies and 60% of rat studies. 80% of all studies lacked a sample size calculation, while 60% of pig and 80% of rat studies omitted a specified primary outcome. CONCLUSION: This study indicates insufficient reporting and methodological shortcomings in animal models of cardiac arrest.

Cytochrome c in patients undergoing coronary artery bypass grafting: A post hoc analysis of a randomized trial.

PURPOSE: To establish whether plasma cytochrome c is detectable in patients undergoing cardiac surgery, whether cytochrome c levels are associated with lactate/inflammatory markers/cellular oxygen consumption, and whether cytochrome c levels are associated with clinical outcomes. MATERIALS AND METHODS: This was an observational sub-study of a randomized trial comparing thiamine to placebo in patients undergoing coronary artery bypass grafting. Patients had blood drawn before, after, and again 6h after surgery. Cytochrome c, inflammatory markers, and cellular oxygen consumption were measured. RESULTS: 64 patients were included. Cytochrome c was detectable in 63 (98%) patients at baseline with a median cytochrome c level of 0.18ng/mL (quartiles: 0.13, 0.55). There was no difference from baseline level to post-surgical level (0.19ng/mL [0.09, 0.51], p=0.36) or between post-surgical level and 6-hour post-surgical level (0.17ng/mL [0.10, 0.57], p=0.61). There was no difference between the thiamine and placebo groups' change in cytochrome c levels from baseline to after surgery (p=0.22). Cytochrome c levels were not associated with lactate, inflammatory markers, cellular oxygen consumption, or clinical outcomes. CONCLUSIONS: Cytochrome c levels did not increase after cardiac surgery and was not associated with the degree of inflammation or clinical outcomes.

Contemporary animal models of cardiac arrest: A systematic review.

AIM OF THE STUDY: Animal models are widely used in cardiac arrest research. This systematic review aimed to provide an overview of contemporary animal models of cardiac arrest. METHODS: Using a comprehensive research strategy, we searched PubMed and EMBASE from March 8, 2011 to March 8, 2016 for cardiac arrest animal models. Two investigators reviewed titles and abstracts for full text inclusion from which data were extracted according to pre-defined definitions. RESULTS: Search criteria yielded 1741 unique titles and abstracts of which 490 full articles were included. The most common animals used were pigs (52%) followed by rats (35%) and mice (6%). Studies favored males (52%) over females (16%); 17% of studies included both sexes, while 14% omitted to report on sex. The most common methods for induction of cardiac arrest were either electrically-induced ventricular fibrillation (54%), asphyxia (25%), or potassium (8%). The median no-flow time was 8min (quartiles: 5, 8, range: 0-37min). The majority of studies used adrenaline during resuscitation (64%), while bicarbonate (17%), vasopressin (8%) and other drugs were used less prevalently. In 53% of the studies, the post-cardiac arrest observation time was ≥24h. Neurological function was an outcome in 48% of studies while 43% included assessment of a cardiac outcome. CONCLUSIONS: Multiple animal models of cardiac arrest exist. The great heterogeneity of these models along with great variability in definitions and reporting make comparisons between studies difficult. There is a need for standardization of animal cardiac arrest research and reporting.