Intraretinal hemorrhages and detailed retinal phenotype of three patients with Alagille syndrome.

PURPOSE: To explore patterns of disease expression in Alagille syndrome (ALGS). METHODS: Patients underwent ophthalmic examination, optical coherence tomography (OCT) imaging, fundus intravenous fluorescein angiography (IVFA), perimetry and full-field electroretinograms (ffERGs). An adult ALGS patient had multimodal imaging and specialized perimetry. RESULTS: The proband (P1) had a heterozygous pathogenic variant in JAG1; (p.Gln410Ter) and was incidentally diagnosed at age 7 with a superficial retinal hemorrhage, vascular tortuosity, and midperipheral pigmentary changes. The hemorrhage recurred 15 months later. Her monozygotic twin sister (P2) had a retinal hemorrhage at the same location at age 11. Visual acuities for both patients were 20/30 in each eye. IVFA was normal. OCT showed thinning of the outer nuclear in the peripapillary retina. A ffERG showed normal cone-mediated responses in P1 (rod-mediated ERGs not documented), normal ffERGs in P2. Coagulation and liver function were normal. An unrelated 42-year-old woman with a de-novo pathogenic variant (p. Gly386Arg) in JAG1 showed a similar pigmentary retinopathy and hepatic vascular anomalies; rod and cone function was normal across large expanses of structurally normal retina that sharply transitioned to a blind atrophic peripheral retina. CONCLUSION: Nearly identical recurrent intraretinal hemorrhages in monozygotic twins with ALGS suggest a shared subclinical microvascular abnormality. We hypothesize that the presence of large areas of functionally and structurally intact retina surrounded by severe chorioretinal degeneration, is against a predominant involvement of JAG1 in the function of the neurosensory retina, and that instead, primary abnormalities of chorioretinal vascular development and/or homeostasis may drive the peculiar phenotypes.

Reduced Expression of TMEM16A Impairs Nitric Oxide-Dependent Cl- Transport in Retinal Amacrine Cells.

Postsynaptic cytosolic Cl- concentration determines whether GABAergic and glycinergic synapses are inhibitory or excitatory. We have shown that nitric oxide (NO) initiates the release of Cl- from acidic internal stores into the cytosol of retinal amacrine cells (ACs) thereby elevating cytosolic Cl-. In addition, we found that cystic fibrosis transmembrane conductance regulator (CFTR) expression and Ca2+ elevations are necessary for the transient effects of NO on cytosolic Cl- levels, but the mechanism remains to be elucidated. Here, we investigated the involvement of TMEM16A as a possible link between Ca2+ elevations and cytosolic Cl- release. TMEM16A is a Ca2+-activated Cl- channel that is functionally coupled with CFTR in epithelia. Both proteins are also expressed in neurons. Based on this and its Ca2+ dependence, we test the hypothesis that TMEM16A participates in the NO-dependent elevation in cytosolic Cl- in ACs. Chick retina ACs express TMEM16A as shown by Western blot analysis, single-cell PCR, and immunocytochemistry. Electrophysiology experiments demonstrate that TMEM16A functions in amacrine cells. Pharmacological inhibition of TMEM16A with T16inh-AO1 reduces the NO-dependent Cl- release as indicated by the diminished shift in the reversal potential of GABAA receptor-mediated currents. We confirmed the involvement of TMEM16A in the NO-dependent Cl- release using CRISPR/Cas9 knockdown of TMEM16A. Two different modalities targeting the gene for TMEM16A (ANO1) were tested in retinal amacrine cells: an all-in-one plasmid vector and crRNA/tracrRNA/Cas9 ribonucleoprotein. The all-in-one CRISPR/Cas9 modality did not change the expression of TMEM16A protein and produced no change in the response to NO. However, TMEM16A-specific crRNA/tracrRNA/Cas9 ribonucleoprotein effectively reduces both TMEM16A protein levels and the NO-dependent shift in the reversal potential of GABA-gated currents. These results show that TMEM16A plays a role in the NO-dependent Cl- release from retinal ACs.

Preliminary Exploration of Weekly Peer Group Discussions as a Strategy for Coping with Feelings Associated with Euthanasia in Dairy Caretakers.

Dairy caretakers experience a variety of occupational risks including stress related to performing euthanasia and making euthanasia-related decisions for cattle in their care. Few supportive interventions exist to help caretakers cope with euthanasia-related stress. The aim of this study was to assess the impact of weekly peer discussion sessions as an intervention to reduce euthanasia-related stress and grief in dairy caretakers. This study utilized scores from a modified pet-based bereavement questionnaire to assess the change in bereavement of caretakers in response to euthanasia-related stress in a non-treatment group (who did not attend peer discussion sessions, n = 7) and a treatment group (who attended peer discussion sessions, n = 15). Key findings of this study were that discussion sessions did not have a direct impact on the study outcomes as measured using a pet bereavement scale, as there was no difference in the change in bereavement scores during the 8 week study period between the treatment and non-treatment groups. Thematic analysis of peer discussions revealed that compassion towards dairy cattle is a prominent factor in areas of decision making, protocols, and training. Further studies should continue to explore how performing euthanasia and making euthanasia-related decisions impacts caretakers and what supportive interventions can reduce stress and grief.

Dairy Caretaker Perspectives on Performing Euthanasia as an Essential Component of Their Job.

The objectives of this study were to identify caretakers' perceptions and stressors related to performing on-farm euthanasia as well as potential ideas for intervention strategies to alleviate negative effects of performing euthanasia on caretaker well-being. Additionally, we aimed to determine how euthanasia methods, procedures, and training correlate with dairy caretakers' attitudes toward performing on-farm euthanasia and their job satisfaction and sense of well-being. Thirty-eight dairy caretakers (19 workers, 15 supervisors, 3 owners, 1 veterinarian) participated in focus groups or interviews conducted and recorded on five Northern Colorado dairies. Thematic analysis of focus group and interview transcripts revealed seven recurring themes. Variation in available training and euthanasia protocols among dairy farms was evident. There was a lack of awareness regarding available mental health resources and little communication between farm personnel about euthanasia-related stress. Training was correlated with caretaker knowledge about euthanasia procedures and the language used to refer to euthanasia. Human-animal bonds and empathy toward animals were evident in participants regardless of training, job position, or dairy experience. Interventions such as training programs, mental health resources, and other support systems should be tested for effectiveness in addressing euthanasia-related stressors, improving euthanasia decision-making and practice, and increasing animal wellbeing on dairy farms.

Preliminary Study Exploring Caretaker Perspectives of Euthanasia on Swine Operations.

The purpose of this study was to gain a better understanding of euthanasia training, caretaker perceptions of euthanasia, and available resources for individuals who perform euthanasia as part of their job on swine operations in the United States. An online survey was distributed via an e-newsletter and in-person recruitment at swine industry events. Survey questions were related to training, attitudes towards performing euthanasia, work environment, and communication. Forty-five responses (17 workers, 21 managers, 7 owners) were recorded and summarized. The majority of workers (n = 14, 82%) agreed that they had "received enough training to euthanize pigs correctly" and that training had made them "confident about performing euthanasia", yet 35% (6) also indicated they would like more euthanasia training. Less than one-third of workers indicated that strategies for dealing with "personal stress" (4, 24%) and "emotional wellness" 29% (5) were included in training programs but the majority (14, 82%) agreed that "trainings included human safety while performing euthanasia". Most caretakers (37, 82%) agreed that they felt they could "communicate with my supervisors" if they felt uncomfortable performing euthanasia. Opportunities for the future include enhancing euthanasia training opportunities and content to include more awareness of strategies to deal with stress related to euthanasia.

Preliminary Exploration of Swine Veterinarian Perspectives of On-Farm Euthanasia.

Euthanasia is a critical component in swine production and veterinarians play an important role in euthanasia protocol development and training. This study aimed to understand veterinarian involvement in and perspectives on euthanasia on pig farms. An online survey was disseminated both at a pig welfare conference and online via a veterinarian e-newsletter. Twenty-five veterinarians participated in the survey. The majority of respondents indicated that caretakers are the individuals making euthanasia decisions and performing the task (n = 17, 68% and 22, 88%, respectively). The majority (22, 88%) of respondents indicated that most of the facilities with which they work have a written euthanasia protocol, and 72% (18) indicated that they assisted in protocol development. Only half of respondents (13, 52%) agreed that "all employees performing euthanasia have been trained adequately", and 80% (20) identified an interest in delivering more training. Less than half the respondents indicated that strategies for coping with "personal stress" and "emotional wellness" (12, 48%) were included in euthanasia training. While the moral stress of performing euthanasia is recognized, there is opportunity for addressing mental well-being in euthanasia resources. Although preliminary, this study supports the need for further euthanasia training on-farm, involving veterinarians in the process.