Sphingosine-1-phosphate signaling regulates the ability of Müller glia to become neurogenic, proliferating progenitor-like cells.

The purpose of these studies is to investigate how Sphingosine-1-phosphate (S1P) signaling regulates glial phenotype, dedifferentiation of Müller glia (MG), reprogramming into proliferating MG-derived progenitor cells (MGPCs), and neuronal differentiation of the progeny of MGPCs. We found that S1P-related genes are highly expressed by retinal neurons and glia, and levels of expression were dynamically regulated following retinal damage. S1PR1 is highly expressed by resting MG and is rapidly downregulated following acute retinal damage. Drug treatments that activate S1PR1 or increase levels of S1P suppressed the formation of MGPCs, whereas treatments that inhibit S1PR1 or decreased levels of S1P stimulated the formation of MGPCs. Inhibition of S1PR1 or SPHK1 significantly enhanced the neuronal differentiation of the progeny of MGPCs. Further, ablation of microglia from the retina, wherein the formation of MGPCs in damaged retinas is impaired, has a significant impact upon expression patterns of S1P-related genes in MG. Inhibition of S1PR1 and SPHK1 partially rescued the formation of MGPCs in damaged retinas missing microglia. Finally, we show that TGFß/Smad3 signaling in the resting retina maintains S1PR1 expression in MG. We conclude that the S1P signaling is dynamically regulated in MG and MGPCs and activation of S1P signaling depends, in part, on signals produced by reactive microglia.

Status-dependent metabolic effects of social interactions in a group-living fish.

Social interactions can sometimes be a source of stress, but social companions can also ameliorate and buffer against stress. Stress and metabolism are closely linked, but the degree to which social companions modulate metabolic responses during stressful situations-and whether such effects differ depending on social rank-is poorly understood. To investigate this question, we studied Neolamprologus pulcher, a group-living cichlid fish endemic to Lake Tanganyika and measured the metabolic responses of dominant and subordinate individuals when they were either visible or concealed from one another. When individuals could see each other, subordinates had lower maximum metabolic rates and tended to take longer to recover following an exhaustive chase compared with dominants. In contrast, metabolic responses of dominants and subordinates did not differ when individuals could not see one another. These findings suggest that the presence of a dominant individual has negative metabolic consequences for subordinates, even in stable social groups with strong prosocial relationships.

Variability of faecal microbiota and antibiotic resistance genes in flocks of migratory gulls and comparison with the surrounding environment.

Gulls commonly rely on human-generated waste as their primary food source, contributing to the spread of antibiotic-resistant bacteria and their resistance genes, both locally and globally. Our understanding of this process remains incomplete, particularly in relation to its potential interaction with surrounding soil and water. We studied the lesser black-backed gull, Larus fuscus, as a model to examine the spatial variation of faecal bacterial communities, antibiotic resistance genes (ARGs), and mobile genetic elements (MGEs) and its relationship with the surrounding water and soil. We conducted sampling campaigns within a connectivity network of different flocks of gulls moving across functional units (FUs), each of which represents a module of highly interconnected patches of habitats used for roosting and feeding. The FUs vary in habitat use, with some gulls using more polluted sites (notably landfills), while others prefer more natural environments (e.g., wetlands or beaches). Faecal bacterial communities in gulls from flocks that visit and spend more time in landfills exhibited higher richness and diversity. The faecal microbiota showed a high compositional overlap with bacterial communities in soil. The overlap was greater when compared to landfill (11%) than to wetland soils (6%), and much lower when compared to bacterial communities in surrounding water (2% and 1% for landfill and wetland water, respectively). The relative abundance of ARGs and MGEs were similar between FUs, with variations observed only for specific families of ARGs and MGEs. When exploring the faecal carriage of ARGs and MGEs in bird faeces relative to soil and water compartments, gull faeces were enriched in ARGs classified as High-Risk. Our results shed light on the complex dynamics of antibiotic resistance spread in wild bird populations, providing insights into the interactions among gull movement and feeding behavior, habitat characteristics, and the dissemination of antibiotic resistance determinants across environmental reservoirs.

Comparative Analysis of the Clinical Presentation of Individuals Who Test Positive or Negative for SARS-CoV-2: Results from a Test Street Study.

The common cold, the flu, and the 2019 coronavirus disease (COVID-19) have many symptoms in common. As such, without testing for severe-acute-respiratory-syndrome-related coronavirus 2 (SARS-CoV-2), it is difficult to conclude whether or not one is infected with SARS-CoV-2. The aim of the current study was to compare the presence and severity of COVID-19-related symptoms among those who tested positive or negative for the beta variant of SARS-CoV-2 (B.1.351) and identify the clinical presentation with the greatest likelihood of testing positive for SARS-CoV-2. n = 925 individuals that were tested for SARS-CoV-2 at Dutch mass testing sites (i.e., test streets) were invited to complete a short online survey. The presence and severity of 17 COVID-19-related symptoms were assessed. In addition, mood, health correlates, and quality of life were assessed for the week before the test. Of the sample, n = 88 tested positive and n = 837 tested negative for SARS-CoV-2. Individuals who tested positive for SARS-CoV-2 reported experiencing a significantly greater number, as well as greater overall symptom severity, compared to individuals who tested negative for SARS-CoV-2. A binary logistic regression analysis revealed that increased severity levels of congestion, coughing, shivering, or loss of smell were associated with an increase in the odds of testing positive for SARS-CoV-2, whereas an increase in the severity levels of runny nose, sore throat, or fatigue were associated with an increase in the odds of testing negative for SARS-CoV-2. No significant differences in mood or health correlates were found between those who tested positive or negative for SARS-CoV-2, except for a significantly higher stress score among those who tested negative for SARS-CoV-2. In conclusion, individuals that tested positive for SARS-CoV-2 experienced a significantly greater number and more severe COVID-19-related symptoms compared to those who tested negative for SARS-CoV-2. Experiencing shivering and loss of smell may be the best indicators for increased likelihood of testing positive for SARS-CoV-2.

Associations between news coverage, social media discussions, and search trends about celebrity deaths, screening, and other colorectal cancer-related events.

OBJECTIVE: Colorectal cancer (CRC) is the third leading cause of cancer death among both men and women in the United States. CRC-related events may increase media coverage and public attention, boosting awareness and prevention. This study examined associations between several types of CRC events (including unplanned celebrity cancer deaths and planned events like national CRC awareness months, celebrity screening behavior, and screening guideline changes) and news coverage, Twitter discussions, and Google search trends about CRC and CRC screening. METHODS: We analyzed data from U.S. national news media outlets, posts scraped from Twitter, and Google Trends on CRC and CRC screening during a three-year period from 2020 to 2022. We used burst detection methods to identify temporal spikes in the volume of news, tweets, and search after each CRC-related event. RESULTS: There is a high level of heterogeneity in the impact of celebrity CRC events. Celebrity CRC deaths were more likely to precede spikes in news and tweets about CRC overall than CRC screening. Celebrity screening preceded spikes in news and tweets about screening but not searches. Awareness months and screening guideline changes did precede spikes in news, tweets, and searches about screening, but these spikes were inconsistent, not simultaneous, and not as large as those events concerning most prominent public figures. CONCLUSIONS: CRC events provide opportunities to increase attention to CRC. Media and public health professionals should actively intervene during CRC events to increase emphasis on CRC screening and evidence-based recommendations.

Minimally invasive nasal infusion (MINI) approach for CNS delivery of protein therapeutics: A case study with ovalbumin.

One of the primary obstacles in treating central nervous system (CNS) disorders lies in the limited ability of disease-modifying drugs to cross the blood-brain barrier (BBB). Our previously described Minimally Invasive Nasal Depot (MIND) technique has proven successful in delivering various drugs to the brain in rat models via a trans-olfactory mucosal approach. In this study, we introduce a novel Minimally Invasive Nasal Infusion (MINI) delivery approach for administering ovalbumin, a model protein, utilizing a programmable infusion pump (iPRECIO SMP-310R) in a mouse model. This research highlights the significant role of olfactory mucosa in nose-to-brain delivery, with an efficacy of nearly 45% compared to intracerebroventricular (ICV) administration. This demonstrates its potential as an alternative procedure for treating CNS diseases, offering a greater safety profile relative to the highly invasive clinical routes traditionally adopted for CNS drug delivery.

Combined JAK inhibition and PD-1 immunotherapy for non-small cell lung cancer patients.

Persistent inflammation driven by cytokines such as type-one interferon (IFN-I) can cause immunosuppression. We show that administration of the Janus kinase 1 (JAK1) inhibitor itacitinib after anti-PD-1 (programmed cell death protein 1) immunotherapy improves immune function and antitumor responses in mice and results in high response rates (67%) in a phase 2 clinical trial for metastatic non-small cell lung cancer. Patients who failed to respond to initial anti-PD-1 immunotherapy but responded after addition of itacitinib had multiple features of poor immune function to anti-PD-1 alone that improved after JAK inhibition. Itacitinib promoted CD8 T cell plasticity and therapeutic responses of exhausted and effector memory-like T cell clonotypes. Patients with persistent inflammation refractory to itacitinib showed progressive CD8 T cell terminal differentiation and progressive disease. Thus, JAK inhibition may improve the efficacy of anti-PD-1 immunotherapy by pivoting T cell differentiation dynamics.

Historical baleen plates indicate that once abundant Antarctic blue and fin whales demonstrated distinct migratory and foraging strategies.

Southern hemisphere blue (Balaenoptera musculus intermedia) and fin (Balaenoptera physalus) whales are the largest predators in the Southern Ocean, with similarities in morphology and distribution. Yet, understanding of their life history and foraging is limited due to current low abundances and limited ecological data. To address these gaps, historic Antarctic blue (n = 5) and fin (n = 5) whale baleen plates, collected in 1947-1948 and recently rediscovered in the Smithsonian National Museum of Natural History, were analyzed for bulk (δ13C and δ15N) stable isotopes. Regular oscillations in isotopic ratios, interpreted as annual cycles, revealed that baleen plates contain approximately 6 years (14.35 ± 1.20 cm year-1) of life history data in blue whales and 4 years (16.52 ± 1.86 cm year-1) in fin whales. Isotopic results suggest that: (1) while in the Southern Ocean, blue and fin whales likely fed at the same trophic level but demonstrated niche differentiation; (2) fin whales appear to have had more regular annual migrations; and (3) fin whales may have migrated to ecologically distinct sub-Antarctic waters annually while some blue whales may have resided year-round in the Southern Ocean. These results reveal differences in ecological niche and life history strategies between Antarctic blue and fin whales during a time period when their populations were more abundant than today, and before major human-driven climatic changes occurred in the Southern Ocean.

Demographic and conservation genomic assessment of the threatened marbled teal (Marmaronetta angustirostris).

Genetic assessment of species that have experienced dramatic population declines provides critical information that is instrumental for the design of conservation recovery programs. Here, we use different sources of molecular data (mtDNA and ddRAD-seq) to evaluate the genetic status of wild and captive populations of marbled teal (Marmaronetta angustirostris), a duck species classified as critically endangered in Spain and near threatened at a global scale. First, we determined the evolutionary and demographic trajectories of the wild population from Spain and the currently much larger population from Iraq, which is also the documented source of European zoo stocks. Second, we evaluated the suitability of the different captive populations for ongoing restocking programs in Spain and assessed their potential impact on the genetic composition of wild populations. Populations from Spain and Iraq were assigned to distinct genetic clusters, albeit with an overall low level of genetic differentiation, in line with their recent divergence (<8000 years ago) and lack of phylogeographic structure in the species. Demogenomic inferences revealed that the two populations have experienced parallel demographic trajectories, with a marked bottleneck during the last glacial period followed by a sudden demographic expansion and stability since the onset of the Holocene. The wild population from Spain presented high levels of inbreeding, but we found no evidence of recent genetic bottlenecks compatible with the human-driven decline of the species during the past century. The captive populations from the two Spanish centers involved in restocking programs showed genetic introgression from European zoos; however, we found limited evidence of introgression from the zoo genetic stock into the wild population from Spain, suggesting captive-bred birds have limited breeding success in the wild. Our study illustrates how ex situ conservation programs should consider the genetic distinctiveness of populations when establishing breeding stocks and highlights the importance of genetically assessing captive populations prior to reinforcement actions.

Effects of structural remodelling on gill physiology.

The complex relationships between the structure and function of fish gills have been of interest to comparative physiologists for many years. Morphological plasticity of the gill provides a dynamic mechanism to reversibly alter its structure in response to changes in the conditions experienced by the fish. The best known example of gill remodelling is the growth or retraction of cell masses between the lamellae, a rapid process that alters the lamellar surface area that is exposed to the water (i.e. the functional lamellar surface area). Decreases in environmental O2 availability and/or increases in metabolic O2 demand stimulate uncovering of the lamellae, presumably to increase the capacity for O2 uptake. This review addresses four questions about gill remodelling: (1) what types of reversible morphological changes occur; (2) how do these changes affect physiological function from the gill to the whole animal; (3) what factors regulate reversible gill plasticity; and (4) is remodelling phylogenetically widespread among fishes? We address these questions by surveying the current state of knowledge of gill remodelling in fishes, with a focus on identifying gaps in our understanding that future research should consider.

Understanding the health system utilisation and reasons for avoidable mortality after fatal injury within a Three-Delays framework in Karonga, Northern Malawi: a retrospective analysis of verbal autopsy data.

OBJECTIVES: To use verbal autopsy (VA) data to understand health system utilisation and the potential avoidability associated with fatal injury. Then to categorise any evident barriers driving avoidable delays to care within a Three-Delays framework that considers delays to seeking (Delay 1), reaching (Delay 2) or receiving (Delay 3) quality injury care. DESIGN: Retrospective analysis of existing VA data routinely collected by a demographic surveillance site. SETTING: Karonga Health and Demographic Surveillance Site (HDSS) population, Northern Malawi. PARTICIPANTS: Fatally injured members of the HDSS. PRIMARY AND SECONDARY OUTCOME MEASURES: The primary outcome was the proportion of fatal injury deaths that were potentially avoidable. Secondary outcomes were the delay stage and corresponding barriers associated with avoidable deaths and the health system utilisation for fatal injuries within the health system. RESULTS: Of the 252 deaths due to external causes, 185 injury-related deaths were analysed. Deaths were predominantly among young males (median age 30, IQR 11-48), 71.9% (133/185). 35.1% (65/185) were assessed as potentially avoidable. Delay 1 was implicated in 30.8% (20/65) of potentially avoidable deaths, Delay 2 in 61.5% (40/65) and Delay 3 in 75.4% (49/65). Within Delay 1, 'healthcare literacy' was most commonly implicated barrier in 75% (15/20). Within Delay 2, 'communication' and 'prehospital care' were the most commonly implicated in 92.5% (37/40). Within Delay 3, 'physical resources' were most commonly implicated, 85.7% (42/49). CONCLUSIONS: VA is feasible for studying pathways to care and health system responsiveness in avoidable deaths following injury and ascertaining the delays that contribute to deaths. A large proportion of injury deaths were avoidable, and we have identified several barriers as potential targets for intervention. Refining and integrating VA with other health system assessment methods is likely necessary to holistically understand an injury care health system.

The subacromial bursa modulates tendon healing after rotator cuff injury in rats.

Rotator cuff injuries result in more than 500,000 surgeries annually in the United States, many of which fail. These surgeries typically involve repair of the injured tendon and removal of the subacromial bursa, a synovial-like tissue that sits between the rotator cuff and the acromion. The subacromial bursa has been implicated in rotator cuff pathogenesis and healing. Using proteomic profiling of bursa samples from nine patients with rotator cuff injury, we show that the bursa responds to injury in the underlying tendon. In a rat model of supraspinatus tenotomy, we evaluated the bursa's effect on the injured supraspinatus tendon, the uninjured infraspinatus tendon, and the underlying humeral head. The bursa protected the intact infraspinatus tendon adjacent to the injured supraspinatus tendon by maintaining its mechanical properties and protected the underlying humeral head by maintaining bone morphometry. The bursa promoted an inflammatory response in injured rat tendon, initiating expression of genes associated with wound healing, including Cox2 and Il6. These results were confirmed in rat bursa organ cultures. To evaluate the potential of the bursa as a therapeutic target, polymer microspheres loaded with dexamethasone were delivered to the intact bursae of rats after tenotomy. Dexamethasone released from the bursa reduced Il1b expression in injured rat supraspinatus tendon, suggesting that the bursa could be used for drug delivery to reduce inflammation in the healing tendon. Our findings indicate that the subacromial bursa contributes to healing in underlying tissues of the shoulder joint, suggesting that its removal during rotator cuff surgery should be reconsidered.

Using narratives to correct politically charged health misinformation and address affective belief echoes.

BACKGROUND: In May 2020, news outlets reported misinformation about the Centers for Disease Control (CDC) related to COVID-19. Correcting misinformation about outbreaks and politics is particularly challenging. Affective belief echoes continue to influence audiences even after successful correction. Narrative and emotional flow scholarship suggest that a narrative corrective with a positive ending could reduce belief echoes. Therefore, this study investigated the efficacy of a narrative corrective with a relief ending for correcting misinformation about the CDC. METHODS: Between 29 May and 4 June 2020, we tested the effectiveness of a narrative to correct this misinformation. Participants in the United States (N = 469) were enrolled via Qualtrics panels in an online message experiment and randomized to receive a narrative corrective, a didactic corrective or no corrective. RESULTS: The narrative corrective resulted in lower endorsement of the misinformation compared with the control and the didactic corrective. The narrative corrective had a positive indirect effect on perceived CDC competence and mask wearing intentions for politically moderate and conservative participants via relief. CONCLUSIONS: Public health institutions, such as the CDC, should consider utilizing narrative messaging with positive emotion endings to correct misinformation. Narratives better address affective belief echoes, particularly for counter-attitudinal audiences.

Evaluating patient factors, operative management and postoperative outcomes in trauma laparotomy patients worldwide: a protocol for a global observational multicentre trauma study.

INTRODUCTION: Trauma contributes to the greatest loss of disability-adjusted life-years for adolescents and young adults worldwide. In the context of global abdominal trauma, the trauma laparotomy is the most commonly performed operation. Variation likely exists in how these patients are managed and their subsequent outcomes, yet very little global data on the topic currently exists. The objective of the GOAL-Trauma study is to evaluate both patient and injury factors for those undergoing trauma laparotomy, their clinical management and postoperative outcomes. METHODS: We describe a planned prospective multicentre observational cohort study of patients undergoing trauma laparotomy. We will include patients of all ages who present to hospital with a blunt or penetrating injury and undergo a trauma laparotomy within 5 days of presentation to the treating centre. The study will collect system, patient, process and outcome data, following patients up until 30 days postoperatively (or until discharge or death, whichever is first). Our sample size calculation suggests we will need to recruit 552 patients from approximately 150 recruiting centres. DISCUSSION: The GOAL-Trauma study will provide a global snapshot of the current management and outcomes for patients undergoing a trauma laparotomy. It will also provide insight into the variation seen in the time delays for receiving care, the disease and patient factors present, and patient outcomes. For current standards of trauma care to be improved worldwide, a greater understanding of the current state of trauma laparotomy care is paramount if appropriate interventions and targets are to be identified and implemented.

DNA methylation of imprint control regions associated with Alzheimer's disease in non-Hispanic Blacks and non-Hispanic Whites.

Alzheimer's disease (AD) prevalence is twice as high in non-Hispanic Blacks (NHBs) as in non-Hispanic Whites (NHWs). The objective of this study was to determine whether aberrant methylation at imprint control regions (ICRs) is associated with AD. Differentially methylated regions (DMRs) were bioinformatically identified from whole-genome bisulfite sequenced DNA derived from brain tissue of 9 AD (5 NHBs and 4 NHWs) and 8 controls (4 NHBs and 4 NHWs). We identified DMRs located within 120 regions defined as candidate ICRs in the human imprintome ( https://genome.ucsc.edu/s/imprintome/hg38.AD.Brain_track ). Eighty-one ICRs were differentially methylated in NHB-AD, and 27 ICRs were differentially methylated in NHW-AD, with two regions common to both populations that are proximal to the inflammasome gene, NLRP1, and a known imprinted gene, MEST/MESTIT1. These findings indicate that early developmental alterations in DNA methylation of regions regulating genomic imprinting may contribute to AD risk and that this epigenetic risk differs between NHBs and NHWs.

Metabolic outcomes in obese mice undergoing one-anastomosis gastric bypass (OAGB) with a long or a short biliopancreatic limb.

One-anastomosis gastric bypass (OAGB) has gained importance as a simple, safe, and effective operation to treat morbid obesity. We previously found that Roux-en-Y gastric bypass surgery with a long compared with a short biliopancreatic limb (BPL) leads to improved weight loss and glucose tolerance in obese mice. However, it is not known whether a long BPL in OAGB surgery also results in beneficial metabolic outcomes. Five-week-old male C57BL/6J mice fed a high-fat diet (HFD) for 8 weeks underwent OAGB surgery with defined BPL lengths (5.5 cm distally of the duodenojejunal junction for short and 9.5 cm for long BPL), or sham surgery combined with caloric restriction. Weight loss, glucose tolerance, obesity-related comorbidities, endocrine effects, gut microbiota, and bile acids were assessed. Total weight loss was independent of the length of the BPL after OAGB surgery. However, a long BPL was associated with lower glucose-stimulated insulin on day 14, and an improved glucose tolerance on day 35 after surgery. Moreover, a long BPL resulted in reduced total cholesterol, while there were no differences in the resolution of metabolic dysfunction-associated steatotic liver disease (MASLD) and adipose tissue inflammation. Tendencies of an attenuated hypothalamic-pituitary-adrenal (HPA) axis and aldosterone were present in the long BPL group. With both the short and long BPL, we found an increase in primary conjugated bile acids (pronounced in long BPL) along with a loss in bacterial Desulfovibrionaceae and Erysipelotrichaceae and simultaneous increase in Akkermansiaceae, Sutterellaceae, and Enterobacteriaceae. In summary, OAGB surgery with a long compared with a short BPL led to similar weight loss, but improved glucose metabolism, lipid, and endocrine outcomes in obese mice, potentially mediated through changes in gut microbiota and related bile acids. Tailoring the BPL length in humans might help to optimize metabolic outcomes after bariatric surgery.NEW & NOTEWORTHY Weight loss following OAGB surgery in obese mice was not influenced by BPL length, but a longer BPL was associated with improved metabolic outcomes, including glucose and lipid homeostasis. These changes could be mediated by bile acids upon altered gut microbiota. Further validation of these findings is required through a randomized human study.

p53 promotes revival stem cells in the regenerating intestine after severe radiation injury.

Ionizing radiation induces cell death in the gastrointestinal (GI) epithelium by activating p53. However, p53 also prevents animal lethality caused by radiation-induced acute GI syndrome. Through single-cell RNA-sequencing of the irradiated mouse small intestine, we find that p53 target genes are specifically enriched in regenerating epithelial cells that undergo fetal-like reversion, including revival stem cells (revSCs) that promote animal survival after severe damage of the GI tract. Accordingly, in mice with p53 deleted specifically in the GI epithelium, ionizing radiation fails to induce fetal-like revSCs. Using intestinal organoids, we show that transient p53 expression is required for the induction of revival stem cells and is controlled by an Mdm2-mediated negative feedback loop. Together, our findings reveal that p53 suppresses severe radiation-induced GI injury by promoting fetal-like reprogramming of irradiated intestinal epithelial cells.

Protein phosphatases regulate the formation of Müller glia-derived progenitor cells in the chick retina.

Different kinase-dependent cell signaling pathways are known to play important roles in glia-mediated neuroprotection and reprogramming of Müller glia (MG) into Müller glia-derived progenitor cells (MGPCs) in the retina. However, very little is known about the phosphatases that regulate kinase-dependent signaling in MG. Using single-cell RNA-sequencing (scRNA-seq) databases, we investigated patterns of expression of Dual Specificity Phosphatases (DUSP1/6) and other protein phosphatases in normal and damaged chick retinas. We found that DUSP1, DUSP6, PPP3CB, PPP3R1 and PPPM1A/B/D/E/G are widely expressed by many types of retinal neurons and are dynamically expressed by MG and MGPCs in retinas during the process of reprogramming. We find that inhibition of DUSP1/6 and PP2C phosphatases enhances the formation of proliferating MGPCs in damaged retinas and in retinas treated with insulin and FGF2 in the absence of damage. By contrast, inhibition of PP2B phosphatases suppressed the formation of proliferating MGPCs, but increased numbers of proliferating MGPCs in undamaged retinas treated with insulin and FGF2. In damaged retinas, inhibition of DUSP1/6 increased levels of pERK1/2 and cFos in MG whereas inhibition of PP2B's decreased levels of pStat3 and pS6 in MG. Analyses of scRNA-seq libraries identified numerous differentially activated gene modules in MG in damaged retinas versus MG in retinas treated with insulin+FGF2 suggesting significant differences in kinase-dependent signaling pathways that converge on the formation of MGPCs. Inhibition of phosphatases had no significant effects upon numbers of dying cells in damaged retinas. We conclude that the activity of different protein phosphatases acting through retinal neurons and MG "fine-tune" the cell signaling responses of MG in damaged retinas and during the reprogramming of MG into MGPCs.

Ibudilast Protects Retinal Bipolar Cells from Excitotoxic Retinal Damage and Activates the mTOR Pathway.

Ibudilast, an inhibitor of macrophage migration inhibitory factor (MIF) and phosphodiesterase (PDE), has been recently shown to have neuroprotective effects in a variety of neurologic diseases. We utilize a chick excitotoxic retinal damage model to investigate ibudilast's potential to protect retinal neurons. Using single cell RNA-sequencing (scRNA-seq), we find that MIF, putative MIF receptors CD74 and CD44, and several PDEs are upregulated in different retinal cells during damage. Intravitreal ibudilast is well tolerated in the eye and causes no evidence of toxicity. Ibudilast effectively protects neurons in the inner nuclear layer from NMDA-induced cell death, restores retinal layer thickness on spectral domain optical coherence tomography, and preserves retinal neuron function, particularly for the ON bipolar cells, as assessed by electroretinography. PDE inhibition seems essential for ibudilast's neuroprotection, as AV1013, the analogue that lacks PDE inhibitor activity, is ineffective. scRNA-seq analysis reveals upregulation of multiple signaling pathways, including mTOR, in damaged Müller glia (MG) with ibudilast treatment compared to AV1013. Components of mTORC1 and mTORC2 are upregulated in both bipolar cells and MG with ibudilast. The mTOR inhibitor rapamycin blocked accumulation of pS6 but did not reduce TUNEL positive dying cells. Additionally, through ligand-receptor interaction analysis, crosstalk between bipolar cells and MG may be important for neuroprotection. We have identified several paracrine signaling pathways that are known to contribute to cell survival and neuroprotection and might play essential roles in ibudilast function. These findings highlight ibudilast's potential to protect inner retinal neurons during damage and show promise for future clinical translation.