HESTEFORSK Forståelse av etiologien bak acquired equine polyneuropathy via studier av Schwanske cellekulturer H-18-47-409

The first year of the project, we succeeded with aim A and now have cultivated Schwann cells (SC) from 3 non-neurological horses. We have a total of 83 million frozen cells from these control-horses. Different sampling-methods have been tested until we now have a procedure that enables sampling in the field, if necessary. In September 2020 we sampled nerves from a horse that had AEP in 2012. Nerve samples examined at Ludwig-Maximilian University confirmed mild, chronic changes compatible with previously described findings in recovered AEP horses. One single, possible inclusion was also described. Inclusions have previously not been described in horses years after recovery of AEP, but this finding is still associated with some uncertainty and probably not possible to recover in the cell cultures. In the laboratory, the cells were successfully cultivated and we have 35 aliquots à 1 million cells stored frozen. In September 2021 we sampled nerves from a horse that had AEP both in 2013 and 2018. Both times the clinical signs were mild and recovery time a few months. The horse was euthanized due to lameness. We have frozen serum from both periods of AEP as well as forage samples. Nerve samples examined at Ludwig-Maximillian University confirmed mild, chronic re- and demyelinating neuropathy, consistent with recovered AEP. No inclusions were described. Cells are growing in the laboratory, ready to be frozen. Cell sorting (aim B) Mixed cultures (Schwann cells and fibroblasts) represent a natural constellation in nerves in vivo, but we think that some experiments may be easier to interpret with pure SC cultures. A few weeks before the pandemic lockdown last year, we had planned to proceed with fluorescens-activated cell sorting (FACS) at Oslo University Hospital. This activity was not possible during the lockdown. After moving into new facilities, the Veterinary Faculty has now access to their own FACS, and during autumn we will perform cell sorting of the cultures that we have accumulated. The goal is to recover as clean SC cultures as possible. Triggering of Schwann cell injury (aim C and D) Initial experiments with adding serum from healthy and affected horses to cultures from healthy and affected horses will be initiated this autumn. The hypothesis is that serum from affected horses contain elements that can affect the SC. One important marker for affected cells is GRP78 BiP, a protein in RER associated with cell stress. Studies of cytoplasmatic inclusions in SC (in situ) in acutely AEP affected horses have shown that the inclusions are positive for this protein. We have acquired an antibody against GRP78 BiP and will use it in immunofluorescence studies to detect a possible increase of the protein in the SC cultivated with serum from affected horses. With our new Leica Stellaris confocal microscope with, among other technically advanced options, Lightning Super Resolution enabling resolution down to 120 nm, we hope to be able to document possible changes in the cells. In addition to GRP78 BiP, we have a panel of antibodies against myelin markers showing the degree of maturity in SC. In non-stimulated SC cultures the cells are mostly in a p75-positive, immature state. By stimulating the maturation of the cells with cAMP, we can monitor the cells` ability to mature. We will also be able to evaluate possible differences between cells from healthy and affected horses, as well as healthy cells added either buffer or serum from affected horses. This year is the first since 1995 without any registered acute cases of AEP in Norway or Sweden. It is of course possible that there have been cases that are not registered, but in our experience, people are well informed of the disease and our aim to register new cases. Since we do not yet have any samples from acute cases, we are lacking access to pathologic nerve samples with obvious typical Schwann cell inclusions. It is still our aim to include such samples in our study, and meanwhile we will work with the collected nerve samples with chronic changes compatible with previous AEP. The material from the horse that was euthanized this September is especially valuable since it was one of few surviving the disease twice, and we have serum from both times as well as forage samples. Regarding C and D. Covid-19 lockdown affected the project severely the whole year until this autumn. Moving and completion of the new pathology facilities in Ås was severely delayed and the cell laboratory was not ready for use until this autumn. It has thus not been possible to perform any of the planned experiments, but we do not expect any more delays in the future.

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Acquired equine polyneuropathy (AEP) er en dramatisk nervesykdom som rammer nordiske hester. Det er påvist karakteristiske histopatologiske forandringer i de perifere nervene på hester avlivet pga sykdommen. Disse inkluderer re- og demyelinisering, samt hypertrofi av perikaria og proteinlignende inklusjoner i de myelinproduserende Schwanske cellene. Hypotesen er at en ukjent faktor, muligens fra fôret, påvirker metabolismen i de Schwanske cellene og fremkaller forandringene. Det er nødvendig med videre studier for å kunne belyse årsaksforholdene, men begrenset tilgang på prøver har vanskeliggjort dette. Prøvene må tas ut i umiddelbar tilknytning til avlivning og mange AEP affiserte hester kan ikke transporteres til patologi-enheter. Prøver som tas ut er dermed svært verdifulle. Ved å dyrke Schwanske celler i kulturer vil dette problemet omgås fordi man kan fryse og tine dem opp igjen til videre undersøkelser. Metoden er alt etablert på NMBU for hund og vil benyttes i studien på hest.