Pagano, Teresa Bruna (2017) “New insights in the etiopathogenesis of muscle inflammation and aging in animals”. [Tesi di dottorato]

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Tipologia del documento:	Tesi di dottorato
Lingua:	English
Titolo:	“New insights in the etiopathogenesis of muscle inflammation and aging in animals”
Autori:	Autore Email Pagano, Teresa Bruna teresabruna@hotmail.it
Data:	11 Novembre 2017
Numero di pagine:	195
Istituzione:	Università degli Studi di Napoli Federico II
Dipartimento:	dep15
Dottorato:	phd095
Ciclo di dottorato:	30
Coordinatore del Corso di dottorato:	nome email Cringoli, Giuseppe giuseppe.cringoli@unina.it
Tutor:	nome email Papparella, Serenella [non definito]
Data:	11 Novembre 2017
Numero di pagine:	195
Parole chiave:	myositis, equine piroplasmosis, sarcocystis, aging, autophagy, cellular prion protein
Settori scientifico-disciplinari del MIUR:	Area 07 - Scienze agrarie e veterinarie > VET/03 - Patologia generale e anatomia patologica veterinaria
Depositato il:	08 Gen 2018 12:45
Ultima modifica:	19 Mar 2019 12:07
URI:	http://www.fedoa.unina.it/id/eprint/12204

Abstract

Muscle pathology has unique relevance in many different fields of veterinary research, ranging from the diagnostics, to the comparative medicine involving animal models of human diseases, to the animal based food safety. The Laboratory of Comparative of Neuromuscular Disorders was established decades ago at the Department Veterinary Medicine of Naples in order to contribute to the knowledge of muscle pathology in domestic animals using modern techniques and undertaking international scientific collaborations. In this context, the project of this doctoral thesis had the main purpose to produce new scientific data in at least two interest area: the etiopathogenesis of inflammatory myopathies and skeletal muscle aging. Thus, the experimental results of four research project conducted in four different species are presented, including: 1) Inflammatory myopathy in horses affected by chronic piroplasmosis; 2) Lymphoplasmacytic myositis and expression of Major Histocompatibility Complex class I and II in ovine muscular sarcocystosis; 3) Age related skeletal muscle atrophy and upregulation of autophagy in dogs; 4) Expression and biochemical properties of cellular Prion protein in skeletal muscle of aged cows. In the first and second chapters of the thesis, two inflammatory myopathies associated with parasites having or not having a specific tropism for skeletal muscle (Sarcocystis tenella and Theileria equi -Babesia caballi, respectively) are explored. In the first study, an inflammatory myopathy characterized by a mainly CD8-CD4+ lymphocytic population and fiber degeneration is described in 16 horses serologically positive for equine piroplasms with clinical signs and serum biochemical evidence of a myopathy. To test the hypothesis of circulating autoantibodies directed against an unknown muscular antigen, the serum from affected horses was tested by indirect immunofluorescence to sections of normal equine muscle. In all cases, distinct sarcolemmal staining was detected in sections incubated with serum from affected horses, in contrast to sections incubated with phosphate-buffered saline or equine control sera. Furthermore, a significant increase of interferon-γ, interleukin-12, and tumor necrosis factor-α gene expression by compared to healthy controls was revealed by Reverse transcription polymerase chain reaction (RT-PCR). Thus, we concluded that inflammatory myopathy associated with equine piroplasmosis may involve an autoimmune pathogenesis with upregulation of inflammatory cytokines that may cause myofiber atrophy and degeneration. The aim of the second chapter, leading with muscle inflammation in ovine muscular sarcosytosis, was to investigate if parasitized muscle fibers could play a role in immune-stimulation, as sporadically described in accidental muscular sarcocystosis in definitive hosts. Skeletal muscle samples from 78 sheep presenting muscular sarcocysts revealed inflammatory changes in 69% of cases, consisting of an endomysial lymphoplasmacellular infiltrate, with attendant myofiber degeneration and necrosis. The predominant T cell populations were CD3+, CD8+ with lesser numbers of CD4+ and CD79α+ cells. Eosinophils were constantly absent. Notably, moderate to strong sarcolemmal labeling to Major histocompatibility complex (MHC) I and II was found both in biopsies with evident inflammatory infiltrate and in cases without inflammation. The wall of the cysts resulted strongly positive to MHC Iand II, and occasionally co-localized with the membrane associated protein Dystrophin. Our data suggest that muscle fibers respond to the presence of cysts by expression of MHC I and II that can play a role in stimulating and maintaining the lymphoplasmacellular inflammation. The possible role of MHC I on the cyst wall is discussed, but basically more research is needed in order to evaluate its possible involvement in inflammation. Moreover we speculated about the possibility that the abnormal expression of MHC I and II on muscle fibers is not only typical of myopathies having an immune-mediated pathogenesis but also of myositis associated with muscular parasites. The last two chapters of the thesis are dedicated to muscle aging, and in particular to the role of autophagy in canine sarcopenia and of cellular prion protein in bovine muscle aging. Sarcopenia, the age related loss of muscle mass and strength, is a multifactorial condition that occurs in a variety of species and represents a major healthcare concern in human medicine. We evaluated the expression of three markers of autophagy, Beclin 1, LC3 and p62, in muscle wasting of 25 geriatric dogs, to establish whether the levels of autophagy change with increasing age. Our results indicated a marked intracytoplasmic staining for Beclin 1 and LC3 in 80% of the muscle samples from the older dogs and a significantly greater expression of LC3 II and Beclin 1, by Western blotting. The results of the study suggest that enhanced autophagy might be one of the factors underlying muscle atrophy in canine age related muscle wasting. Finally, we explored the expression and biochemical properties of cellular prion protein (PrPC) in skeletal muscle of cows as well as their possible changes in geriatric animals. PrPC over expression has been described in many different human neuromuscular disorders but little is known about PrPC in health and disease in bovine muscle tissue. Skeletal muscle and brain samples of 12 aged and 8 young Podolica cows were collected and compared by histomorphological techniques as well as several experiments involving immunoblot and specifically analyzing the amount of PrPC, its glycosylation profile, proteinase resistance, solubility levels and tendency to aggregate. Histologically, muscle biopsies of aged cows showed typical myopathic features already described in bovine sarcopenia. Immunohistochemistry for PrP revealed a distinct granular positivity in intramuscular nerve branches and muscle spindles in all cases. Membrane immunopositivity was more prominent on angular atrophic fibers. Increased levels of positivity were found in degenerated fibers (desmin depleted) with a positive labeling of rimmed vacuoles. Prion protein positivity levels was also significantly increased in cases displaying inflammatory changes. No differences between muscles and brains of young and old animals were detected by quantitative immunoblot and Proteinase K titration. The glycoform profile and the molecular mass of PrPC in muscle samples appeared slightly different compared to the brain. Sucrose gradient velocity sedimentation gave variable results, with a generally lower amount of PrP in high density sucrose fractions in the muscle compared to the brain. After high speed centrifugation the majority of muscle PrPC was found in the soluble fraction, without significant differences between young and old animals. Our preliminary data suggest that: 1) the amount and the main biochemical properties of prion protein do not change with age, 2) PrPC is overexpressed in degenerated, vacuolated and angular muscle fibers; 3) The glycoform profile of PrPC differs between brain and muscle tissue. In conclusion, the main innovative aspects of this thesis are: 1) The first description in the literature of a likely immune-mediated myositis occurring in chronic equine piroplasmosis 2) The demonstration of high prevalence of lymphoplasmacellular myositis in ovine muscular sarcocystosis implying the overexpression of MHC I on muscle fibers as well as on the cyst wall. 3) The report of enhanced autophagy in age-related muscle atrophy in dogs. 4) The first detailed description of PrPC localization and biochemical properties in bovine muscle tissue and its possible involvement in neurogenic atrophy, muscle degeneration and chronic inflammation. Collectively, these results enhance the relevance of morphological studies as starting point to investigate the pathogenesis of muscle diseases in veterinary and comparative pathology.

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