Neutrophil Extracellular Traps Release following Hypoxic-Ischemic Brain Injury in Newborn Rats Treated with Therapeutic Hypothermia.

The peripheral immune system plays a critical role in neuroinflammation of the central nervous system after an insult. Hypoxic-ischemic encephalopathy (HIE) induces a strong neuroinflammatory response in neonates, which is often associated with exacerbated outcomes. In adult models of ischemic stroke, neutrophils infiltrate injured brain tissue immediately after an ischemic insult and aggravate inflammation via various mechanisms, including neutrophil extracellular trap (NETs) formation. In this study, we used a neonatal model of experimental hypoxic-ischemic (HI) brain injury and demonstrated that circulating neutrophils were rapidly activated in neonatal blood. We observed an increased infiltration of neutrophils in the brain after exposure to HI. After treatment with either normothermia (NT) or therapeutic hypothermia (TH), we observed a significantly enhanced expression level of the NETosis marker Citrullinated H3 (Cit-H3), which was significantly more pronounced in animals treated with TH than in those treated with NT. NETs and NLR family pyrin domain containing 3 (NLRP-3) inflammasome assembly are closely linked in adult models of ischemic brain injury. In this study, we observed an increase in the activation of the NLRP-3 inflammasome at the time points analyzed, particularly immediately after TH, when we observed a significant increase in NETs structures in the brain. Together, these results suggest the important pathological functions of early arriving neutrophils and NETosis following neonatal HI, particularly after TH treatment, which is a promising starting point for the development of potential new therapeutic targets for neonatal HIE.

Proteomic profiling of carbonic anhydrase CA3 in skeletal muscle.

INTRODUCTION: Carbonic anhydrase (CA) is a key enzyme that mediates the reversible hydration of carbon dioxide. Skeletal muscles contain high levels of the cytosolic isoform CA3. This enzyme has antioxidative function and plays a crucial role in the maintenance of intracellular pH homeostasis. AREAS COVERED: Since elevated levels of serum CA3, often in combination with other muscle-specific proteins, are routinely used as a marker of general muscle damage, it was of interest to examine recent analyses of this enzyme carried out by modern proteomics. This review summarizes the mass spectrometry-based identification and evaluation of CA3 in normal, adapting, dystrophic, and aging skeletal muscle tissues. EXPERT OPINION: The mass spectrometric characterization of CA3 confirmed this enzyme as a highly useful marker of both physiological and pathophysiological alterations in skeletal muscles. Cytosolic CA3 is clearly enriched in slow-twitching type I fibers, which makes it an ideal marker for studying fiber type shifting and muscle adaptations. Importantly, neuromuscular diseases feature distinct alterations in CA3 in skeletal muscle tissues versus biofluids, such as serum. Characteristic changes of CA3 in age-related muscle wasting and dystrophinopathy established this enzyme as a suitable biomarker candidate for differential diagnosis and monitoring of disease progression and therapeutic impact.

Proteomic profiling of fatty acid binding proteins in muscular dystrophy.

Introduction: Duchenne muscular dystrophy is a neuromuscular disorder, which is caused by abnormalities in the DMD gene that encodes the membrane cytoskeletal protein dystrophin. Besides progressive skeletal muscle wasting, dystrophinopathy also affects non-skeletal muscle tissues, including cells in the cardio-respiratory system, the central nervous system, the liver and the kidney.Areas covered: This review summarizes the proteomic characterization of a key class of lipid chaperones, the large family of fatty acid binding proteins, and their potential role in muscular dystrophy. Recent proteomic surveys using animal models and patient specimens are reviewed. Pathobiochemical changes in specific proteoforms of fatty acid binding protein in the multi-system pathology of dystrophinopathy are discussed.Expert opinion: The mass spectrometric identification of distinct changes in fatty acid binding proteins in muscle, heart, liver, kidney and serum demonstrates that considerable alterations occur in key steps of metabolite transport and fat metabolism in muscular dystrophy. These new findings might be helpful to further develop a comprehensive biomarker signature of metabolic changes in X-linked muscular dystrophy, which should improve (i) our understanding of complex pathobiochemical changes due to dystrophin deficiency, (ii) the identification of novel therapeutic targets, and (iii) the design of differential diagnostic, prognostic and therapy-monitoring approaches.

Proteomic profiling of giant skeletal muscle proteins.

INTRODUCTION: Distinct subtypes of contractile fibres are highly diverse in their proteomic profile and greatly adaptable to physiological or pathological challenges. A striking biochemical feature of heterogeneous skeletal muscle tissues is the presence of a considerable number of extremely large protein species, which often present a bioanalytical challenge for the systematic separation and identification of muscle proteomes during large-scale screening surveys. Areas covered: This review outlines the proteomic characterization of skeletal muscles with a special focus on giant proteins of the sarcomere, the cytoskeleton and the sarcoplasmic reticulum. This includes an overview of the involvement of large muscle proteins, such as titin, nebulin, obscurin, plectin, dystrophin and the ryanodine receptor calcium release channel, during normal muscle functioning, swift adaptations to changed physiological demands and changes in relation to pathobiochemical insults. Expert commentary: The proteomic screening and characterization of total muscle extracts and various subcellular fractions has confirmed the critical role of large skeletal muscle proteins in the regulation of ion homeostasis, the maintenance of contraction-relaxation cycles and fibre elasticity, and the stabilisation of supramolecular complexes of the muscle periphery and cytoskeletal networks of contractile fibres. These findings will be helpful for the future functional systems analysis of giant muscle proteins.

Proteomic profiling of the mouse diaphragm and refined mass spectrometric analysis of the dystrophic phenotype.

The diaphragm is a crucial muscle involved in active inspiration and whole body homeostasis. Previous biochemical, immunochemical and cell biological investigations have established the distribution and fibre type-specific expression of key diaphragm proteins. Building on these findings, it was of interest to establish the entire experimentally assessable diaphragm proteome and verify the presence of specific protein isoforms within this specialized subtype of skeletal muscle. A highly sensitive Orbitrap Fusion Tribrid mass spectrometer was used for the systematic identification of the mouse diaphragm-associated protein population. Proteomics established 2925 proteins by high confidence peptide identification. Bioinformatics was used to determine the distribution of the main protein classes, biological processes and subcellular localization within the diaphragm proteome. Following the establishment of the respiratory muscle proteome with special emphasis on protein isoform expression in the contractile apparatus, the extra-sarcomeric cytoskeleton, the extracellular matrix and the excitation-contraction coupling apparatus, the mass spectrometric analysis of the diaphragm was extended to the refined identification of proteome-wide changes in X-linked muscular dystrophy. The comparative mass spectrometric profiling of the dystrophin-deficient diaphragm from the mdx-4cv mouse model of Duchenne muscular dystrophy identified 289 decreased and 468 increased protein species. Bioinformatics was employed to analyse the clustering of changes in protein classes and potential alterations in interaction patterns of proteins involved in metabolism, the contractile apparatus, proteostasis and the extracellular matrix. The detailed pathoproteomic profiling of the mdx-4cv diaphragm suggests highly complex alterations in a variety of crucial cellular processes due to deficiency in the membrane cytoskeletal protein dystrophin.

Proteomic serum biomarkers for neuromuscular diseases.

INTRODUCTION: The clinical evaluation of neuromuscular symptoms often includes the assessment of altered blood proteins or changed enzyme activities. However, the blood concentration of many muscle-derived serum markers is not specific for different neuromuscular disorders and also shows alterations in the course of these diseases. Thus, the establishment of more reliable biomarker signatures for improved muscle diagnostics is required. Areas covered: To address the lack of muscle disease-specific marker molecules, mass spectrometry-based proteomics was applied to the systematic identification and biochemical characterization of new serum biomarker candidates. This article outlines serum proteomics in relation to neuromuscular disorders and reviews the bioanalytical results from recent proteomic profiling studies of representative neuromuscular disorders, including motor neuron disease, muscular dystrophies and sarcopenia of old age. Pathophysiological changes in the skeletal muscle proteome are reflected by serum alterations in a variety of sarcomeric proteins, metabolic enzymes and signaling proteins. Expert commentary: Based on the proteomic identification of actively secreted or passively released skeletal muscle proteins following pathophysiological insults, new biomarker candidates can now be used to develop liquid biopsy procedures for superior diagnostic approaches, design novel prognostic tools and establish more reliable methods for the systematic evaluation of experimental therapies to treat neuromuscular disease.

Proteomic profiling of liver tissue from the mdx-4cv mouse model of Duchenne muscular dystrophy.

BACKGROUND: Duchenne muscular dystrophy is a highly complex multi-system disease caused by primary abnormalities in the membrane cytoskeletal protein dystrophin. Besides progressive skeletal muscle degeneration, this neuromuscular disorder is also associated with pathophysiological perturbations in many other organs including the liver. To determine potential proteome-wide alterations in liver tissue, we have used a comparative and mass spectrometry-based approach to study the dystrophic mdx-4cv mouse model of dystrophinopathy. METHODS: The comparative proteomic profiling of mdx-4cv versus wild type liver extracts was carried out with an Orbitrap Fusion Tribrid mass spectrometer. The distribution of identified liver proteins within protein families and potential protein interaction patterns were analysed by systems bioinformatics. Key findings on fatty acid binding proteins were confirmed by immunoblot analysis and immunofluorescence microscopy. RESULTS: The proteomic analysis revealed changes in a variety of protein families, affecting especially fatty acid, carbohydrate and amino acid metabolism, biotransformation, the cellular stress response and ion handling in the mdx-4cv liver. Drastically increased protein species were identified as fatty acid binding protein FABP5, ferritin and calumenin. Decreased liver proteins included phosphoglycerate kinase, apolipoprotein and perilipin. The drastic change in FABP5 was independently verified by immunoblotting and immunofluorescence microscopy. CONCLUSIONS: The proteomic results presented here indicate that the intricate and multifaceted pathogenesis of the mdx-4cv model of dystrophinopathy is associated with secondary alterations in the liver affecting especially fatty acid transportation. Since FABP5 levels were also shown to be elevated in serum from dystrophic mice, this protein might be a useful indicator for monitoring liver changes in X-linked muscular dystrophy.

Comparative gel-based proteomic analysis of chemically crosslinked complexes in dystrophic skeletal muscle.

Duchenne muscular dystrophy is a highly progressive muscle wasting disease with a complex pathophysiology that is based on primary abnormalities in the dystrophin gene. In order to study potential changes in the oligomerization of high-molecular-mass protein complexes in dystrophic skeletal muscle, chemical crosslinking was combined with mass spectrometric analysis. The biochemical stabilization of protein interactions was carried out with the homo-bifunctional and amine-reactive agent bis[sulfosuccinimidyl]suberate, followed by protein shift analysis in one-dimensional gels. The proteomic approach identified 11 and 15 protein species in wild type versus dystrophic microsomal fractions, respectively, as well as eight common proteins, with an electrophoretic mobility shift to very high molecular mass following chemical crosslinking. In dystrophin-deficient preparations, several protein species with an increased tendency of oligomerisation were identified as components of the sarcolemma and its associated intra- and extracellular structures, as well as mitochondria. This included the sarcolemmal proteins myoferlin and caveolin, the cytoskeletal components vimentin and tubulin, extracellular collagen alpha-1(XII) and the mitochondrial trifunctional enzyme and oxoglutarate dehydrogenase. These changes are probably related to structural and metabolic adaptations, especially cellular repair processes, which agrees with the increased oligomerisation of myosin-3, myosin-9 and actin, and their role in cellular regeneration and structural adjustments in dystrophinopathy.

Label-free mass spectrometric analysis of the mdx-4cv diaphragm identifies the matricellular protein periostin as a potential factor involved in dystrophinopathy-related fibrosis.

Proteomic profiling plays a decisive role in the identification of novel biomarkers of muscular dystrophy and the elucidation of new pathobiochemical mechanisms that underlie progressive muscle wasting. Building on the findings of recent comparative analyses of tissue samples and body fluids from dystrophic animals and patients afflicted with Duchenne muscular dystrophy, we have used here label-free MS to study the severely dystrophic diaphragm from the not extensively characterized mdx-4cv mouse. This animal model of progressive muscle wasting exhibits less dystrophin-positive revertant fibers than the conventional mdx mouse, making it ideal for the future monitoring of experimental therapies. The pathoproteomic signature of the mdx-4cv diaphragm included a significant increase in the fibrosis marker collagen and related extracellular matrix proteins (asporin, decorin, dermatopontin, prolargin) and cytoskeletal proteins (desmin, filamin, obscurin, plectin, spectrin, tubulin, vimentin, vinculin), as well as decreases in proteins of ion homeostasis (parvalbumin) and the contractile apparatus (myosin-binding protein). Importantly, one of the most substantially increased proteins was identified as periostin, a matricellular component and apparent marker of fibrosis and tissue damage. Immunoblotting confirmed a considerable increase of periostin in the dystrophin-deficient diaphragm from both mdx and mdx-4cv mice, suggesting an involvement of this matricellular protein in dystrophinopathy-related fibrosis.

Label-free mass spectrometric analysis reveals complex changes in the brain proteome from the mdx-4cv mouse model of Duchenne muscular dystrophy.

BACKGROUND: X-linked muscular dystrophy is a primary disease of the neuromuscular system. Primary abnormalities in the Dmd gene result in the absence of the full-length isoform of the membrane cytoskeletal protein dystrophin. Besides progressive skeletal muscle wasting and cardio-respiratory complications, developmental cognitive deficits and behavioural abnormalities are clinical features of Duchenne muscular dystrophy. In order to better understand the mechanisms that underlie impaired brain functions in Duchenne patients, we have carried out a proteomic analysis of total brain extracts from the mdx-4cv mouse model of dystrophinopathy. RESULTS: The comparative proteomic profiling of the mdx-4cv brain revealed a significant increase in 39 proteins and a decrease in 7 proteins. Interesting brain tissue-associated proteins with an increased concentration in the mdx-4cv animal model were represented by the glial fibrillary acidic protein GFAP, the neuronal Ca(2+)-binding protein calretinin, annexin AnxA5, vimentin, the neuron-specific enzyme ubiquitin carboxyl-terminal hydrolase isozyme L1, the dendritic spine protein drebrin, the cytomatrix protein bassoon of the nerve terminal active zone, and the synapse-associated protein SAP97. Decreased proteins were identified as the nervous system-specific proteins syntaxin-1B and syntaxin-binding protein 1, as well as the plasma membrane Ca(2+)-transporting ATPase PMCA2 that is mostly found in the brain cortex. The differential expression patterns of GFAP, vimentin, PMCA2 and AnxA5 were confirmed by immunoblotting. Increased GFAP levels were also verified by immunofluorescence microscopy. CONCLUSIONS: The large number of mass spectrometrically identified proteins with an altered abundance suggests complex changes in the mdx-4cv brain proteome. Increased levels of the glial fibrillary acidic protein, an intermediate filament component that is uniquely associated with astrocytes in the central nervous system, imply neurodegeneration-associated astrogliosis. The up-regulation of annexin and vimentin probably represent compensatory mechanisms involved in membrane repair and cytoskeletal stabilization in the absence of brain dystrophin. Differential alterations in the Ca(2+)-binding protein calretinin and the Ca(2+)-pumping protein PMCA2 suggest altered Ca(2+)-handling mechanisms in the Dp427-deficient brain. In addition, the proteomic findings demonstrated metabolic adaptations and functional changes in the central nervous system from the dystrophic phenotype. Candidate proteins can now be evaluated for their suitability as proteomic biomarkers and their potential in predictive, diagnostic, prognostic and/or therapy-monitoring approaches to treat brain abnormalities in dystrophinopathies.

Comparative proteomic analysis of the contractile-protein-depleted fraction from normal versus dystrophic skeletal muscle.

In basic and applied myology, gel-based proteomics is routinely used for studying global changes in the protein constellation of contractile fibers during myogenesis, physiological adaptations, neuromuscular degeneration, and the natural aging process. Since the main proteins of the actomyosin apparatus and its auxiliary sarcomeric components often negate weak signals from minor muscle proteins during proteomic investigations, we have here evaluated whether a simple prefractionation step can be employed to eliminate certain aspects of this analytical obstacle. To remove a large portion of highly abundant contractile proteins from skeletal muscle homogenates without the usage of major manipulative steps, differential centrifugation was used to decisively reduce the sample complexity of crude muscle tissue extracts. The resulting protein fraction was separated by two-dimensional gel electrophoresis, and 2D-landmark proteins were identified by mass spectrometry. To evaluate the suitability of the contractile-protein-depleted fraction for comparative proteomics, normal versus dystrophic muscle preparations were examined. The mass spectrometric analysis of differentially expressed proteins, as determined by fluorescence difference in-gel electrophoresis, identified 10 protein species in dystrophic mdx hindlimb muscles. Interesting new biomarker candidates included Hsp70, transferrin, and ferritin, whereby their altered concentration levels in dystrophin-deficient muscle were confirmed by immunoblotting.

Proteomic reference map for sarcopenia research: mass spectrometric identification of key muscle proteins of organelles, cellular signaling, bioenergetic metabolism and molecular chaperoning.

During the natural aging process, frailty is often associated with abnormal muscular performance. Although inter-individual differences exit, in most elderly the tissue mass and physiological functionality of voluntary muscles drastically decreases. In order to study age-related contractile decline, animal model research is of central importance in the field of biogerontology. Here we have analyzed wild type mouse muscle to establish a proteomic map of crude tissue extracts. Proteomics is an advanced and large-scale biochemical method that attempts to identify all accessible proteins in a given biological sample. It is a technology-driven approach that uses mass spectrometry for the characterization of individual protein species. Total protein extracts were used in this study in order to minimize the potential introduction of artefacts due to excess subcellular fractionation procedures. In this report, the proteomic survey of aged muscles has focused on organellar marker proteins, as well as proteins that are involved in cellular signaling, the regulation of ion homeostasis, bioenergetic metabolism and molecular chaperoning. Hence, this study has establish a proteomic reference map of a highly suitable model system for future aging research.

Proteomic reference map for sarcopenia research: mass spectrometric identification of key muscle proteins located in the sarcomere, cytoskeleton and the extracellular matrix.

Sarcopenia of old age is characterized by the progressive loss of skeletal muscle mass and concomitant decrease in contractile strength. Age-related skeletal muscle dysfunctions play a key pathophysiological role in the frailty syndrome and can result in a drastically diminished quality of life in the elderly. Here we have used mass spectrometric analysis of the mouse hindlimb musculature to establish the muscle protein constellation at advanced age of a widely used sarcopenic animal model. Proteomic results were further analyzed by systems bioinformatics of voluntary muscles. In this report, the proteomic survey of aged muscles has focused on the expression patterns of proteins involved in the contraction-relaxation cycle, membrane cytoskeletal maintenance and the formation of the extracellular matrix. This includes proteomic markers of the fast versus slow phenotypes of myosin-containing thick filaments and actin-containing thin filaments, as well as proteins that are associated with the non-sarcomeric cytoskeleton and various matrisomal layers. The bioanalytical usefulness of the newly established reference map was demonstrated by the comparative screening of normal versus dystrophic muscles of old age, and findings were verified by immunoblot analysis.

Proteomic profiling of cardiomyopathic tissue from the aged mdx model of Duchenne muscular dystrophy reveals a drastic decrease in laminin, nidogen and annexin.

The majority of patients afflicted with Duchenne muscular dystrophy develop cardiomyopathic complications, warranting large-scale proteomic studies of global cardiac changes for the identification of new protein markers of dystrophinopathy. The aged heart from the X-linked dystrophic mdx mouse has been shown to exhibit distinct pathological aspects of cardiomyopathy. In order to establish age-related alterations in the proteome of dystrophin-deficient hearts, cardiomyopathic tissue from young versus aged mdx mice was examined by label-free LC-MS/MS. Significant age-dependent alterations were established for 67 proteins, of which 28 proteins were shown to exhibit a lower abundance and 39 proteins were found to be increased in their expression levels. Drastic changes were demonstrated for 17 proteins, including increases in Ig chains and transferrin, and drastic decreases in laminin, nidogen and annexin. An immunblotting survey of young and old wild-type versus mdx hearts confirmed these proteomic findings and illustrated the effects of natural aging versus dystrophin deficiency. These proteome-wide alterations suggest a disintegration of the basal lamina structure and cytoskeletal network in dystrophin-deficient cardiac fibres, increased levels of antibodies in a potential autoimmune reaction of the degenerating heart, compensatory binding of excess iron and a general perturbation of metabolic pathways in dystrophy-associated cardiomyopathy.

Verification of Protein Changes Determined by 2D-DIGE Based Proteomics Using Immunofluorescence Microscopy.

Fluorescence two-dimensional difference gel electrophoresis (2D-DIGE) is a key biochemical method for the comparative analysis of complex protein mixtures. The technique focuses on the identification and characterization of individual protein species following gel electrophoretic separation making it an important analytical tool of top-down proteomics. In order to verify changes in the expression levels of a particular protein, as determined by 2D-DIGE analysis, and evaluate the subcellular localization of the proteoform of interest, immunofluorescence microscopy is very well suited. This chapter describes in detail the preparation of tissue specimens and the process of cryo-sectioning, as well as incubation with primary antibodies and fluorescently labeled secondary antibodies, followed by image analysis. As illustrative examples, the co-detection of immuno-labeled dystrophin and the Y-chromosome in skeletal muscle are shown, and the localization of calbindin in the cerebellum is presented.

Histological and Histochemical Microscopy Used to Verify 2D-DIGE Pathoproteomics.

Comparative gel electrophoretic analyses of normal versus pathological specimens can swiftly identify proteome-wide changes in the concentration of specific protein isoforms. The application of fluorescence two-dimensional difference gel electrophoresis (2D-DIGE) can be employed for the characterization of complex protein populations in health and disease. In order to verify pathoproteomic findings and correlate them to histopathological alterations, standardized histological and histochemical methodology can be applied for the cell biological analysis of normal versus pathological tissue samples. This chapter outlines the usage of histochemical ATPase staining of fast and slow fiber types in normal versus dystrophic skeletal muscles, as well as the application of hematoxylin and eosin staining of nuclei and the cellular body in kidney cells, and Sudan black staining of lipids in cryo-sections.

Bioinformatic Analysis of the Subproteomic Profile of Cardiomyopathic Tissue.

Following large-scale protein separation by two-dimensional gel electrophoresis or liquid chromatography, mass spectrometry-based proteomics can be used for the swift identification and characterization of cardiac proteins and their various proteoforms. Comparative cardiac proteomics has been widely applied for the systematic analysis of heart disease and the establishment of novel diagnostic protein biomarkers. The X-linked neuromuscular disorder Duchenne muscular dystrophy is a multisystemic disease that is characterized by late-onset cardiomyopathy. This chapter outlines the bioinformatic analysis of the subproteomic profile of cardiac tissue from wild-type versus the dystrophic mdx-4cv mouse model of dystrophinopathy.

Extracellular Matrix Proteomics: The mdx-4cv Mouse Diaphragm as a Surrogate for Studying Myofibrosis in Dystrophinopathy.

The progressive degeneration of the skeletal musculature in Duchenne muscular dystrophy is accompanied by reactive myofibrosis, fat substitution, and chronic inflammation. Fibrotic changes and reduced tissue elasticity correlate with the loss in motor function in this X-chromosomal disorder. Thus, although dystrophinopathies are due to primary abnormalities in the DMD gene causing the almost-complete absence of the cytoskeletal Dp427-M isoform of dystrophin in voluntary muscles, the excessive accumulation of extracellular matrix proteins presents a key histopathological hallmark of muscular dystrophy. Animal model research has been instrumental in the characterization of dystrophic muscles and has contributed to a better understanding of the complex pathogenesis of dystrophinopathies, the discovery of new disease biomarkers, and the testing of novel therapeutic strategies. In this article, we review how mass-spectrometry-based proteomics can be used to study changes in key components of the endomysium, perimysium, and epimysium, such as collagens, proteoglycans, matricellular proteins, and adhesion receptors. The mdx-4cv mouse diaphragm displays severe myofibrosis, making it an ideal model system for large-scale surveys of systematic alterations in the matrisome of dystrophic fibers. Novel biomarkers of myofibrosis can now be tested for their appropriateness in the preclinical and clinical setting as diagnostic, pharmacodynamic, prognostic, and/or therapeutic monitoring indicators.

Mass spectrometry-based proteomic characterization of the middle-aged mouse brain for animal model research of neuromuscular diseases.

Neuromuscular diseases with primary muscle wasting symptoms may also display multi-systemic changes in the body and exhibit secondary pathophysiological alterations in various non-muscle tissues. In some cases, this includes proteome-wide alterations and/or adaptations in the central nervous system. Thus, in order to provide an improved bioanalytical basis for the comprehensive evaluation of animal models that are routinely used in muscle research, this report describes the mass spectrometry-based proteomic characterization of the mouse brain. Crude tissue extracts were examined by bottom-up proteomics and detected 4558 distinct protein species. The detailed analysis of the brain proteome revealed the presence of abundant cellular proteoforms in the neuronal cytoskeleton, as well as various brain region enriched proteins, including markers of the cerebral cortex, cerebellum, hippocampus and the olfactory bulb. Neuroproteomic markers of specific cell types in the brain were identified in association with various types of neurons and glia cells. Markers of subcellular structures were established for the plasmalemma, nucleus, endoplasmic reticulum, mitochondria and other crucial organelles, as well as synaptic components that are involved in presynaptic vesicle docking, neurotransmitter release and synapse remodelling.

Comparing the efficacy in reducing brain injury of different neuroprotective agents following neonatal hypoxia-ischemia in newborn rats: a multi-drug randomized controlled screening trial.

Intrapartum hypoxia-ischemia leading to neonatal encephalopathy (NE) results in significant neonatal mortality and morbidity worldwide, with > 85% of cases occurring in low- and middle-income countries (LMIC). Therapeutic hypothermia (HT) is currently the only available safe and effective treatment of HIE in high-income countries (HIC); however, it has shown limited safety or efficacy in LMIC. Therefore, other therapies are urgently required. We aimed to compare the treatment effects of putative neuroprotective drug candidates following neonatal hypoxic-ischemic (HI) brain injury in an established P7 rat Vannucci model. We conducted the first multi-drug randomized controlled preclinical screening trial, investigating 25 potential therapeutic agents using a standardized experimental setting in which P7 rat pups were exposed to unilateral HI brain injury. The brains were analysed for unilateral hemispheric brain area loss after 7 days survival. Twenty animal experiments were performed. Eight of the 25 therapeutic agents significantly reduced brain area loss with the strongest treatment effect for Caffeine, Sonic Hedgehog Agonist (SAG) and Allopurinol, followed by Melatonin, Clemastine, ß-Hydroxybutyrate, Omegaven, and Iodide. The probability of efficacy was superior to that of HT for Caffeine, SAG, Allopurinol, Melatonin, Clemastine, ß-hydroxybutyrate, and Omegaven. We provide the results of the first systematic preclinical screening of potential neuroprotective treatments and present alternative single therapies that may be promising treatment options for HT in LMIC.