Neurotrauma investigation through spatial omics guided by mass spectrometry imaging: Target identification and clinical applications.

Traumatic brain injury (TBI) represents one of the major public health concerns worldwide due to the increase in TBI incidence as a result of injuries from daily life accidents such as sports and motor vehicle transportation as well as military-related practices. This type of central nervous system trauma is known to predispose patients to several neurological disorders such as Parkinson's disease, Alzheimer's disease, chronic trauamatic encephalopathy, and age-related Dementia. Recently, several proteomic and lipidomic platforms have been applied on different TBI studies to investigate TBI-related mechanisms that have broadened our understanding of its distinct neuropathological complications. In this study, we provide an updated comprehensive overview of the current knowledge and novel perspectives of the spatially resolved microproteomics and microlipidomics approaches guided by mass spectrometry imaging used in TBI studies and its applications in the neurotrauma field. In this regard, we will discuss the use of the spatially resolved microproteomics and assess the different microproteomic sampling methods such as laser capture microdissection, parafilm assisted microdissection, and liquid microjunction extraction as accurate and precise techniques in the field of neuroproteomics. Additionally, we will highlight lipid profiling applications and their prospective potentials in characterizing molecular processes involved in the field of TBI. Specifically, we will discuss the phospholipid metabolism acting as a precursor for proinflammatory molecules such as eicosanoids. Finally, we will survey the current state of spatial neuroproteomics and microproteomics applications and present the various studies highlighting their findings in these fields.

Canine Mammary Tumors: Classification, Biomarkers, Traditional and Personalized Therapies.

In recent years, many studies have focused their attention on the dog as a proper animal model for human cancer. In dogs, mammary tumors develop spontaneously, involving a complex interplay between tumor cells and the immune system and revealing several molecular and clinical similarities to human breast cancer. In this review, we summarized the major features of canine mammary tumor, risk factors, and the most important biomarkers used for diagnosis and treatment. Traditional therapy of mammary tumors in dogs includes surgery, which is the first choice, followed by chemotherapy, radiotherapy, or hormonal therapy. However, these therapeutic strategies may not always be sufficient on their own; advancements in understanding cancer mechanisms and the development of innovative treatments offer hope for improved outcomes for oncologic patients. There is still a growing interest in the use of personalized medicine, which should play an irreplaceable role in the research not only in human cancer therapy, but also in veterinary oncology. Moreover, immunotherapy may represent a novel and promising therapeutic option in canine mammary cancers. The study of novel therapeutic approaches is essential for future research in both human and veterinary oncology.

Impact of Canine Amniotic Mesenchymal Stem Cell Conditioned Media on the Wound Healing Process: In Vitro and In Vivo Study.

The aim of this study was to provide a beneficial treatment effect of mesenchymal stem cell products derived from the canine amniotic membrane (AM-MSC) on the complicated wound healing process in dogs. AM-MSCs were characterized in terms of morphology, phenotypic profile, and multilineage differentiation potential. The in vitro study of the effect of canine amniotic mesenchymal stem cell conditioned media (AMMSC-CM) on a primary skin fibroblast cell culture scratch assay showed a decrease in the measured scratch area of about 66.39% against the negative control (Dulbecco's Modified Eagle's Medium-32.55%) and the positive control (Dulbecco's Modified Eagle's Medium supplemented with FGF2, N2, B27, and EGF-82.077%) after 72 h treatment. In the experimental study, seven dogs with complicated nonhealing wounds were treated with a combination of antibiotics, NSAIDs, and local AMMSC-CM application. After 15 days of therapy, we observed a 98.47% reduction in the wound surface area as opposed to 57.135% in the control group treated by conventional therapy based on debridement of necrotic tissue, antibiotic therapy, pain management, and change of wound dressing.

Canine Bone Marrow-derived Mesenchymal Stem Cells: Genomics, Proteomics and Functional Analyses of Paracrine Factors.

Adult stem cells have become prominent candidates for treating various diseases in veterinary practice. The main goal of our study was therefore to provide a comprehensive study of canine bone marrow-derived mesenchymal stem cells (BMMSC) and conditioned media, isolated from healthy adult dogs of different breeds. Under well-defined standardized isolation protocols, the multipotent differentiation and specific surface markers of BMMSC were supplemented with their gene expression, proteomic profile, and their biological function. The presented data confirm that canine BMMSC express important genes for differentiation toward osteo-, chondro-, and tendo-genic directions, but also genes associated with angiogenic, neurotrophic, and immunomodulatory properties. Furthermore, using proteome profiling, we identify for the first time the dynamic release of various bioactive molecules, such as transcription and translation factors and osteogenic, growth, angiogenic, and neurotrophic factors from canine BMMSC conditioned medium. Importantly, the relevant genes were linked to their proteins as detected in the conditioned medium and further associated with angiogenic activity in chorioallantoic membrane (CAM) assay. In this way, we show that the canine BMMSC release a variety of bioactive molecules, revealing a strong paracrine component that may possess therapeutic potential in various pathologies. However, extensive experimental or preclinical trials testing canine sources need to be performed in order to better understand their paracrine action, which may lead to novel therapeutic strategies in veterinary medicine.

Improved tissue integrity after alginate treatment in rat spinal cord injury: evidence from ex vivo diffusion tensor imaging.

Diffusion tensor imaging (DTI) is a magnetic resonance imaging technique used to characterize fibrous structures such as white matter in the central nervous system, including normal and spinal cord injury (SCI) conditions. Our aim was to evaluate the effect of alginate treatment in the rat SCI by DTI parametric measures. Ex vivo DTI data were collected by spin echo sequence with following parameters TR/TE: 2500 ms/32 ms and b-value of 1500 s/mm2. Main significant changes were found in fractional anisotropy (FA), and radial diffusivity (RD), between the saline- and alginatetreated group at the level of individual sections and whole spinal cord. Results indicate that ex vivo DTI can be used as a tool for tissue structure characterisation and both FA and RD as promising prognostic parameters of SCI treatment.

Impact of mesenchymal stem cells derived conditioned media on neural progenitor cells.

Neurodegenerative diseases are common problem for companion animals. Due to the limited ability of injured axons to regenerate, innovative therapies combined with rehabilitation have been applied and evaluated. Among them, stem cells and their conditioned media implantation, which can ameliorate damaged tissue has been suggested as a promising treatment strategy. The main goal of our study was to characterize mesenchymal stem cells (MSC) derived from canine adipose tissue (AT-MSC) and umbilical cord (UC-MSC) and analyse effect of their conditioned media (CM) on neurite outgrowth of neural progenitor cells isolated from the brain cortex of neonatal rats. MSC from both sources showed high osteogenic and chondrogenic potential and expression of CD90 and CD29. Furthermore, both UC-MSCCM and AT-MSCCM stimulated neurite growth. Interestingly, this effect was more pronounced with UC-MSCCM when compared to AT-MSCCM in vitro, which may be related to the different content of neurotrophic factors included in the CM.

Diffusion Tensor Imaging: Tool for Tracking Injured Spinal Cord Fibres in Rat.

Spinal cord injury (SCI) is a severe disorder of the CNS leading to tissue damage and disability. Because it is critical to understand the pathological processes, it is important to find efficient ways to diagnose the severity of injured spinal cord tracts in situ from beginning up to a certain level of recovery following therapeutic interventions. In the current study, we set-up the criteria for diffusion tensor imaging (DTI) in order to capture changes of nerve fibre tracts in rat spinal cord compression injury. We tested four DTI parameters, such as fractional anisotropy, mean diffusivity, axial diffusivity and radial diffusivity at the lesion site, in time course of 7 weeks. Afterwards, we compared DTI data with histological results and locomotor outcomes to examine their consistency and capability of reflecting the lesion development in time. Our data confirm that DTI is a valuable in vivo imaging tool capable to distinguish damaged white matter tracts after mild SCI in rat. Fractional anisotropy showed decreased values for injury site, while the mean diffusivity had higher values, with increased both axial and radial diffusivity in comparison to control subjects. Thus, the combination of DTI parameters can reflect the severity of lesion in time and may correlate with histological evaluation of spared tissue, but not with locomotor recovery following mild injury associated with spontaneous recovery.

Spinal Cord Injury: Animal Models, Imaging Tools and the Treatment Strategies.

Spinal cord injury (SCI) often leads to irreversible neuro-degenerative changes with life-long consequences. While there is still no effective therapy available, the results of past research have led to improved quality of life for patients suffering from partial or permanent paralysis. In this review we focus on the need, importance and the scientific value of experimental animal models simulating SCI in humans. Furthermore, we highlight modern imaging tools determining the location and extent of spinal cord damage and their contribution to early diagnosis and selection of appropriate treatment. Finally, we focus on available cellular and acellular therapies and novel combinatory approaches with exosomes and active biomaterials. Here we discuss the efficacy and limitations of adult mesenchymal stem cells which can be derived from bone marrow, adipose tissue or umbilical cord blood and its Wharton's jelly. Special attention is paid to stem cell-derived exosomes and smart biomaterials due to their special properties as a delivery system for proteins, bioactive molecules or even genetic material.

Paclitaxel Treatment and Proprotein Convertase 1/3 (PC1/3) Knockdown in Macrophages is a Promising Antiglioma Strategy as Revealed by Proteomics and Cytotoxicity Studies.

High grade gliomas are the most common brain tumors in adult. These tumors are characterized by a high infiltration in microglial cells and macrophages. The immunosuppressive tumor environment is known to orient immune cells toward a pro-tumoral and anti-inflammatory phenotype. Therefore, the current challenge for cancer therapy is to find a way to reorient macrophages toward an antitumoral phenotype. Previously, we demonstrated that macrophages secreted antitumoral factors when they were invalidated for the proprotein converstase 1/3 (PC1/3) and treated with LPS. However, achieving an activation of macrophages via LPS/TLR4/Myd88-dependent pathway appears yet unfeasible in cancer patients. On the contrary, the antitumor drug Paclitaxel is also known to activate the TLR4 MyD88-dependent signaling pathway and mimics LPS action. Therefore, we evaluated if PC1/3 knock-down (KD) macrophages could be activated by Paclitaxel and efficient against glioma. We report here that such a treatment of PC1/3 KD macrophages drove to the overexpression of proteins mainly involved in cytoskeleton rearrangement. In support of this finding, we found that these cells exhibited a Ca2+ increase after Paclitaxel treatment. This is indicative of a possible depolymerization of microtubules and may therefore reflect an activation of inflammatory pathways in macrophages. In such a way, we found that PC1/3 KD macrophages displayed a repression of the anti-inflammatory pathway STAT3 and secreted more pro-inflammatory cytokines. Extracellular vesicles isolated from these PC1/3 KD cells inhibited glioma growth. Finally, the supernatant collected from the coculture between glioma cells and PC1/3 KD macrophages contained more antitumoral factors. These findings unravel the potential value of a new therapeutic strategy combining Paclitaxel and PC1/3 inhibition to switch macrophages toward an antitumoral immunophenotype.

Stem Cell Conditioned Medium Treatment for Canine Spinal Cord Injury: Pilot Feasibility Study.

Spinal cord injury (SCI) involves nerve damage and often leads to motor, sensory and autonomic dysfunctions. In the present study, we have designed a clinical protocol to assess the feasibility of systemic delivery of allogenic canine bone marrow tissue-derived mesenchymal stem cell conditioned medium (BMMSC CM) to dogs with SCI. Four client-owned dogs with chronic SCI lasting more than six months underwent neurological and clinical evaluation, MRI imaging and blood tests before being enrolled in this study. All dogs received four intravenous infusions with canine allogenic BMMSC CM within one month. Between the infusions the dogs received comprehensive physiotherapy, which continued for three additional months. No adverse effects or complications were observed during the one, three and six months follow-up periods. Neither blood chemistry panel nor hematology profile showed any significant changes. All dogs were clinically improved as assessed using Olby locomotor scales after one, three and six months of BMMSC CM treatment. Furthermore, goniometric measurements revealed partial improvement in the range of joint motion. Bladder function improved in two disabled dogs. We conclude that multiple delivery of allogenic cell-derived conditioned medium to dogs with chronic SCI is feasible, and it might be clinically beneficial in combination with physiotherapy.

RhoA Inhibitor Treatment At Acute Phase of Spinal Cord Injury May Induce Neurite Outgrowth and Synaptogenesis.

The therapeutic use of RhoA inhibitors (RhoAi) has been experimentally tested in spinal cord injury (SCI). In order to decipher the underlying molecular mechanisms involved in such a process, an in vitro neuroproteomic-systems biology platform was developed in which the pan-proteomic profile of the dorsal root ganglia (DRG) cell line ND7/23 DRG was assessed in a large array of culture conditions using RhoAi and/or conditioned media obtained from SCI ex vivo derived spinal cord slices. A fine mapping of the spatio-temporal molecular events of the RhoAi treatment in SCI was performed. The data obtained allow a better understanding of regeneration/degeneration induced above and below the lesion site. Results notably showed a time-dependent alteration of the transcription factors profile along with the synthesis of growth cone-related factors (receptors, ligands, and signaling pathways) in RhoAi treated DRG cells. Furthermore, we assessed in a rat SCI model the in vivo impact of RhoAi treatment administered in situ via alginate scaffold that was combined with FK506 delivery. The improved recovery of locomotion was detected only at the early postinjury time points, whereas after overall survival a dramatic increase of synaptic contacts on outgrowing neurites in affected segments was observed. We validate these results by in vivo proteomic studies along the spinal cord segments from tissue and secreted media analyses, confirming the increase of the synaptogenesis expression factors under RhoAi treatment. Taken together, we demonstrate that RhoAi treatment seems to be useful to stimulate neurite outgrowth in both in vitro as well in vivo environments. However, for in vivo experiments there is a need for sustained delivery regiment to facilitate axon regeneration and promote synaptic reconnections with appropriate target neurons also at chronic phase, which in turn may lead to higher assumption for functional improvement.

3D Reconstruction and Evaluation of Accessory Hepatic Veins in Right Hemilivers in Laboratory Animals by Metrotomography: Implications for Surgery.

BACKGROUND The current study investigated the detection of accessory hepatic veins and their vascular territories in the right hemiliver in rats, guinea pigs, and rabbits, which has become a prerequisite for newly developed clinical procedures. We compared the anatomical continuity of accessory hepatic veins with accessory hepatic veins existing in human livers. MATERIAL AND METHODS The analysis of accessory hepatic veins was performed using a corrosion cast method in combination with computer tomography (CT). RESULTS In normal livers, accessory hepatic veins were regularly found. The length of these veins was 0.88±0.29 (cm ±SD) in rats, 1.10±0.39 in guinea pigs, and 1.28±0.48 in rabbits. Accessory hepatic veins became a part of the draining vessel draining into segment VI and VII; represented by interpolating and following Chouinard's segmental concept. CONCLUSIONS The importance of detecting accessory hepatic veins lies in the identification of structures requiring special attention during surgery, in reduction of surgical complications, and in choosing the best approach to maintain the vitality of a drainage segment. The vascular reconstruction should be done during surgical interventions.

Extracellular vesicles: pathogenetic, diagnostic and therapeutic value in traumatic brain injury.

INTRODUCTION: Traumatic brain injury (TBI) is a leading cause of death and disability worldwide. Accurate classification according to injury-specific and patient-specific characteristics is critical to help informed clinical decision-making and to the pursuit of precision medicine in TBI. Reliable biomarker signatures for improved TBI diagnostics are required but still an unmet need. Areas covered: Extracellular vesicles (EVs) represent a new class of biomarker candidates in TBI. These nano-sized vesicles have key roles in cell signaling profoundly impacting pathogenic pathways, progression and long-term sequelae of TBI. As such EVs might provide novel neurobiological insights, enhance our understanding of the molecular mechanisms underlying TBI pathophysiology and recovery, and serve as biomarker signatures and therapeutic targets and delivery systems. Expert commentary: EVs are fast gaining momentum in TBI research, paving the way for new transformative diagnostic and treatment approaches. Their potential to sort out TBI variability and active involvement in the mechanisms underpinning different clinical phenotypes point out unique opportunities for improved classification, risk-stratification ad intervention, harboring promise of predictive, personalized, and even preemptive therapeutic strategies. Although a great deal of progress has been made, substantial efforts are still required to ensure the needed rigorous validation and reproducibility for clinical implementation of EVs.

Brain-Cortex Microglia-Derived Exosomes: Nanoparticles for Glioma Therapy.

The function and integrity of the nervous system require interactive exchanges among neurons and glial cells. Exosomes and other extracellular vesicles (EVs) are emerging as a key mediator of intercellular communication, capable of transferring nucleic acids, proteins and lipids influencing numerous functional and pathological aspects of both donor and recipient cells. The immune response mediated by microglia-derived exosomes is most prominently involved in the spread of neuroinflammation, neurodegenerative disorders, and brain cancer. Therefore, in the present study we describe a reproducible and highly efficient method for yielding purified primary microglia cells, followed by exosome isolation and their characterization. An in vitro biological assay demonstrates that microglia-derived exosomes tested on a 3D spheroid glioma culture were able to inhibit tumor invasion in time course. These results evidence that brain microglia-derived exosomes could be used as nanotherapeutic agents against glioma cells.

Proteomic Analysis of the Spatio-temporal Based Molecular Kinetics of Acute Spinal Cord Injury Identifies a Time- and Segment-specific Window for Effective Tissue Repair.

Spinal cord injury (SCI) represents a major debilitating health issue with a direct socioeconomic burden on the public and private sectors worldwide. Although several studies have been conducted to identify the molecular progression of injury sequel due from the lesion site, still the exact underlying mechanisms and pathways of injury development have not been fully elucidated. In this work, based on OMICs, 3D matrix-assisted laser desorption ionization (MALDI) imaging, cytokines arrays, confocal imaging we established for the first time that molecular and cellular processes occurring after SCI are altered between the lesion proximity, i.e. rostral and caudal segments nearby the lesion (R1-C1) whereas segments distant from R1-C1, i.e. R2-C2 and R3-C3 levels coexpressed factors implicated in neurogenesis. Delay in T regulators recruitment between R1 and C1 favor discrepancies between the two segments. This is also reinforced by presence of neurites outgrowth inhibitors in C1, absent in R1. Moreover, the presence of immunoglobulins (IgGs) in neurons at the lesion site at 3 days, validated by mass spectrometry, may present additional factor that contributes to limited regeneration. Treatment in vivo with anti-CD20 one hour after SCI did not improve locomotor function and decrease IgG expression. These results open the door of a novel view of the SCI treatment by considering the C1 as the therapeutic target.

Localized Intrathecal Delivery of Mesenchymal Stromal Cells Conditioned Medium Improves Functional Recovery in a Rat Model of Spinal Cord Injury.

It was recently shown that the conditioned medium (CM) of mesenchymal stem cells can enhance viability of neural and glial cell populations. In the present study, we have investigated a cell-free approach via CM from rat bone marrow stromal cells (MScCM) applied intrathecally (IT) for spinal cord injury (SCI) recovery in adult rats. Functional in vitro test on dorsal root ganglion (DRG) primary cultures confirmed biological properties of collected MScCM for production of neurosphere-like structures and axon outgrowth. Afterwards, rats underwent SCI and were treated with IT delivery of MScCM or vehicle at postsurgical Days 1, 5, 9, and 13, and left to survive 10 weeks. Rats that received MScCM showed significantly higher motor function recovery, increase in spared spinal cord tissue, enhanced GAP-43 expression and attenuated inflammation in comparison with vehicle-treated rats. Spared tissue around the lesion site was infiltrated with GAP-43-labeled axons at four weeks that gradually decreased at 10 weeks. Finally, a cytokine array performed on spinal cord extracts after MScCM treatment revealed decreased levels of IL-2, IL-6 and TNFα when compared to vehicle group. In conclusion, our results suggest that molecular cocktail found in MScCM is favorable for final neuroregeneration after SCI.

Oral administration of inosine promotes recovery after experimental spinal cord injury in rat.

Inosine, a purine nucleoside, is one of the novel substances, which can preserve the neuronal and glial viability and stimulate intact neurons to extend axons. We, herein, evaluated the effect of oral inosine treatment on spinal cord injury (SCI) recovery by means of locomotor and bladder function, quantification of neurons and spinal cord tissue sparing. Rats after compression SCI were divided into groups-SCI-Aqua and SCI-Inosine (daily application of aqua for injection or inosine)-locomotion of hind limbs (BBB score) and urinary bladder function were evaluated from day 1 to 28 after SCI. The neuronal profile was determined by immunohistochemistry with NeuN antibodies and tissue sparing by Luxol fast blue staining method. SCI affected the functional movement of hind limbs in both groups with gradual improvement (increased BBB score) during survival. However, we found a significant difference in BBB score and recovery of bladder function between SCI-Aqua and SCI-Inosine groups during the second week of survival following SCI. In addition, the number of NeuN positive cells and percentage of tissue sparing was also significantly higher in SCI-Inosine group when compared with the SCI-Aqua group. Daily oral administration of inosine after SCI throughout the survival was beneficial for locomotion and micturition, neuronal survival and tissue sparing. This indicates that inosine may represent one of the co-stimulatory factors for treatment strategies to promote neuronal plasticity after SCI.

Three-Dimensional Cultivation a Valuable Tool for Modelling Canine Mammary Gland Tumour Behaviour In Vitro.

Cell cultivation has been one of the most popular methods in research for decades. Currently, scientists routinely use two-dimensional (2D) and three-dimensional (3D) cell cultures of commercially available cell lines and primary cultures to study cellular behaviour, responses to stimuli, and interactions with their environment in a controlled laboratory setting. In recent years, 3D cultivation has gained more attention in modern biomedical research, mainly due to its numerous advantages compared to 2D cultures. One of the main goals where 3D culture models are used is the investigation of tumour diseases, in both animals and humans. The ability to simulate the tumour microenvironment and design 3D masses allows us to monitor all the processes that take place in tumour tissue created not only from cell lines but directly from the patient's tumour cells. One of the tumour types for which 3D culture methods are often used in research is the canine mammary gland tumour (CMT). The clinically similar profile of the CMT and breast tumours in humans makes the CMT a suitable model for studying the issue not only in animals but also in women.

Brain-derived neurotrophic factor blood levels in two models of transient brain ischemia in rats.

We monitored possible influence of transient focal and global brain ischemia on BDNF blood level. In both models noticeable fluctuation of BDNF concentration mainly in reperfusion was observed. During the first 90 min, BDNF in total blood and in blood cells continuously decreased in both models but plasma BDNF raised at 40 min and peaked at 90 min of reperfusion. Our data confirm the impact of transient brain ischemia on BDNF levels in the circulatory system, suggest blood cells as a possible source of BDNF and demonstrate the interdependence of blood compartments and physiological state of an affected organism.