Principal Investigator:

Simone Piva, Department of Medical and Surgical Specialties, Radiological Sciences and Public Health, University of Brescia, Italy.

Fabio Turla, Department of Medical and Surgical Specialties, Radiological Sciences and Public Health, University of Brescia, Italy.

Nicola Latronico, Department of Medical and Surgical Specialties, Radiological Sciences and Public Health, University of Brescia, Italy.

Gasparotti Roberto, Department of Medical and Surgical Specialties, Radiological Sciences and Public Health, University of Brescia, Italy.

Daniele Corbo, Department of Medical and Surgical Specialties, Radiological Sciences and Public Health, University of Brescia, Italy.

Co-Investigator:

Eiji Kawamoto, MD, PhD, Associate Professor, Department of Disaster and Emergency Medicine, Mie University School of Medicine

Yoshiyuki Nakamori, MD, Assistant Professor Department of Clinical Anesthesiology, Mie University School of Medicine

Masataka Kamei, MD, PhD, Professor Department of Clinical Anesthesiology, Mie University School of Medicine

Kaoru Ikejiri, MD. Instructor, Department of Disaster and Emergency Medicine, Mie University School of Medicine

Yuichi Akama, MD. Instructor, Department of Disaster and Emergency Medicine, Mie University School of Medicine

Motomu Shimaoka (Laboratory hosting), Professor and Chairman Dept of Molecular Pathobiology and Cell Adhesion Biology Mie University Graduate School of Medicine, Mie University School of Medicine

Overview

Sepsis (and septic shock) is a multi-organ failure consequent to an uncontrolled immune system response to infection, affecting more than 31.5 million people in high-income countries, causing about 5.3 million deaths annually.¹ The central nervous system is one of the first organs to be affected by sepsis. The resulting clinical entity, namely delirium (sometimes referred as sepsis-associated encephalopathy, SAE, or sepsis-associated delirium, SAD), affects approximately a third of septic patients and is a risk factor for long term disability and mortality. Although delirium is nosologically and clinically well-characterized, its pathophysiology is still under debate.

Intensive care unit acquired weakness (ICU-AW) is the expression of another target organ of sepsis: muscles and nerves. ICU-AW  is detected up to 67% in critically ill patients with sepsis,² and is associated with difficulty in weaning from the ventilator, prolonged ICU stay, higher hospitalization charges and increases long-term morbidity and mortality.³

The purpose of this study is to elucidate the rule of the cross-talk between muscle and brain in the pathogenesis of SAD.

The study will be carried out in two different steps:

STEP1 (Neuro-ICU step: ITALY): patients with SAD will be studied using functional brain MRI (fMRI), and exosomes from muscle biopsy will be isolated. The correlation between the failure of brain-muscle cross-talk and the presence of SAD will be clarified by cytotoxic activity.

STEP2 (Animal model step: JAPAN): the human muscle exosomes isolated from patients with SAD will be transfected in an animal model (rat) of sepsis, and micro fMRI of rats will be performed, eventually confirming the possible rule of muscle-derived exosomes in the brain-muscle crass-talk alteration.

This international joint research is also expected to have a ripple effect that strengthens the network between EU and Japanese scientists.

INTRODUCTION

Sepsis is one of the most significant challenges in intensive care units (ICU) patients.⁴ Data from high­income countries suggests that 31.5 million cases of sepsis and 19.4 million cases of severe sepsis occur each year globally, with potentially 5.3 million deaths annually.¹ The central nervous system is one of the first organs to be affected by sepsis. The resulting clinical entity, namely delirium (sometimes referred as sepsis-associated encephalopathy, SAE, or sepsis-associated delirium, SAD), is transient and reversible brain dysfunction, occurring when the source of sepsis is located outside of the central nervous system. SAE affects approximately a third of septic patients and is a risk factor for long term disability and mortality. ^5,6 Delirium manifests itself with a disturbance of attention, awareness, and cognition that develops over a short period (hours to days) and fluctuates over time.^7–9 Although delirium is nosologically and clinically well-characterized, its pathophysiology is still under debate. The mechanism underlying SAD involves inflammatory and noninflammatory processes that affect endothelial cells, glial cells and neurons; these processes also disrupt intracellular metabolism and induce a breakdown of the blood-brain barrier.¹⁰ Moreover, at present, there is no effective treatment method, and it is an urgent task to elucidate the pathologic mechanism that leads to target identification for new treatment development.

DDgraphy

tmaoaboration with the oration with ine layer Muscle and nerves are also interested during sepsis. Intensive care unit–acquired weakness (ICU-AW), defined as “clinically detected weakness in critically ill patients in whom there is no plausible etiology other than critical illness,” is the clinical manifestation of the muscle and nerve involvement during sepsis.  ICU-AW can be ascribed to a critical illness polyneuropathy (CIP), a critical illness myopathy (CIM), or severe muscle disuse atrophy. These three conditions often coexist, and the combination of CIP and CIM – indicated as critical illness myopathy and neuropathy (CRIMYNE) or critical illness polyneuromyopathy (CIPNM) – is the most common overlap syndrome. ICU-AW is detected in 30 to 50% of patients, and the incidence is even higher (up to 67%) in critically ill patients with sepsis. ICU-AW is associated with difficulty in weaning from the ventilator, prolonged ICU stay, and higher hospitalization charges and increases long-term morbidity and mortality.³

In this study, we are going to explore the crass-talk between muscle and brain during sepsis, focusing on the rule of muscle-derived exosomes in determining the abnormality in brain function clinically manifested as delirium.

 Muscle-Brain Connection and cognitive function

Aerobic exercise has the effect of improving the cognitive function of the elderly.¹¹ By exercising, skeletal muscle promotes the secretion of soluble molecules called myokines and biological nanoparticles called exosomes, and they are supposed to act on the brain as circulating molecules.¹² Furthermore, increased expression in skeletal muscle of enzymes that break down metabolites that are toxic to the central nervous system may be involved.¹³ Healthy skeletal muscles under appropriate exercise load are thought to play a role in protecting CNS function, and the importance of skeletal muscle-brain communication is related to cognitive decline due to aging and dementia. Moreover, pathological neuronal loss and increased apoptosis have been reported in various models of sepsis in rodents;¹⁴ however, the involvement of skeletal muscle-brain communication in sepsis-related encephalopathy is unknown.

 ICU-AW

Muscles are also often interested during sepsis. ICU-AW, defined as a “clinically detected weakness in critically ill patients in whom there is no plausible etiology other than critical illness,” ³ is the most common neuromuscular impairment that affects the clinical course and outcomes of ICU patients.¹⁵ ICU-AW is detected up to 67% in critically ill patients with sepsis,² and is associated with difficulty in weaning from the ventilator, prolonged ICU stay, higher hospitalization charges and increases long-term morbidity and mortality.³

Although they are clearly distinct entities, SAD and ICU-AW are possibly related and may even interact negatively with each other. Both are influenced by the severity of illness, are aggravated by the treatment adopted in the ICU, and may share some predisposing and trigger factors.³ In particular, inflammation is a hallmark for both entities.

Dr. Kawamoto and Dr. Ikejiri are currently working on a research project aimed at elucidating the pathological state of ICU-AW at the cellular level, and the exosome-mediated neighborhood of skeletal muscle cells and muscle tissue-resident macrophages. We have obtained preliminary data indicating the importance of intercellular communication (Ikejiri, Kaken Young Researcher 19K18318).

Abnormal skeletal muscle-brain communication

In the present study, we would like to propose a new model for SAD. According to our hypothesis, during sepsis, exosomes containing miR-133a, a skeletal muscle-specific microRNA (myo-miRNA), are over-secreted and resident macrophages become M1 (pro-inflammatory phenotype). M1 macrophages are able to induce myopathy, and skeletal muscle cells damaged by myopathy have reduced mitochondrial function,¹⁶ depleted energy and reduced myokine expression and secretion, which has an effect of improving cognitive function. Moreover, inflamed muscle produces an excess of exosomes containing miR-133a that flow in the circulating blood and reach the blood-brain barrier vascular endothelium and microglial cells; activation of TLR-9-dependent signals by exosomes and epigenetic interference with gene expression by myo-miRNA induces hyperpermeability of the brain blood-brain barrier and microglial cell activation, resulting in cerebral inflammation / white matter disorder during sepsis.

OUTLINE OF INTERNATIONAL JOINT RESEARCH

The neuro-ICU in Brescia University (Dr. Piva) has important expertise in SAD and ICU-AW. An ongoing study (see STEP1) is correlating neuroinflammation and fMRI alterations. The focus of Brescia University will be:

1. Enrollment of septic patients and evaluation of central nervous function in patients with sepsis by resting-state fMRI.
2. Needle muscle biopsy required for skeletal muscle exosome isolation and analysis (fresh sample collection).

The neuro-ICU in Brescia has a very rare and valuable research environment that has established a system that systematically performs both resting-state fMRI and needle muscle biopsy. Therefore, Dr. Kawamoto and Dr. Nakamori will conduct an experimental study using fresh clinical muscle. They need to go directly to the neuro-ICU in Brescia University and collaborate with Dr. Simon Piva, the director of Neuro-ICU, for international collaboration for a period of  6 months.

The step to be completed at neuro-ICU at Brescia University will be as follow:

 STEP1.  Dr. Kawamoto and Dr. Nakamori will move directly to the neuro-ICU in Brescia University, Italy, and conduct research in cooperation with Dr. Piva (from January 2020 to June 2020).

Dr. Piva is leading a study on the correlation between neuroinflammatory biomarkers and resting-state fMRI (ClinicalTrial.gov ID: NP3468, Title: Neuroinflammation During ICU-associated Delirium in Critically Ill Patients and Its Association with Structural and Functional Brain Alterations: a Nested Case-control Study; for brevity in the present protocol the study will be referred as fMRI / Sepsis project, preliminary results in Figure 1). Moreover, as per the study protocol, Dr. Piva is performing needle muscle biopsy on patients with SAD. Fresh muscle samples will be processed directly in Brescia University by Dr. Kawamoto and Dr. Nakamori that have expertise in muscle processing (Brescia University does not have such expertise), in order to isolate exosomes, microRNAs, and myokines. The extraction process needs to be performed within few minutes after biopsy. When exosomes, microRNAs, and myokines are harvest they could be safely stored at -80°C and sent by dry ice via FedEx at the proper time (see STEP2).

The objectives of STEP1 are:

1. Objective evaluation of CNS dysfunction performed using the connectivity of the default mode network (DMN) measured by resting state-fMRI.

1B. For abnormal skeletal muscle secretion, a skeletal muscle biopsy will be performed, and exosomes will be isolated and analyzed to measure specific microRNA levels, mitochondrial DNA levels, and skeletal muscle exosome cytotoxic activity in vitro.

1C. To investigate the correlation between the above CNS dysfunction level and abnormal skeletal muscle secretion level.

STEP2. In order to further develop the correlation research results shown in STEP1, the activity of clinical samples (skeletal muscle-derived exosomes) will be evaluated at Mie University (Japan), using animal models and micro MRI equipment. The purposes of STEP2 are:

2A. To prove the causal relationship of “muscle-brain crass-talk failure”. As described above, a fresh biopsy sample is essential for exosome separation from skeletal muscle, but once separated, exosomes can be stored frozen with little loss of activity. Part of the exosomes isolated at the University of Brescia will be transported to Mie University by Fedex in dry ice and analyzed for biological activity using a rat model in a molecular pathology laboratory. Research at Mie University is centered on Dr. Kamei, Dr. Akama, Dr. Ikejiri at Mie University and Dr. Piva from Italy that will move in Japan for 6 months (from September 2020 to December 2020).

2B. Performing resting fMRI analysis in rats using animal MRI (micro MRI) equipment installed at Mie University, by establishing an experimental system that can reproduce the resting fMRI findings (DMN activity disorder) similar to those of patients with SAD by optimizing conditions (severity of sepsis and fMRI imaging timing and conditions) in a sepsis model.

2C. Demonstrate that administration of skeletal muscle-derived exosomes from patients with SAD to rats can reproduce (part of) the same abnormal fMRI findings as patients. These in vivo studies can experimentally show the causal relationship that pathogenic factors secreted by skeletal muscles damaged by sepsis contribute to determining CNS dysfunction during sepsis. In our hypothesis, “Cross-talk failure is at the heart of the pathology of sepsis-related encephalopathy.” (Figure 2)

Research purpose and method of international joint research

Neuroimaging of sepsis-related encephalopathy: One of the factors hindering the elucidation of the pathogenesis of sepsis-related encephalopathy is the fact that the diagnosis relies mainly on clinical observation, and the development of effective imaging and biomarkers is fundamental especially in those patients where a clinical evaluation is not possible (i.e. come, deep sedation, use of paralytic agents).

Dr. Piva uses the resting fMRI to focus on the need for more objective diagnostic methods in the treatment of patients with SAD at the Brescia University Hospital Neuro-ICU, Italy. The patient’s neuroimaging data is currently being collected (fMRI / Sepsis Project).

Preliminary results from the ongoing study show an alteration of DMN activity in patients with SAD (figure 1) Therefore, fMRI findings may be used for objective assessment of SAD.

On the other hand, Dr. Kawamoto has the expertise of conducting research on MRI tissue metabolism and organ blood flow in a mouse sepsis model using a micro MRI apparatus manufactured by MR Solutions, Inc., which was recently set up in Mie University. Currently, fMRI protocol optimization for rat sepsis models larger than mice is being carried out in cooperation with MR Solutions. Using the patient fMRI data as a reference, we will establish a protocol for measuring DMN activity in a rat sepsis model, and take a challenging approach to construct a system for evaluating sepsis-related encephalopathy in an animal model. Therefore, in this joint research, it is possible to accelerate the process of elucidating the pathophysiology by comparing both the neuroimaging data in the clinical field and the data in the rat model.

ICU-AW and skeletal muscle-brain communication: At first glance, skeletal muscle does not seem to be directly related to sepsis, but aerobic exercise using skeletal muscle improves cognitive function in aging and Alzheimer’s disease. As a molecular mechanism, the involvement of skeletal muscle-brain communication via exosomes and myokines has been clarified.

In patients who have been admitted to the ICU due to sepsis, myopathy is caused not only by bed-rest disuse atrophy but also by systemic inflammation and metabolic abnormalities. In recent years, muscle weakness (ICU-AW) has attracted attention as an important condition. Furthermore, it has been shown that other dynamic exercises of skeletal muscles such as early rehabilitation are effective in preventing ICU-AW. Until now, little attention has been paid on the causal relationship between the pathophysiology of SAD and the pathophysiology of septic myopathy. In this study, we focus on the possibility of this causal relationship, and examine the academic question, “Abnormal skeletal muscle-brain crass-talk failure plays an important role in the pathogenesis of sepsis-related encephalopathy.”

Dr. Kawamoto and Dr. Ikejiri, have already conducted research on the interaction between resident macrophages residing in muscle tissue and skeletal muscle cells as a mechanism that causes ICU-AW. A model system using muscle tissue collected from a rat model and resident macrophages is under study.

  OUTLINE OF RESEARCH PLAN

[STEP1] Outline of Research Plan at the University of Brescia, Italy

Fresh skeletal muscle biopsy sample analysis from patients with sepsis-related encephalopathy. Dr. Piva conducts a skeletal muscle biopsy with the consent of the patients with sepsis included in the fMRI / Sepsis project who perform DMN activity measurement with fMRI. Kawamoto and Nakamori perform the following analysis using fresh biopsy samples.

1. Skeletal muscle disorder level assessment: It is known that skeletal muscle cell disorders in sepsis are mainly characterized by mitochondrial dysfunction. To asses mitochondrial ATP production ability representing skeletal muscle mitochondrial function, we will measure mitochondrial DNA / RNA amount quantified by real-time qPCR and DNA oxidation state evaluated by LC-MS / MS (liquid chromatography tandem mass spectrometry).
2. Skeletal muscle-derived exosomes: exosomes secreted into skeletal muscle tissues will be separated from biopsy sample homogenates from fractions precipitated by ultracentrifugation, and myokines will be detected from soluble fractions (see below).
3. Analyze active substances contained in isolated exosomes, focusing on nucleic acids (DNA / RNA): Comprehensive profiling of pro-inflammatory mitochondrial DNA fragments and microRNAs (miR-133a) and microglial activation will be assessed using deep sequencing or arrays. Dr. Kawamoto has extensive experience in a comprehensive analysis of exosomal RNA.¹⁷
4. Secretome analysis (the measurement of myokines level): The analysis of the secretome of skeletal muscle cells from the homogenate soluble fraction of the biopsy sample will be performed by proteomics, that will allowed to quantify the expression of myokines (myostatin, cathepsin G, iricin), and the expression level of the enzyme that degrades neurotoxic kynurenic acid. The myocine that has undergone extensive detection compared to the control will be quantitatively measured by ELISA method separately.
5. Bioassay: we will examine the biological activity of exosomes and myokines secreted by skeletal muscle using cultured cells (microglia cell line, brain blood-brain barrier / vascular endothelial cell line). In particular, the bioassay will allow us to detect and measure the biological activity of microglia and the increased permeability of vascular endothelial cells.
6. Correlation evaluation with fMRI: Expression level and biological activity level of microRNA and myokine secreted by skeletal muscle as described above, will be correlated to CNS function evaluation data collected by fMRI / Sepsis project (DMN activity evaluation by fMRI).

 

[STEP2]  Outline of the research plan at Mie University

A fresh biopsy sample is essential for exosome separation from skeletal muscle, but once separated exosomes can be cryo-preserved with almost no loss of activity. Part of the exosomes isolated at the University of Brescia, Italy, will be transferred to Mie University by FedEx dry-ice and analyzed for biological activity using an in-vivo rat model and a micro MRI apparatus.

1. Establishment of an animal model of sepsis-related encephalopathy: Since the diagnosis of SAD is based on clinical symptoms, it is difficult to establish it in an animal model. Even in the cecal ligation and puncture (CLP) model, it is very difficult to assess delirium development. The fMRI / Sepsis project collects fMRI data for patients with SAD and an objective and quantitative diagnostic index is being constructed. In addition, it has recently been found that it is technically possible to evaluate neural network integration using fMRI in rats and mice.¹⁸

Therefore, in this study, fMRI analysis will be performed on rats using the MRI (micro MRI) device for small animals installed at Mie University, and the following points will be examined.

1. To investigate whether the same findings (DMN activity disorder) will be observed between human and rat during sepsis. We will use a rat sepsis model (small amount of endotoxin injection) as control; rats will be injected wit exosomes (case) derived from human muscle as previously explained. Case and control rats will receive micro fMRI in order to establish whether the alteration in human and case rats will be similar.
2. Effect of myokine: Following examination of exosomes derived from skeletal muscle, we will administer myokines (harvest in patients with SAD) and subsequently its antibody to the rats. This will be important to establish a possible rule of antibody in preventing SAD development.

These in vivo animal studies reproduce experimentally the causal relationship between pathogenic factors secreted by skeletal muscles damaged by sepsis and central nervous system dysfunction. Our experiment could clarify the rule of brain-muscle crosstalk in SAD pathophysiology.

Expected problems and countermeasures

(1) The amount of exosomes that can be separated from skeletal muscle biopsy samples may not be sufficient for rat experiments. In this case, skeletal muscle samples can be cultured for a short period,¹⁹ and exosomes secreted into the supernatant can be harvested. Changes in the exosome properties secreted by skeletal muscle in culture will be evaluated by comparing fresh samples with microRNA expression profiles. Another approach is to reduce the amount of exosomes required for the experiment by using mice that are smaller in size than rats. As a trade-off, the size of the brain is reduced, so the resolution of fMRI is slightly reduced. However, a previous paper demonstrated its feasibility.¹⁸

(2) It may be difficult to reproduce the resting fMRI findings (DMN activity disorder) similar to those of delirium patients in rats (or mice). Systematically identify the type and severity of sepsis model (degree of CLP treatment, adjustment of endotoxin level, etc.), stage (elapsed time after onset, several hours to several days), and type and presence of anesthesia for immobilization. It is necessary to examine and optimize. Since fMRI can be performed with the animals alive, optimization can be performed in a short period of time.

Specific roles for the study contributors and travel Plan

The facilities for the present study will be placed as follow:

• University of Brescia: in the Brescia University ICU, patient’s enrollment will take place and all the biological specimens will be collected: blood and muscle biopsy. Drs. Kawamoto and Nakamori (during their 6 months stay in Italy), and Dr. Piva, will enroll all the patients and will perform proposed experiments using fresh biopsy samples in the laboratory space at the Brescia University. Moreover, in Brescia University Prof. Gasparotti and Dr. Corbo will perform fMRI as per protocol.
• Tsu University, Nagoya (Japan): in Prof. Motomu’s Laboratory, Dr. Piva (during his 6 months stay in Japan), Dr. Kamei, Dr. Ikejiri, and Dr. Akama will perform relevant experiments using human exosomes and animal models as previously explained.

ROLES AND PREPARATIONS OF OVERSEAS COLLABORATORS

 

Simone Piva, M.D. (Director of Neuro-ICU, Brescia University Hospital, Italy) is also a visiting associate professor at Mie University, Regional Disaster Medical Leader Development Center and Department of Molecular Pathology, Graduate School of Medicine. Dr. Piva was trained in basic medical research at the laboratory of Harvard Medical School, Professor Shimaoka Molecular Pathology.

Dr. Piva is an expert in the intensive care field of the central nervous system and serves as the representative researcher of the clinical study “fMRI / Sepsis project”. The fMRI / Sepsis project is a clinical study aimed at establishing an objective and quantitative diagnosis method for SAD. The present study makes the best use of joint Italian-Japanese research, obtained skeletal muscle biopsy samples from patient cohorts participating in the fMRI / Sepsis project, and biochemical and biological data on skeletal muscle-derived exosomes and myokines. It provides an invaluable opportunity to examine the correlation with sepsis-related encephalopathy severity data by fMRI evaluation.

As the principal researcher of the Italian side, Dr. Piva’s

• Ensure access to fMRI / Sepsis project patient data.
• Get consent form from patients and perform the skeletal muscle biopsies.
• Providing resources (approval of research performance from the research space, equipment, and facility manager) for in vitro experiments using skeletal muscle biopsy samples by Japanese researchers (Kawamoto / Nakamori) in the Department of Anesthesiology, University of Brescia University of Medicine.
• Participate in discussions on data analysis and interpretation.

Dr. Piva is carrying out the fMRI / Sepsis project and is currently collecting fMRI data for septic and non-septic patients who have not developed encephalopathy as a comparison target with the septic-related encephalopathy patients at the Brescia University Hospital Neuro-ICU (Figure 1). Dr. Piva stayed at Mie University Regional Disaster Medical Leader Development Center for 3 weeks in February 2019 and conducted preliminary research for this study.

Mie University has a micro-MRI facility manufactured by MR Solutions and has established a research environment for MRI imaging of small animals. Kawamoto has been conducting research to evaluate organ damage in a rat sepsis model over time using micro MRI but is currently optimizing imaging conditions for fMRI with the support of MR Solutions engineers. Dr. Piva advised fMRI experimental conditions during his stay at Mie University.

The separation of exosomes and biochemical and biological analysis methods have been established by Kawamoto.¹⁷ Dr. Piva will stay in Mie University directly to observe experiments related to exosome separation and analysis, and by meeting with Kawamoto and Nakamori, he has enough equipment and reagents to set up in the Department of Anesthesiology, Brescia University School of Medicine. I understood.

 

Furthermore, Kawamoto and Ikejiri conducted bioinformatics analysis based on the microRNA database as a preliminary study, and identified miR-133a as a skeletal muscle-specific microRNA (myomiRNA) whose expression is increased by sepsis. We are conducting research on functions and their involvement in sepsis. Ikejiri’s research has focused on the effects of miR-133a on macrophages, but at the time of a meeting with Dr. Piva, the possibility of an effect on the central nervous system of miR-133a was verified. We examined the method to do this.

 

 

 

 

 

Figure 1: Preliminary data from fMRI / Sepsis Project. After enrollment of 15 patients, we have build a new hypothesis based on default mode network alteration during delirium (see text in the figure).

 

Figure 2: Illustration of STEP1 and STEP2. In STEP1 (Brescia University), critically ill patients will be evaluated for the presence of both SAD and ICU-AW. Once the diagnosis of SAD is made, muscle biopsy will be performed. Fresh muscle will be immediately processed for exosomes extraction and cytotoxic activity will be caracterized. After refrigeration exosomes will be sended in dry ice to Mie University and injected in rats. Micro-fMRI will be then performed in order to compare human and rats findings.

 

 

 

 

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