Role of Cerebral Vasculature and Effect of Circulating Exosomes in Propagation of Systemic Inflammatory Responses into the Central Nervous System

Sepsis-associated encephalopathy (SAE) is an acutely progressing brain dysfunction induced by systemic inflammation. The mechanism of initiation of neuroinflammation during SAE, which ultimately leads to delirium and cognitive dysfunction, remains elusive. The goal of this project was to study the molecular events of SAE to capture its onset and progression into the central nervous system (CNS), and further identify the cellular players involved in mediating acute inflammatory signaling. Gene expression profiling on the cerebral vessels isolated from the brains of the mice treated with peripheral lipopolysaccharide (LPS) revealed that the cerebral vasculature responds within minutes to acute systemic inflammation by upregulating the expression of immediate early response genes, followed by activation of the NF-κB pathway. To identify the earliest responding cell type, fluorescence-activated cell sorting (FACS) was utilized to sort the immunolabelled glial and vascular cells from the brains of the mice treated with LPS at different time points and gene expression profiling was performed. Bioinformatic analysis of the sequencing data followed by further validation revealed that the cerebral endothelial cells (CECs) activation is the earliest event in the CNS and that they are the most likely source of proinflammatory mediators that could further initiate glial cell activation. This is further followed by the activation of apoptotic signaling in the CECs which is known to lead to blood brain barrier (BBB) disruption and allow the peripheral cytokines to leak into the CNS, exacerbate the gliosis and result in neuroinflammatory cascade. Together, these results model the sequential events during the advancement of systemic inflammation into the CNS, and facilitate better understanding of the interplay between the vascular and glial cells in initiating and driving acute neuroinflammation during SAE. Systemic inflammation does lead to neuroinflammation, thereby linking the peripheral inflammatory conditions to the CNS. However, the mechanisms through which systemic inflammation exerts its effect on the CNS are poorly understood. Exosomes are small (30 to 100 nanometers) membrane bound extracellular vesicles released by most of the mammalian cells. Exosomes play a vital role in cell to cell communication. This includes regulation of inflammatory responses by shuttling mRNAs, miRNAs and cytokines both locally and systemically to the neighboring as well as distant cells to further modulate their transcriptional and/or translational states and affect the functional phenotype of those cells which have taken up these exosomes. The role of circulating blood exosomes in mediating neuroinflammation during systemic inflammation was thus studied. Serum derived exosomes from LPS-challenged mice (SDEL) were freshly isolated from the sera of the mice which were earlier treated with LPS and used to study SDEL effects on neuroinflammation. Exosomes isolated from the sera of the mice injected with saline were used as control. In vitro studies showed that the SDEL upregulate pro-inflammatory cytokine gene expression in the cell lines of microglia (BV2), astrocytes (C8-D1A) and cerebral microvascular endothelial cells (Bend.3). To further study their effects in vivo, SDEL were then intravenously injected into normal adult mice. The recipient mice that received SDEL exhibited elevated microglial activation. Moreover, increased astrogliosis, and elevated CNS expression of pro-inflammatory cytokine mRNA were observed in SDEL recipient mice. Additionally, SDEL injected directly into the cerebral ventricles resulted in significant microgliosis as well as, astrogliosis. Together, these results demonstrate a novel role of peripheral circulating exosomes in causing neuroinflammation during systemic inflammatory conditions

Identification and Quantification of the Collagen Type I, III and V in Rabbit Patellar Tendons

Tendon injuries pose a great clinical challenge to orthopedic surgeons. Over 200,000 patients undergo tendon repair every year in the United States alone. The role of progression of a tendon injury is multifactorial as a lot of factors come into play during and after the injury at various phases of healing process. There is a still a vast requirement for thorough elucidation and understanding of the pathophysiology and the factors involved in the progression of tendon injury. Although the degenerative role of several MMPs and ADAMTs have been reported, yet there is very less information on the actual role and function of the various types of collagen in tendons and their response to the above factors, particularly after an injury and during the healing and repair phases. Our results indicate that there is a lot of variability in the levels of collagen types I, III and V in relation to the age of the animal, injury and the healing period. This might not be any surprising as the fluctuation in the levels indicate that there might be a different mechanisms involved in progression and healing of the injuries in young and old rabbits, at various phases of healing

In vivo evidence for the contribution of peripheral circulating inflammatory exosomes to neuroinflammation

Abstract Background Neuroinflammation is implicated in the development and progression of many neurodegenerative diseases. Conditions that lead to a peripheral immune response are often associated with inflammation in the central nervous system (CNS), suggesting a communication between the peripheral immune system and the neuroimmune system. The underlying mechanism of this relationship remains largely unknown; however, experimental studies have demonstrated that exposure to infectious stimuli, such as lipopolysaccharide (LPS) or high-fat diet (HFD) feeding, result in profound peripheral- and neuro-inflammation. Methods Using the model of endotoxemia with LPS, we studied the role of serum-derived exosomes in mediating neuroinflammation. We purified circulating exosomes from the sera of LPS-challenged mice, which were then intravenously injected into normal adult mice. Results We found that the recipient mice that received serum-derived exosomes from LPS-challenged mice exhibited elevated microglial activation. Moreover, we observed astrogliosis, increased systemic pro-inflammatory cytokine production, and elevated CNS expression of pro-inflammatory cytokine mRNA and the inflammation-associated microRNA (miR-155) in these recipient mice. Gene expression analysis confirmed that many inflammatory microRNAs were significantly upregulated in the purified exosomes under LPS-challenged conditions. We observed accumulated signaling within the microglia of mice that received tail-vein injections of fluorescently labeled exosomes though the percentage of those microglial cells was found low. Finally, purified LPS-stimulated exosomes from blood when infused directly into the cerebral ventricles provoked significant microgliosis and, to a lesser extent, astrogliosis. Conclusions The experimental results suggest that circulating exosomes may act as a neuroinflammatory mediator in systemic inflammation