Neuropathic pain represents a possible outcome of neural tissue injury; it occurs also as a concomitant symptom of different diseases or as a side effect of several treatments. Up to date, it constitutes a great challenge in clinical practice, as currently available treatments are still unsatisfactory. Mechanism-based treatment approaches are promising strategy in neuropathic pain management. However, there is still a lack of information about the exact mechanisms involved in the development and/or maintenance of neuropathic pain. This Doctoral Thesis is aimed to explore the mechanisms underlying the development of neuropathic pain states in different models. The principal part of this work is focused on the study of anti-inflammatory effect of Angiotensin II receptor type 1 (AT1R) blocker, losartan, in two different models of peripheral neuropathy: paclitaxel-induced peripheral neuropathy (PIPN) and spinal nerve ligation (SNL). The work also aimed to access the involvement of spinal transient receptor potential vanilloid type 1 (TRPV1) channels in the process of neuronal activation induced by paclitaxel (PAC) and chemokine CCL2 treatment. In order to fulfil the abovementioned aims, behavioral, immunohistochemical and molecular methods were used. For every model of peripheral neuropathy, the...

The brain is capable of plasticity, so that the structural and functional loss that are caused by cortical injury may recover. Neuroinflammatory response can greatly influence post-injury recovery by modulating synaptic plasticity. In our previous work, mesenchymal derived exosomes were found to promote functional recovery by converting microglia from a pro-inflammatory state to an anti-inflammatory state in aged rhesus monkeys after cortical injury in the primary motor cortex. In the present project, we demonstrated the effects of exosomes on synaptic changes and synapse-microglia interactions after lesion in the same monkeys. To further investigate the effects of modulating neuroinflammation on synaptic changes after injury, we also investigated dietary curcumin, an anti-inflammatory substance, in a separate group of monkeys. Both treatments showed an effect as neuroinflammatory modulators that reduced the density of microglial markers, Iba- 1/P2RY12. However, the cortical injury induced synaptic loss was reversed by the exosome treatment, whereas the other anti-inflammatory treatment, curcumin, did not show the same effect. Our results are consistent with previous study that cortical injury induced synaptic loss and microglia activation. Exosomes can both reduce inflammation and synapse loss after injury, but curcumin only showed anti-inflammatory effects. Overall, these data suggested that exosomes and curcumin had different mechanisms of how to modulate inflammation and synaptic properties to promote recovery after cortical injury.

Chronic heart failure (HF) is estimated to affect 23 million people worldwide, and many patients show minimal improvement after treatment with high-potency medications. HF with reduced left ventricular ejection fraction makes up approximately half of cases and is associated with high mortality: a 5-year survival rate of only 25% after hospitalization. This disease is marked by autonomic and cardiac dysfunction, as well as increased inflammatory markers both in the brain and microbiota of the gastrointestinal tract. As a main component of the autonomic nervous system, the vagus nerve has been identified as a potential treatment target for HF. Vagus nerve stimulation (VNS) is thought to help re-balance the autonomic system and has shown promising results in clinical trials for treatment of HF. Although the mechanism of action for VNS remains partially understood, anti-inflammatory pathways have been shown to play a significant role, and these pathways may be enhanced by microbiota signaling via the vagus nerve. The goal of the current study is to provide insight into VNS treatment for HF with reduced ejection fraction via a pressure overload (PO) model. Male Sprague-Dawley rats were randomly divided into age-matched control (n=7), PO (n=6), and PO+VNS (n=11). PO rats underwent aortic constriction (~40%) to induce HF, and a subset of these had VNS leads implanted around the left cervical vagus nerve. Treatment was initiated for PO+VNS rats after reaching a 20% drop in left ventricular relative ejection fraction (EF, p<0.001). VNS was delivered using 1.0 mA pulses at 20 Hz, with 14 sec on-time followed by 66 sec off-time for 2 months to model settings used in successful clinical studies. Echocardiography to image the heart and fecal samples to assess microbiota were collected at regular intervals for all rats. Hearts were weighed at termination for a final heart to body weight ratio, and brains were processed to assess neuroinflammation. Findings indicate that while PO reduced EF ~40% at termination (p<0.05), VNS treatment restored EF back to control levels (p<0.0001 compared to study midpoint). Further, the heart/body weight ratio was increased for PO rats (p<0.05) compared to controls and PO+VNS rats. These data demonstrate that physiological markers of heart failure can be mitigated using these VNS settings. Notably, 66% of microbiota populations altered by PO were prevented with VNS treatment. Further, prolonged VNS significantly affected microbiota populations involved in inflammatory processes. Neuroinflammation was assessed in two key autonomic nuclei: paraventricular nucleus of the hypothalamus and locus coeruleus. PO displayed increased neuroinflammation as measured by microglial density in both regions, and VNS attenuated this effect (p<0.001). These findings indicate relevant contributions of inflammatory mechanisms and microbiome alterations for beneficial VNS effects leading to improved cardiac function in HF.

Neuroinflammation is prevalent in neurodegenerative diseases and plays a significant role in the central nervous system (CNS) innate immunity, which is the body’s first line of defense mechanisms against invading pathogens and injuries to maintain homeostasis. However, in neurodegenerative diseases, neuroinflammation becomes persistent alongside the subsequent damage to nearby neurons and affects CNS-resident immune glial cells, such as microglia and astrocytes. Accumulating evidence suggests that neuroinflammation is mainly characterized by the excessive activation of glial cells, thus causing abnormal changes in their microenvironment and release soluble factors that can promote or inhibit neuroinflammation. Currently, there is no effective treatment to cure these progressive neurological disorders. Therefore, it is critical to understand how neuroinflammation affects astroglia cell function and their biomechanical properties that change their behavior throughout disease progression. Astrocytes are the most predominant glial cell in the CNS and are critical in the development and maintenance of neuroinflammatory disorders. To date, very little is known regarding the role and specific function of Piezo1 mechanosensitive ion channel (MSC) in the CNS. Recently, Piezo1 expression was found to be upregulated in Lipopolysaccharide (LPS)-induced neuroinflammation in mouse astrocyte cultures. However, it is unknown whether the aberrant mechanical environment in astrocytes interplay with the mechanosensory function of Piezo1 and its current activity in neuroinflammatory conditions. In this study, we investigated Piezo1 mechanosensitive ionic currents by performing in vitro patch-clamp electrophysiology and calcium imaging. Our preliminary studies revealed that astrocytes derived from the mouse cerebellum stimulated with LPS or Piezo1 agonist, Yoda1, increased Ca2+ influx and further augmented when treated concurrently. We also found that electrophysiology recordings showed changes in mechanosensitive ionic currents and were comparable with our calcium imaging data indicating that MSCs are involved in neuroinflammation. Therefore, we postulated that Piezo1, a non-selective cation MSC that opens in response to mechanical force is a key mechanosensor involved in neuroinflammation by altered mechanical signals in C8-S astrocytes. Using an in vitro system of Mouse C8-S (Astrocyte type II clone), the goal of this study was to investigate if neuroinflammatory conditions upregulate Piezo1 calcium influx and current activity. We show that astrocytic Piezo1 regulates mechanotransducive release of ATP by controlling the mechanically induced calcium influx and current activation in LPS-induced astrocytes. Additionally, Piezo1 antagonist, GsMTx4 and Piezo1 siRNA significantly reduced the LPS-induced current, indicating that Piezo1 is involved in neuroinflammation. Our findings demonstrate that the activity of Piezo1 stimulated by neuroinflammatory conditions may be significant for the development of therapeutics to prevent or treat neuroinflammatory disorders and diseases.

Indiana University-Purdue University Indianapolis (IUPUI) / Non-invasive brain stimulation is valuable for studying neural circuits and treating various neurological disorders in human. However, current technologies of noninvasive brain stimulation usually have low spatial and temporal precision and poor brain penetration, which greatly limit their application. A new class of nanoparticles known as magneto-electric nanoparticles (MENs) is highly efficient in coupling an externally applied magnetics wave with generating local electric fields for neuronal activity modulation. Here, a new type of MENs was developed that consisted of CoFe2O4- BaTiO3 and had excellent magneto-electrical coupling properties. Calcium imaging technique was used to demonstrate their efficacy in evoking neuronal activity in organotyic and acute cortical slices that expressed GCaMP6 protein. For in vivo noninvasive delivery of MENs to brain, fluorescently labeled MENs were intravenously injected and attracted to pass through blood brain barrier to a targeted brain region by applying a focal magnet field. Magnetic wave (~450 G at 10 Hz) applied to mouse brain was able to activate cortical network activity, as revealed by in vivo two-photon and mesoscopic imaging of calcium signals at both cellular and global network levels. The effect was further confirmed by the increased number of c-Fos expressing cells after magnetic stimulation. Histological analysis indicated that neither brain delivery of MENs nor the subsequent magnetic stimulation caused any significant increases in the numbers of GFAP and IBA1 positive astrocytes and microglia in the brain. MENs stimulation also show high efficacy in short-term pain relieve when tested with a tibial nerve injury mouse model. The study demonstrates the feasibility of using MENs as a novel efficient and non-invasive technique of brain stimulation, which may have great potential for translation.

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Our laboratory has invented a series of oxime acetylcholinesterase (AChE) reactivators (US Patent 9,227,937) that enter the brain, reduce time to cessation of seizure-like activities, and prevent organophosphorus compound (OP) neuropathology, not seen with the current U.S. approved AChE reactivator, pralidoxime (2-PAM). Thus, 2-PAM fails to protect the brain against damage and long-term cognitive and behavioral deficits seen in humans after OP exposure. However, the mechanisms by which these novel oximes provide central neuroprotection through preservation of neuronal cell structures from damage in a rat model are not fully understood by AChE reactivation alone. This dissertation investigated neurotoxic mechanisms of NIMP as potential targets for additional direct and indirect neuroprotection by our lead in vivo AChE reactivator, Oxime 20. Male Sprague Dawley rats exposed to NIMP experienced neurotoxic effects in areas critical to OP-induced seizure generation (e.g., hippocampus and piriform cortex) such as the inhibition of multiple serine hydrolases (i.e., fatty acid amide hydrolase (FAAH), monoacylglycerol lipase (MAGL)), necrotic cell death evident by increased necrotic receptor-interacting serine/threonine-protein kinase 1 (RIPK1) levels and no apoptotic caspase-3 activity, and increased levels of neuroinflammation via elevated levels of pro-inflammatory oxylipins 4 days post lethal exposure. However, due to the lack of statistical significance, NIMP exposure did not definitively affect the subcellular location of either phosphorylated excitatory N-methyl-D-aspartate (NMDA) receptor or inhibitory γ-aminobutyric acid (GABA) receptor subunits. Results suggested that Oxime 20 therapy provided neuroprotection after NIMP exposure, such as limited reactivation of other serine hydrolase targets, significantly decreased RIPK1 levels (i.e., necrotic environment) in the hippocampus, and significantly decreased inflammatory oxylipins 4 days post-NIMP exposure. Thus, reducing OP-induced neuroinflammation might be the main contributor to the neuroprotection (i.e., neuronal cell structure preservation) previously observed in our laboratory.

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BACKGROUND A subarachnoid hemorrhage (SAH) is a bleed into the subarachnoid space surrounding the brain. This disease affects roughly 30,000 Americans each year and approximately one in six affected individuals die at the time of the ictal event. Individuals that do survive suffer from many complications including delayed cerebral vasospasm (DCV), cerebral edema, fever, and increased intracranial pressure (ICP) amongst others. These patients often suffer from brain damage due to neuronal apoptosis as a consequence of excess neuroinflammation. Microglia, the resident macrophage of the central nervous system, and Toll-like Receptor-4 (TLR4), a pro-inflammatory transmembrane receptor, have both been shown to play a role in the neuroinflammation seen in SAH. RBC components have been shown to activate microglial TLR4, and this event is suggested to trigger downstream mechanisms leading to neuronal apoptosis. The presented research takes a closer look at the role of microglial TLR4 in early neuronal apoptosis seen in an SAH model. METHODS All mice used were 10- to 12-week-old males on a C57BL/6 background: TLR4−/−, MyD88−/−, TRIF−/− and wild type (WT). To induce an SAH, a total of 60 ul of arterial blood from a donor WT mouse was injected for over 30 seconds into another mouse. For in vitro experiments, either primary microglia (PMG) or murine microglial BV2 cells were used. Microglia were separated from murine neuronal HT22 cells by 3um cell culture inserts or transwells, before being stimulated with lipopolysaccharide (LPS), red blood cells (RBCs), or RBC components including hemin (structurally similar to heme) and hemoglobin. In vivo samples were studied using either immunohistochemistry (IHC) or Fluorescence Activated Cell Sorting (FACS), and in vitro cells were studied using IHC and Light Microscopy. Neuronal cell death was measured using TUNEL and/or FloroJade C (FJC) assays. Cognitive function after SAH was measured using the Barnes Maze protocol. RESULTS In a 24-hour time course, more death occurred in the HT22 cells associated with BV2s treated with RBCs for 12-hour and 24-hour incubation time points as compared to 1-hour and 3-hour time points. Similar results were seen in the WT PMGs, as HT22 apoptosis increased in the RBC treated WT groups as the incubation time points increased. The WT PMG and MyD88−/− RBC treated PMGs showed significant death as compared to a WT untreated control (p<0.05) using a FJC assay, and both showed more death in a TUNEL assay as compared to an untreated control. WT mice treated with whole blood and hemoglobin had significantly more apoptosis as compared with a normal saline (NS)-treated control mouse (p<0.05). WT PMGs treated with whole blood and hemoglobin had more apoptosis as compared with an untreated control. MyD88-/- treated with RBC, hemoglobin, and hemin had more HT22 cell death compared with other genotypes treated with the same component. For the Barnes Maze, TLR4−/− mice performed significantly less total errors than WT mice on POD5 and 6 (p<0.01), and took significantly less time to reach the goal chamber on POD4, POD5 (p<0.05), and POD6 (p<0.01). CONCLUSION Our experimental results suggest that a microglial TLR4-dependent, MyD88-independent pathway is involved in neuronal apoptosis very early in an SAH model via RBC and hemoglobin activation, and that neuronal cell apoptosis due to TLR4 expression may be related to SAH-related cognitive and behavioral deficits. Our results suggest that TRIF may be the intracellular adaptor that is involved in this mechanism, but further experiments are needed to confirm this.