Systemic inflammation, for example as a result of infection, often contributes to long-term complications. Neuroinflammation and cognitive decline are key hallmarks of several neurological conditions, including advance age. The contribution of systemic inflammation to the central nervous system (CNS) remains not fully understood. Using a model of peripheral endotoxemia with lipopolysaccharide (LPS) we investigated the role of nuclear factor-κB (NF-κB) activity in mediating long-term neuroinflammation and cognitive dysfunction in aged rats. Herein we describe the anti-inflammatory effects of pyrrolidine dithiocarbamate (PDTC), a selective NF-κB inhibitor, in modulating systemic cytokines including tumor necrosis factor (TNF)-α and interleukin-1β (IL-1β) and CNS markers after LPS exposure in aged rats. In the hippocampus, PDTC not only reduced neuroinflammation by modulating canonical NF-κB activity but also affected IL-1β expression in astrocytes. Parallel effects were observed on behavior and postsynaptic density-95 (PSD95), a marker of synaptic function. Taken together these changes improved acute and long-term cognitive function in aged rats after LPS exposure.

Mechanisms of pain resolution are largely unclear. Increasing evidence suggests that specialized pro-resolving mediators (SPMs), derived from fish oil docosahexaenoic acid (DHA), promote the resolution of acute inflammation and potently inhibit inflammatory and neuropathic pain. In this study, we examined the analgesic impact of DHA and DHA-derived SPMs in a mouse model of post-operative pain induced by tibial bone fracture (fPOP). Intravenous perioperative treatment with DHA (500 μg), resolvin D1 (RvD1, 500 ng) and maresin 1 (MaR1, 500 ng), 10 min and 24 h after the surgery, delayed the development of fPOP (mechanical allodynia and cold allodynia). In contrast, post-operative intrathecal (IT) administration of DHA (500 μg) 2 weeks after the surgery had no effects on established mechanical and cold allodynia. However, by direct comparison, IT post-operative treatment (500 ng) with neuroprotectin D1 (NPD1), MaR1, and D-resolvins, RvD1 and RvD5, but not RvD3 and RvD4, effectively reduced mechanical and cold allodynia. ELISA analysis showed that perioperative DHA treatment increased RvD1 levels in serum and spinal cord samples after bone fracture. Interestingly, sham surgery resulted in transient allodynia and increased RvD1 levels, suggesting a correlation of enhanced SPM levels with acute pain resolution after sham surgery. Our findings suggest that (1) perioperative treatment with DHA is effective in preventing and delaying the development of fPOP and (2) post-treatment with some SPMs can attenuate established fPOP. Our data also indicate that orthopedic surgery impairs SPM production. Thus, DHA and DHA-derived SPMs should be differentially supplemented for treating fPOP and improving recovery.

The complement system plays an important role in many neurological disorders. Complement modulation, including C3/C3a receptor signaling, shows promising therapeutic effects on cognition and neurodegeneration. Yet, the implications for this pathway in perioperative neurocognitive disorders (PND) are not well established. Here, we evaluated the possible role for C3/C3a receptor signaling after orthopedic surgery using an established mouse model of PND.

The present work evaluates the relationship between postoperative immune and neurovascular changes and the pathogenesis of surgery-induced delirium superimposed on dementia.

Delirium is a common postoperative neurologic complication among older adults. Despite its prevalence (14%-50%) and likely association with inflammation, the exact mechanisms that underpin postoperative delirium are unclear. This project aimed to characterize systemic and central nervous system (CNS) inflammatory changes following surgery in mice and humans. Matched plasma and cerebrospinal fluid (CSF) samples from the "Investigating Neuroinflammation Underlying Postoperative Brain Connectivity Changes, Postoperative Cognitive Dysfunction, Delirium in Older Adults" (INTUIT; NCT03273335) study were compared to murine endpoints. Delirium-like behavior was evaluated in aged mice using the 5-Choice Serial Reaction Time Test (5-CSRTT). Using a well established orthopedic surgical model in the FosTRAP reporter mouse we detected neuronal changes in the prefrontal cortex, an area implicated in attention, but notably not in the hippocampus. In aged mice, plasma interleukin-6 (IL-6), chitinase-3-like protein 1 (YKL-40), and neurofilament light chain (NfL) levels increased after orthopedic surgery, but hippocampal YKL-40 expression was decreased. Given the growing evidence for a YKL-40 role in delirium and other neurodegenerative conditions, we assayed human plasma and CSF samples. Plasma YKL-40 levels were similarly increased after surgery, with a trend toward a greater postoperative plasma YKL-40 increase in patients with delirium. However, YKL-40 levels in CSF decreased following surgery, which paralleled the findings in the mouse brain. Finally, we confirmed changes in the blood-brain barrier (BBB) as early as 9 h after surgery in mice, which warrants more detailed and acute evaluations of BBB integrity following surgery in humans. Together, these results provide a nuanced understanding of neuroimmune interactions underlying postoperative delirium in mice and humans, and highlight translational biomarkers to test potential cellular targets and mechanisms.

Postoperative delirium is a frequent and debilitating complication, especially amongst high risk procedures such as orthopedic surgery, and its pathogenesis remains unclear. Inattention is often reported in the clinical diagnosis of delirium, however limited attempts have been made to study this cognitive domain in preclinical models. Here we implemented the 5-choice serial reaction time task (5-CSRTT) to evaluate attention in a clinically relevant mouse model following orthopedic surgery. The 5-CSRTT showed a time-dependent impairment in the number of responses made by the mice acutely after orthopedic surgery, with maximum impairment at 24 h and returning to pre-surgical performance by day 5. Similarly, the latency to the response was also delayed during this time period but returned to pre-surgical levels within several days. While correct responses decreased following surgery, the accuracy of the response (e.g., selection of the correct nose-poke) remained relatively unchanged. In a separate cohort we evaluated neuroinflammation and blood-brain barrier (BBB) dysfunction using clarified brain tissue with light-sheet microscopy. CLARITY revealed significant changes in microglial morphology and impaired astrocytic-tight junction interactions using high-resolution 3D reconstructions of the neurovascular unit. Deposition of IgG, fibrinogen, and autophagy markers (TFEB and LAMP1) were also altered in the hippocampus 24 h after surgery. Together, these results provide translational evidence for the role of peripheral surgery contributing to delirium-like behavior and disrupted neuroimmunity in adult mice.

Postoperative pain is a major clinical problem imposing a significant burden on our patients and society. Up to 57% of patients experience persistent postoperative pain 2 years after orthopedic surgery [49]. Although many studies have contributed to the neurobiological foundation of surgery-induced pain sensitization, we still lack safe and effective therapies to prevent the onset of persistent postoperative pain. We have established a clinically relevant orthopedic trauma model in mice that recapitulates common insults associated with surgery and ensuing complications. Using this model, we have started to characterize how induction of pain signaling contributes to neuropeptides changes in dorsal root ganglia (DRG) and sustained neuroinflammation in the spinal cord [62]. Here we have extended the characterization of pain behaviors for >3 months after surgery, describing a persistent deficit in mechanical allodynia in both male and female C57BL/6J mice after surgery. Notably, we have applied a novel minimally invasive bioelectronic approach to percutaneously stimulate the vagus nerve (termed pVNS) [24] and tested its anti-nociceptive effects in this model. Our results show that surgery induced a strong bilateral hind-paw allodynia with a slight decrease in motor coordination. However, treatment with pVNS for 30-minutes at10 Hz weekly for 3 weeks prevented pain behavior compared to naïve controls. pVNS also improved locomotor coordination and bone healing compared to surgery without treatment. In the DRGs, we observed that vagal stimulation fully rescued activation of GFAP positive satellite cells but did not affect microglial activation. Overall, these data provide novel evidence for the use of pVNS to prevent postoperative pain and may inform translational studies to test anti-nociceptive effects in the clinic.

Patients with pre-existing neurodegenerative disease commonly experience fractures that require orthopedic surgery. Perioperative neurocognitive disorders (PND), including delirium and postoperative cognitive dysfunction, are serious complications that can result in increased 1-year mortality when superimposed on dementia. Importantly, there are no disease-modifying therapeutic options for PND. Our lab developed the "broad spectrum" mixed-lineage kinase 3 inhibitor URMC-099 to inhibit pathological innate immune responses that underlie neuroinflammation-associated cognitive dysfunction. Here, we test the hypothesis that URMC-099 can prevent surgery-induced neuroinflammation and cognitive impairment.

Immune-related events in the periphery can remotely affect brain function, contributing to neurodegenerative processes and cognitive decline. In mice, peripheral surgery induces a systemic inflammatory response associated with changes in hippocampal synaptic plasticity and transient cognitive decline, however, the underlying mechanisms remain unknown. Here we investigated the effect of peripheral surgery on neuronal-glial function within hippocampal neuronal circuits of relevance to cognitive processing in male mice at 6, 24, and 72 h postsurgery. At 6 h we detect the proinflammatory cytokine IL-6 in the hippocampus, followed up by alterations in the mRNA and protein expression of astrocytic and neuronal proteins necessary for optimal energy supply to the brain and for the reuptake and recycling of glutamate in the synapse. Similarly, at 24 h postsurgery the mRNA expression of structural proteins (GFAP and AQP4) was compromised. At this time point, functional analysis in astrocytes revealed a decrease in resting calcium signaling. Examination of neuronal activity by whole-cell patch-clamp shows elevated levels of glutamatergic transmission and changes in AMPA receptor subunit composition at 72 h postsurgery. Finally, lactate, an essential energy substrate produced by astrocytes and critical for memory formation, decreases at 6 and 72 h after surgery. Based on temporal parallels with our previous studies, we propose that the previously reported cognitive decline observed at 72 h postsurgery in mice might be the consequence of temporal hippocampal metabolic, structural, and functional changes in astrocytes that lead to a disruption of the neuroglial metabolic coupling and consequently to a neuronal dysfunction.SIGNIFICANCE STATEMENT A growing body of evidence suggests that surgical trauma launches a systemic inflammatory response that reaches the brain and associates with immune activation and cognitive decline. Understanding the mechanisms by which immune-related events in the periphery can influence brain processes is essential for the development of therapies to prevent or treat postoperative cognitive dysfunction and other forms of cognitive decline related to immune-to-brain communication, such as Alzheimer's and Parkinson's diseases. Here we describe the temporal orchestration of a series of metabolic, structural, and functional changes after aseptic trauma in mice related to astrocytes and later in neurons that emphasize the role of astrocytes as key intermediaries between peripheral immune events, neuronal processing, and potentially cognition.

Despite limited evidence for infection of SARS-CoV-2 in the central nervous system, cognitive impairment is a common complication reported in "recovered" COVID-19 patients. Identification of the origins of these neurological impairments is essential to inform therapeutic designs against them. However, such studies are limited, in part, by the current status of high-fidelity probes to visually investigate the effects of SARS-CoV-2 on the system of blood vessels and nerve cells in the brain, called the neurovascular unit. Here, we report that nanocrystal quantum dot micelles decorated with spike protein (COVID-QDs) are able to interrogate neurological damage due to SARS-CoV-2. In a transwell co-culture model of the neurovascular unit, exposure of brain endothelial cells to COVID-QDs elicited an inflammatory response in neurons and astrocytes without direct interaction with the COVID-QDs. These results provide compelling evidence of an inflammatory response without direct exposure to SARS-CoV-2-like nanoparticles. Additionally, we found that pretreatment with a neuro-protective molecule prevented endothelial cell damage resulting in substantial neurological protection. These results will accelerate studies into the mechanisms by which SARS-CoV-2 mediates neurologic dysfunction.

Postoperative cognitive dysfunction (POCD) is a common complication after surgery, especially amongst elderly patients. Neuroinflammation and iron homeostasis are key hallmarks of several neurological disorders. In this study, we investigated the role of deferoxamine (DFO), a clinically used iron chelator, in a mouse model of surgery-induced cognitive dysfunction and assessed its neuroprotective effects on neuroinflammation, oxidative stress, and memory function.

Postoperative neurocognitive disorders are common complications in elderly patients following surgery or critical illness. High mobility group box 1 protein (HMGB1) is rapidly released after tissue trauma and critically involved in response to sterile injury. Herein, we assessed the role of HMGB1 after liver surgery in aged rats and explored the therapeutic potential of a neutralizing anti-HMGB1 monoclonal antibody in a clinically relevant model of postoperative neurocognitive disorders. Nineteen to twenty-two months Sprague-Dawley rats were randomly assigned as: (1) control with saline; (2) surgery, a partial hepatolobectomy under sevoflurane anesthesia and analgesia, + immunoglobulin G as control antibody; (3) surgery + anti-HMGB1. A separate cohort of animals was used to detect His-tagged HMGB1 in the brain. Systemic anti-HMGB1 antibody treatment exerted neuroprotective effects preventing postoperative memory deficits and anxiety in aged rats by preventing surgery-induced reduction of phosphorylated cyclic AMP response element-binding protein in the hippocampus. Although no evident changes in the intracellular distribution of HMGB1 in hippocampal cells were noted after surgery, HMGB1 levels were elevated on day 3 in rat plasma samples. Experiments with tagged HMGB1 further revealed a critical role of systemic HMGB1 to enable an access to the brain and causing microglial activation. Overall, these data demonstrate a pivotal role for systemic HMGB1 in mediating postoperative neuroinflammation. This may have direct implications for common postoperative complications like delirium and postoperative cognitive dysfunction.

Postoperative cognitive dysfunction (POCD), a syndrome of cognitive deficits occurring 1-12 months after surgery primarily in older patients, is associated with poor postoperative outcomes. POCD is hypothesized to result from neuroinflammation; however, the pathways involved remain unclear. Unbiased proteomic analyses have been used to identify neuroinflammatory pathways in multiple neurologic diseases and syndromes but have not yet been applied to POCD.

Neuroinflammation is a growing hallmark of perioperative neurocognitive disorders (PNDs), including delirium and longer-lasting cognitive deficits. We have developed a clinically relevant orthopedic mouse model to study the impact of a common surgical procedure on the vulnerable brain. The mechanism underlying PNDs remains unknown. Here we evaluated the impact of surgical trauma on the NLRP3 inflammasome signaling, including the expression of apoptosis-associated speck-like protein containing a CARD (ASC), caspase-1, and IL-1β in the hippocampus of C57BL6/J male mice, adult (3-months) and aged (>18-months). Surgery triggered ASC specks formation in CA1 hippocampal microglia, but without inducing significant morphological changes in NLRP3 and ASC knockout mice. Since no therapies are currently available to treat PNDs, we assessed the neuroprotective effects of a biomimetic peptide derived from the endogenous inflammation-ending molecule, Annexin-A1 (ANXA1). We found that this peptide (ANXA1sp) inhibited postoperative NLRP3 inflammasome activation and prevented microglial activation in the hippocampus, reducing PND-like memory deficits. Together our results reveal a previously under-recognized role of hippocampal ANXA1 and NLRP3 inflammasome dysregulation in triggering postoperative neuroinflammation, offering a new target for advancing treatment of PNDs through the resolution of inflammation.

Inflammation is a hallmark of several disease states ranging from neurodegeneration to sepsis but is also implicated in physiological processes like ageing. Non-resolving inflammation and prolonged neuroinflammation are unclear processes implicated in several conditions, including ageing. In this study we studied the long-term effects of endotoxemia, as systemic lipopolysaccharide (LPS) injection, focusing on the role of astrocyte activation and cytokine release in the brain of aged rats. A single dose of LPS (2 mg/kg) or 0.9% saline was injected intraperitoneally in aged rats. Levels of pro-inflammatory cytokines (TNFα and IL-1β) and NF-κB p65 activation were measured systemically and in hippocampal tissue. Astrocytes and cytokines release in the CNS were detected via double immunofluorescence staining at different time-points up to day 30. Serum levels of TNFα and IL-1β were significantly increased acutely after 30 minutes (p<0.001) and up to 6 hours (p<0.001) following LPS-injection. Centrally, LPS-treated rats showed up-regulated mRNA expression and protein levels of pro-inflammatory cytokines in the hippocampus. These changes associated with astrogliosis in the hippocampus dentate gyrus (DG), IL-1β immunoreactivity and elevated NF-κB p65 expression up to day 30 post LPS exposure. Overall, these data demonstrate that LPS induces prolonged neuroinflammation and astrocyte activation in the hippocampus of aged rats. Hippocampal NF-κB p65 and excessive astrocytes-derived IL-1β release may play a pivotal role in regulating long-lasting neuroinflammation.

Resolution agonists, including lipid mediators and peptides such as annexin A1 (ANXA1), are providing novel approaches to treat inflammatory conditions. Surgical trauma exerts a significant burden on the immune system that can affect and impair multiple organs. Perioperative cerebral injury after cardiac surgery is associated with significant adverse neurological outcomes such as delirium and postoperative cognitive dysfunction. Using a clinically relevant rat model of cardiopulmonary bypass (CPB) with deep hypothermic circulatory arrest (DHCA), we tested the pro-resolving effects of a novel bioactive ANXA1 tripeptide (ANXA1sp) on neuroinflammation and cognition. Male rats underwent 2 h CPB with 1 h DHCA at 18°C, and received vehicle or ANXA1sp followed by timed reperfusion up to postoperative day 7. Immortalized murine microglial cell line BV2 were treated with vehicle or ANXA1sp and subjected to 2 h oxygen-glucose deprivation followed by timed reoxygenation. Microglial activation, cell death, neuroinflammation, and NF-κB activation were assessed in tissue samples and cell cultures. Rats exposed to CPB and DHCA had evident neuroinflammation in various brain areas. However, in ANXA1sp-treated rats, microglial activation and cell death (apoptosis and necrosis) were reduced at 24 h and 7 days after surgery. This was associated with a reduction in key pro-inflammatory cytokines due to inhibition of NF-κB activation in the brain and systemically. Treated rats also had improved neurologic scores and shorter latency in the Morris water maze. In BV2 cells treated with ANXA1sp, similar protective effects were observed including decreased pro-inflammatory cytokines and cell death. Notably, we also found increased expression of ANXA1, which binds to NF-κB p65 and thereby inhibits its transcriptional activity. Our findings provide evidence that treatment with a novel pro-resolving ANXA1 tripeptide is neuroprotective after cardiac surgery in rats by attenuating neuroinflammation and may prevent postoperative neurologic complications.

The impact of pro-inflammatory cytokines on neuroinflammation and cognitive function after lipopolysaccharide (LPS) challenge remains elusive. Herein we provide evidence that there is a temporal correlation between high-mobility group box 1 (HMGB-1), microglial activation, and cognitive dysfunction. Disabling the interleukin (IL)-1 signaling pathway is sufficient to reduce inflammation and ameliorate the disability.

Neuroinflammation initiated by damage-associated molecular patterns, including high mobility group box 1 protein (HMGB1), has been implicated in adverse neurological outcomes following lethal hemorrhagic shock and polytrauma. Emergency preservation and resuscitation (EPR) is a novel method of resuscitation for victims of exsanguinating cardiac arrest, shown in preclinical studies to improve survival with acceptable neurological recovery. Sirtuin 3 (SIRT3), the primary mitochondrial deacetylase, has emerged as a key regulator of metabolic and energy stress response pathways in the brain and a pharmacological target to induce a neuronal pro-survival phenotype. This study aims to examine whether systemic administration of an Annexin-A1 bioactive peptide (ANXA1sp) could resolve neuroinflammation and induce sirtuin-3 regulated cytoprotective pathways in a novel rat model of exsanguinating cardiac arrest and EPR. Adult male rats underwent hemorrhagic shock and ventricular fibrillation, induction of profound hypothermia, followed by resuscitation and rewarming using cardiopulmonary bypass (EPR). Animals randomly received ANXA1sp (3 mg/kg, in divided doses) or vehicle. Neuroinflammation (HMGB1, TNFα, IL-6, and IL-10 levels), cerebral cell death (TUNEL, caspase-3, pro and antiapoptotic protein levels), and neurologic scores were assessed to evaluate the inflammation resolving effects of ANXA1sp following EPR. Furthermore, western blot analysis and immunohistochemistry were used to interrogate the mechanisms involved. Compared to vehicle controls, ANXA1sp effectively reduced expression of cerebral HMGB1, IL-6, and TNFα and increased IL-10 expression, which were associated with improved neurological scores. ANXA1sp reversed EPR-induced increases in expression of proapoptotic protein Bax and reduction in antiapoptotic protein Bcl-2, with a corresponding decrease in cerebral levels of cleaved caspase-3. Furthermore, ANXA1sp induced autophagic flux (increased LC3II and reduced p62 expression) in the brain. Mechanistically, these findings were accompanied by upregulation of the mitochondrial protein deacetylase Sirtuin-3, and its downstream targets FOXO3a and MnSOD in ANXA1sp-treated animals. Our data provide new evidence that engaging pro-resolving pharmacological strategies such as Annexin-A1 biomimetic peptides can effectively attenuate neuroinflammation and enhance the neuroprotective effects of EPR after exsanguinating cardiac arrest.

Systemic perturbations can drive a neuroimmune cascade after surgical trauma, including affecting the blood-brain barrier (BBB), activating microglia, and contributing to cognitive deficits such as delirium. Delirium superimposed on dementia (DSD) is a particularly debilitating complication that renders the brain further vulnerable to neuroinflammation and neurodegeneration, albeit these molecular mechanisms remain poorly understood. Here, we have used an orthopedic model of tibial fracture/fixation in APPSwDI/mNos2-/- AD (CVN-AD) mice to investigate relevant pathogenetic mechanisms underlying DSD. We conducted the present study in 6-month-old CVN-AD mice, an age at which we speculated amyloid-β pathology had not saturated BBB and neuroimmune functioning. We found that URMC-099, our brain-penetrant anti-inflammatory neuroprotective drug, prevented inflammatory endothelial activation, breakdown of the BBB, synapse loss, and microglial activation in our DSD model. Taken together, our data link post-surgical endothelial activation, microglial MafB immunoreactivity, and synapse loss as key substrates for DSD, all of which can be prevented by URMC-099.

Links between acute lung injury (ALI), infectious disease, and neurological outcomes have been frequently discussed over the past few years, especially due to the COVID-19 pandemic. Yet, much of the cross-communication between organs, particularly the lung and the brain, has been understudied. Here, we have focused on the role of neutrophils in driving changes to the brain endothelium with ensuing microglial activation and neuronal loss in a model of ALI.