Human birth tissue products as a regenerative medicine to inhibit post-surgical pain through multi-modal action.

Pain after surgery causes significant suffering. Opioid analgesics cause severe side effects and accidental death. Therefore, there is an urgent need to develop non-opioid therapies for managing post-surgical pain and, more importantly, preventing its transition to a chronic state. In a mouse model of post-surgical pain, local application of Clarix Flo (FLO), a human amniotic membrane (AM) product, attenuated established post-surgical pain hypersensitivity without exhibiting known side effects of opioid use in mice. Importantly, preemptive drug treatment also inhibited the transition of post-surgical pain to a prolonged state. This effect was achieved through direct inhibition of nociceptive dorsal root ganglion (DRG) neurons via CD44-dependent pathways, and indirect pain relief by attenuating immune cell recruitment. We further purified the major matrix component, the heavy chain-hyaluronic acid/pentraxin 3 (HC-HA/PTX3) from human AM that has greater purity and water solubility than FLO. HC-HA/PTX3 replicated FLO-induced neuronal and pain inhibition. Mechanistically, HC-HA/PTX3 induced cytoskeleton rearrangements to inhibit sodium current and high-voltage activated calcium current on nociceptive neurons, suggesting it is a key bioactive component mediating pain relief. Collectively, our findings highlight the potential of naturally derived biologics from human birth tissues as an effective non-opioid treatment for post-surgical pain and unravel the underlying mechanisms.

Potential therapeutic targets for intracerebral hemorrhage-associated inflammation: An update.

Intracerebral hemorrhage (ICH) is a subtype of stroke with high mortality and disability but no specific or effective treatment. In the last two decades, much has been learned about the pathologic mechanisms of ICH. It is now known that after ICH onset, immune and inflammatory responses contribute to blood-brain barrier disruption, edema development, and cell death processes, jointly resulting in secondary brain injury. However, the translation of potential therapies from preclinical to clinical success has been disappointing. With the development of new laboratory technology, recent progress has been made in the understanding of ICH pathomechanisms, and promising therapeutic targets have been identified. This review provides an update of recent progress on ICH and describes the prospects for further preclinical studies in this field. Our goal is to discuss new therapeutic targets and directions for the treatment of ICH and promote the effective transformation from preclinical to clinical trials.

Iron toxicity, lipid peroxidation and ferroptosis after intracerebral haemorrhage.

Intracerebral haemorrhage (ICH) is a devastating type of stroke with high mortality and morbidity. However, we have few options for ICH therapy and limited knowledge about post-ICH neuronal death and related mechanisms. In the aftermath of ICH, iron overload within the perihaematomal region can induce lethal reactive oxygen species (ROS) production and lipid peroxidation, which contribute to secondary brain injury. Indeed, iron chelation therapy has shown efficacy in preclinical ICH studies. Recently, an iron-dependent form of non-apoptotic cell death known as ferroptosis was identified. It is characterised by an accumulation of iron-induced lipid ROS, which leads to intracellular oxidative stress. The ROS cause damage to nucleic acids, proteins and lipid membranes, and eventually cell death. Recently, we and others discovered that ferroptosis does occur after haemorrhagic stroke in vitro and in vivo and contributes to neuronal death. Inhibition of ferroptosis is beneficial in several in vivo and in vitro ICH conditions. This minireview summarises current research on iron toxicity, lipid peroxidation and ferroptosis in the pathomechanisms of ICH, the underlying molecular mechanisms of ferroptosis and the potential for combined therapeutic strategies. Understanding the role of ferroptosis after ICH will provide a vital foundation for cell death-based ICH treatment and prevention.

Cerebroprotection by salvianolic acid B after experimental subarachnoid hemorrhage occurs via Nrf2- and SIRT1-dependent pathways.

Salvianolic acid B (SalB), a natural polyphenolic compound extracted from the herb of Salvia miltiorrhiza, possesses antioxidant and neuroprotective properties and has been shown to be beneficial for diseases that affect vasculature and cognitive function. Here we investigated the protective effects of SalB against subarachnoid hemorrhage (SAH)-induced oxidative damage, and the involvement of underlying molecular mechanisms. In a rat model of SAH, SalB inhibited SAH-induced oxidative damage. The reduction in oxidative damage was associated with suppressed reactive oxygen species generation; decreased lipid peroxidation; and increased glutathione peroxidase, glutathione, and superoxide dismutase activities. Concomitant with the suppressed oxidative stress, SalB significantly reduced neurologic impairment, brain edema, and neural cell apoptosis after SAH. Moreover, SalB dramatically induced nuclear factor-erythroid 2-related factor 2 (Nrf2) nuclear translocation and increased expression of heme oxygenase-1 and NADPH: quinine oxidoreductase-1. In a mouse model of SAH, Nrf2 knockout significantly reversed the antioxidant effects of SalB against SAH. Additionally, SalB activated sirtuin 1 (SIRT1) expression, whereas SIRT1-specific inhibitor sirtinol pretreatment significantly suppressed SalB-induced SIRT1 activation and Nrf2 expression. Sirtinol pretreatment also reversed the antioxidant and neuroprotective effects of SalB. In primary cultured cortical neurons, SalB suppressed oxidative damage, alleviated neuronal degeneration, and improved cell viability. These beneficial effects were associated with activation of the SIRT1 and Nrf2 signaling pathway and were reversed by sirtinol treatment. Taken together, these in vivo and in vitro findings suggest that SalB provides protection against SAH-triggered oxidative damage by upregulating the Nrf2 antioxidant signaling pathway, which may be modulated by SIRT1 activation.

Neuroprotection of brain-permeable iron chelator VK-28 against intracerebral hemorrhage in mice.

Iron overload plays a key role in the secondary brain damage that develops after intracerebral hemorrhage (ICH). The significant increase in iron deposition is associated with the generation of reactive oxygen species (ROS), which leads to oxidative brain damage. In this study, we examined the protective effects of VK-28, a brain-permeable iron chelator, against hemoglobin toxicity in an ex vivo organotypic hippocampal slice culture (OHSC) model and in middle-aged mice subjected to an in vivo, collagenase-induced ICH model. We found that the effects of VK-28 were similar to those of deferoxamine (DFX), a well-studied iron chelator. Both decreased cell death and ROS production in OHSCs and in vivo, decreased iron-deposition and microglial activation around hematoma in vivo, and improved neurologic function. Moreover, compared with DFX, VK-28 polarized microglia to an M2-like phenotype, reduced brain water content, deceased white matter injury, improved neurobehavioral performance, and reduced overall death rate after ICH. The protection of VK-28 was confirmed in a blood-injection ICH model and in aged-male and young female mice. Our findings indicate that VK-28 is protective against iron toxicity after ICH and that, at the dosage tested, it has better efficacy and less toxicity than DFX does.

Alternative activation-skewed microglia/macrophages promote hematoma resolution in experimental intracerebral hemorrhage.

Microglia/macrophages (MMΦ) are highly plastic phagocytes that can promote both injury and repair in diseased brain through the distinct function of classically activated and alternatively activated subsets. The role of MMΦ polarization in intracerebral hemorrhage (ICH) is unknown. Herein, we comprehensively characterized MMΦ dynamics after ICH in mice and evaluated the relevance of MMΦ polarity to hematoma resolution. MMΦ accumulated within the hematoma territory until at least 14days after ICH induction. Microglia rapidly reacted to the hemorrhagic insult as early as 1-1.5h after ICH and specifically presented a "protective" alternatively activated phenotype. Substantial numbers of activated microglia and newly recruited monocytes also assumed an early alternatively activated phenotype, but the phenotype gradually shifted to a mixed spectrum over time. Ultimately, markers of MMΦ classic activation dominated at the chronic stage of ICH. We enhanced MMΦ alternative activation by administering intraperitoneal injections of rosiglitazone, and subsequently observed elevations in CD206 expression on brain-isolated CD11b+ cells and increases in IL-10 levels in serum and perihematomal tissue. Enhancement of MMΦ alternative activation correlated with hematoma volume reduction and improvement in neurologic deficits. Intraventricular injection of alternative activation signature cytokine IL-10 accelerated hematoma resolution, whereas microglial phagocytic ability was abolished by IL-10 receptor neutralization. Our results suggest that MMΦ respond dynamically to brain hemorrhage by exhibiting diverse phenotypic changes at different stages of ICH. Alternative activation-skewed MMΦ aid in hematoma resolution, and IL-10 signaling might contribute to regulation of MMΦ phagocytosis and hematoma clearance in ICH.

Pinocembrin protects hemorrhagic brain primarily by inhibiting toll-like receptor 4 and reducing M1 phenotype microglia.

Neuroinflammation is a major contributor to intracerebral hemorrhage (ICH) progression, but no drug is currently available to reduce this response and protect against ICH-induced injury. Recently, the natural product pinocembrin has been shown to ameliorate neuroinflammation and is undergoing a phase II clinical trial for ischemic stroke treatment. In this study, we examined the efficacy of pinocembrin in an ICH model, and further examined its effect on microglial activation and polarization. In vivo, pinocembrin dose-dependently reduced lesion volume by ∼47.5% and reduced neurologic deficits of mice at 72h after collagenase-induced ICH. The optimal dose of pinocembrin (5mg/kg) suppressed microglial activation as evidenced by decreases in CD68-positive microglia and reduced proinflammatory cytokines tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6. Pinocembrin also reduced the number of classically activated M1-like microglia without affecting M2-like microglia in the perilesional region. Additionally, pinocembrin decreased the expression of toll-like receptor (TLR)4 and its downstream target proteins TRIF and MyD88. The protection by pinocembrin was lost in microglia-depleted mice and in TLR4lps-del mice, and pinocembrin failed to decrease the number of M1-like microglia in TLR4lps-del mice. In lipopolysaccharide-stimulated BV-2 cells or primary microglia, pinocembrin decreased M1-related cytokines and markers (IL-1ß, IL-6, TNF-α, and iNOS), NF-κB activation, and TLR4 expression, but it did not interfere with TLR4/MyD88 and TLR4/TRIF interactions or affect microglial phagocytosis of red blood cells. Inhibition of the TLR4 signaling pathway and reduction in M1-like microglial polarization might be the major mechanism by which pinocembrin protects hemorrhagic brain. With anti-inflammatory properties, pinocembrin could be a promising new drug candidate for treating ICH and other acute brain injuries.

NLRP3 inflammasome activation contributes to long-term behavioral alterations in mice injected with lipopolysaccharide.

Lipopolysaccharide (LPS) might affect the central nervous system by causing neuroinflammation, which subsequently leads to brain damage and dysfunction. In this study, we evaluated the role of nod-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome activation in long-term behavioral alterations of 8-week-old male C57BL/6 mice injected intraperitoneally with LPS (5mg/kg). At different time points after injection, we assessed locomotor function with a 24-point neurologic deficit scoring system and the rotarod test; assessed recognition memory with the novel object recognition test; and assessed emotional abnormality (anhedonia and behavioral despair) with the tail suspension test, forced swim test, and sucrose preference test. We also assessed protein expression of NLRP3, apoptosis-associated speck-like protein (ASC), and caspase-1 p10 in hippocampus by Western blotting; measured levels of interleukin (IL)-1ß, IL-18, tumor necrosis factor α (TNFα), and IL-10 in hippocampus; measured TNFα and IL-1ß in serum by ELISA; and evaluated microglial activity in hippocampus by Iba1 immunofluorescence. We found that LPS-injected mice displayed long-term depression-like behaviors and recognition memory deficit; elevated expression of NLRP3, ASC, and caspase-1 p10; increased levels of IL-1ß, IL-18, and TNFα; decreased levels of IL-10; and increased microglial activation. These effects were blocked by the NLRP3 inflammasome inhibitor Ac-Tyr-Val-Ala-Asp-chloromethylketone. The results demonstrate proof of concept that NLRP3 inflammasome activation contributes to long-term behavioral alterations in LPS-exposed mice, probably through enhanced inflammation, and that NLRP3 inflammasome inhibition might alleviate peripheral and brain inflammation and thereby ameliorate long-term behavioral alterations in LPS-exposed mice.

Progesterone exerts neuroprotective effects and improves long-term neurologic outcome after intracerebral hemorrhage in middle-aged mice.

In this study, we examined the effect of progesterone on histopathologic and functional outcomes of intracerebral hemorrhage (ICH) in 10- to 12-month-old mice. Progesterone or vehicle was administered by intraperitoneal injection 1 hour after collagenase-induced ICH and then by subcutaneous injections at 6, 24, and 48 hours. Oxidative and nitrosative stress were assayed at 12 hours post-ICH. Injury markers were examined on day 1, and lesion was examined on day 3. Neurologic deficits were examined for 28 days. Progesterone posttreatment reduced lesion volume, brain swelling, edema, and cell degeneration and improved long-term neurologic function. These protective effects were associated with reductions in protein carbonyl formation, protein nitrosylation, and matrix metalloproteinase-9 activity and attenuated cellular and molecular inflammatory responses. Progesterone also reduced vascular endothelial growth factor expression, increased neuronal-specific Na(+)/K(+) ATPase ɑ3 subunit expression, and reduced protein kinase C-dependent Na(+)/K(+) ATPase phosphorylation. Furthermore, progesterone reduced glial scar thickness, myelin loss, brain atrophy, and residual injury volume on day 28 after ICH. With multiple brain targets, progesterone warrants further investigation for its potential use in ICH therapy.

Autologous Bone Marrow Mononuclear Cell Transplantation Delays Progression of Carotid Atherosclerosis in Rabbits.

Bone marrow mononuclear cells (BMMNCs) can counteract oxidative stress and inhibit the inflammatory response in focal ischemic stroke models. However, the effect of BMMNC transplantation on carotid atherosclerosis needs to be determined. The carotid atherosclerotic plaque model was established in New Zealand White rabbits by balloon injury and 8 weeks of high-fat diet. Rabbits were randomized to receive an intravenous injection of autologous bromodeoxyuridine (BrdU)-labeled BMMNCs or an equal volume of phosphate-buffered saline. Plaques were evaluated for expression of proinflammatory and anti-inflammatory cytokines, anti-oxidant proteins, and markers of cell death. BMMNCs migrated into atherosclerotic plaque on the first day after cell transplantation. BMMNC-treated rabbits had smaller plaques and more collagen deposition than did the vehicle-treated controls on day 28 (p < 0.05). BMMNC treatment significantly increased endothelial nitric oxide synthase and the anti-oxidant enzymes glutathione peroxidase and superoxide dismutase in plaques compared to vehicle treatment on day 7. BMMNC-treated rabbits also had lower levels of cleaved caspase-3 expression; lower levels of proinflammatory cytokines interleukin-1ß, tumor necrosis factor alpha, and matrix metalloproteinase 9; and higher levels of insulin-like growth factor-1 and its receptor (p < 0.05). Autologous BMMNC transplantation can suppress the process of atherosclerotic plaque formation and is associated with enhanced anti-oxidative effect, reduced levels of inflammatory cytokines and cleaved caspase-3, and increased expression of insulin-like growth factor-1 and its receptor. BMMNC transplantation represents a novel approach for the treatment of carotid atherosclerosis.