Model Of Severe Injury Research Articles

Multi-Drug Cocktail Therapy Improves Survival and Neurological Function after Asphyxial Cardiac Arrest in Rodents.

Cardiac arrest (CA) can lead to neuronal degeneration and death through various pathways, including oxidative, inflammatory, and metabolic stress. However, current neuroprotective drug therapies will typically target only one of these pathways, and most single drug attempts to correct the multiple dysregulated metabolic pathways elicited following cardiac arrest have failed to demonstrate clear benefit. Many scientists have opined on the need for novel, multidimensional approaches to the multiple metabolic disturbances after cardiac arrest. In the current study, we have developed a therapeutic cocktail that includes ten drugs capable of targeting multiple pathways of ischemia-reperfusion injury after CA. We then evaluated its effectiveness in improving neurologically favorable survival through a randomized, blind, and placebo-controlled study in rats subjected to 12 min of asphyxial CA, a severe injury model. 14 rats were given the cocktail and 14 received the vehicle after resuscitation. At 72 h post-resuscitation, the survival rate was 78.6% among cocktail-treated rats, which was significantly higher than the 28.6% survival rate among vehicle-treated rats (log-rank test; p = 0.006). Moreover, in cocktail-treated rats, neurological deficit scores were also improved. These survival and neurological function data suggest that our multi-drug cocktail may be a potential post-CA therapy that deserves clinical translation. Our findings demonstrate that, with its ability to target multiple damaging pathways, a multi-drug therapeutic cocktail offers promise both as a conceptual advance and as a specific multi-drug formulation capable of combatting neuronal degeneration and death following cardiac arrest. Clinical implementation of this therapy may improve neurologically favorable survival rates and neurological deficits in patients suffering from cardiac arrest.

Synergistic Enhancement of Adeno‐Associated Virus‐Mediated In Vivo Direct Neuronal Reprogramming by Spatially Aligned Fibrous Matrices in Spinal Cord Injury Models

AbstractIn vivo direct neuronal reprogramming approaches, which convert host glial cells into neurons without using exogenous cellular supplies, have become a powerful strategy for neuronal regeneration. However, their effectiveness in severe spinal cord injury (SCI) models with a physical gap between the injured sites is unclear. This study highlights the important role of physical guidance in increasing the therapeutic efficacy of in vivo reprogramming technologies in SCI hemisection models. Reactive astrocytes are selected as host targets for conversion into neurons. An astrocyte‐tropic adeno‐associated viral (AAV) vector, AAVShH19, is used to deliver the neuronal transcription factor NeuroD1 to reactive astrocytes. The infusion of AAVShH19‐NeuroD1 induces astrocyte‐to‐neuron conversion, but improvement of locomotive function is not observed due to the physical distance between the rostral and caudal sides, which impedes axonal extension. To address this issue, electrospun fibrous matrices with adhesive and aligned structural features are implanted into the hemisection cavity and designed to release AAVShH19‐NeuroD1. The combination of the AAV‐mediated reprogramming approach and the matrices promoted astrocyte‐to‐neuron conversion and coordinated the elongation of neuronal axons along the fibers, leading to improved synaptic connections and motor function. This study demonstrates the potential of physical support to enhance the efficacy of direct neuronal reprogramming.

Lipoxin A4 (LXA4) Reduces Alkali-Induced Corneal Inflammation and Neovascularization and Upregulates a Repair Transcriptome.

To investigate the anti-inflammatory and anti-angiogenic effects of the bioactive lipid mediator LXA4 on a rat model of severe corneal alkali injury. To induce a corneal alkali injury in the right eyes of anesthetized Sprague Dawley rats. They were injured with a Φ 4 mm filter paper disc soaked in 1 N NaOH placed on the center of the cornea. After injury, the rats were treated topically with LXA4 (65 ng/20 μL) or vehicle three times a day for 14 days. Corneal opacity, neovascularization (NV), and hyphema were recorded and evaluated in a blind manner. Pro-inflammatory cytokine expression and genes involved in cornel repair were assayed by RNA sequencing and capillary Western blot. Cornea cell infiltration and monocytes isolated from the blood were analyzed by immunofluorescence and by flow cytometry. Topical treatment with LXA4 for two weeks significantly reduced corneal opacity, NV, and hyphema compared to the vehicle treatment. RNA-seq and Western blot results showed that LXA4 decreased the gene and protein expression of pro-inflammatory cytokines interleukin (IL)-1β and IL-6 and pro-angiogenic mediators matrix metalloproteinase (MMP)-9 and vascular endothelial growth factor (VEGFA). It also induces genes involved in keratinization and ErbB signaling and downregulates immune pathways to stimulate wound healing. Flow cytometry and immunohistochemistry showed significantly less infiltration of neutrophils in the corneas treated with LXA4 compared to vehicle treatment. It also revealed that LXA4 treatment increases the proportion of type 2 macrophages (M2) compared to M1 in blood-isolated monocytes. LXA4 decreases corneal inflammation and NV induced by a strong alkali burn. Its mechanism of action includes inhibition of inflammatory leukocyte infiltration, reduction in cytokine release, suppression of angiogenic factors, and promotion of corneal repair gene expression and macrophage polarization in blood from alkali burn corneas. LXA4 has potential as a therapeutic candidate for severe corneal chemical injuries.

Single-cell transcriptomic atlas of lung microvascular regeneration after targeted endothelial cell ablation

We sought to define the mechanism underlying lung microvascular regeneration in a model of severe acute lung injury (ALI) induced by selective lung endothelial cell ablation. Intratracheal instillation of DT in transgenic mice expressing human diphtheria toxin (DT) receptor targeted to ECs resulted in ablation of >70% of lung ECs, producing severe ALI with near complete resolution by 7 days. Using single-cell RNA sequencing, eight distinct endothelial clusters were resolved, including alveolar aerocytes (aCap) ECs expressing apelin at baseline and general capillary (gCap) ECs expressing the apelin receptor. At 3 days post-injury, a novel gCap EC population emerged characterized by de novo expression of apelin, together with the stem cell marker, protein C receptor. These stem-like cells transitioned at 5 days to proliferative endothelial progenitor-like cells, expressing apelin receptor together with the pro-proliferative transcription factor, Foxm1, and were responsible for the rapid replenishment of all depleted EC populations by 7 days post-injury. Treatment with an apelin receptor antagonist prevented ALI resolution and resulted in excessive mortality, consistent with a central role for apelin signaling in EC regeneration and microvascular repair. The lung has a remarkable capacity for microvasculature EC regeneration which is orchestrated by newly emergent apelin-expressing gCap endothelial stem-like cells that give rise to highly proliferative, apelin receptor-positive endothelial progenitors responsible for the regeneration of the lung microvasculature.

VPA improves ferroptosis in tubular epithelial cells after cisplatin-induced acute kidney injury.

Background: As a novel non-apoptotic cell death, ferroptosis has been reported to play a crucial role in acute kidney injury (AKI), especially cisplatin-induced AKI. Valproic acid (VPA), an inhibitor of histone deacetylase (HDAC) 1 and 2, is used as an antiepileptic drug. Consistent with our data, a few studies have demonstrated that VPA protects against kidney injury in several models, but the detailed mechanism remains unclear. Results: In this study, we found that VPA prevents against cisplatin-induced renal injury via regulating glutathione peroxidase 4 (GPX4) and inhibiting ferroptosis. Our results mainly indicated that ferroptosis presented in tubular epithelial cells of AKI humans and cisplatin-induced AKI mice. VPA or ferrostatin-1 (ferroptosis inhibitor, Fer-1) reduced cisplatin-induced AKI functionally and pathologically, which was characterized by reduced serum creatinine, blood urea nitrogen, and tissue damage in mice. Meanwhile, VPA or Fer-1 treatment in both in vivo and in vitro models, decreased cell death, lipid peroxidation, and expression of acyl-CoA synthetase long-chain family member 4 (ACSL4), reversing downregulation of GPX4. In addition, our study in vitro indicated that GPX4 inhibition by siRNA significantly weakened the protective effect of VPA after cisplatin treatment. Conclusion: Ferroptosis plays an essential role in cisplatin-induced AKI and inhibiting ferroptosis through VPA to protect against renal injury is a viable treatment in cisplatin-induced AKI.

MTORC1 signaling pathway regulates tooth repair

Tooth germ injury can lead to abnormal tooth development and even tooth loss, affecting various aspects of the stomatognathic system including form, function, and appearance. However, the research about tooth germ injury model on cellular and molecule mechanism of tooth germ repair is still very limited. Therefore, it is of great importance for the prevention and treatment of tooth germ injury to study the important mechanism of tooth germ repair by a tooth germ injury model. Here, we constructed a Tg(dlx2b:Dendra2-NTR) transgenic line that labeled tooth germ specifically. Taking advantage of the NTR/Mtz system, the dlx2b+ tooth germ cells were depleted by Mtz effectively. The process of tooth germ repair was evaluated by antibody staining, in situ hybridization, EdU staining and alizarin red staining. The severely injured tooth germ was repaired in several days after Mtz treatment was stopped. In the early stage of tooth germ repair, the expression of phosphorylated 4E-BP1 was increased, indicating that mTORC1 is activated. Inhibition of mTORC1 signaling in vitro or knockdown of mTORC1 signaling in vivo could inhibit the repair of injured tooth germ. Normally, mouse incisors were repaired after damage, but inhibition/promotion of mTORC1 signaling inhibited/promoted this repair progress. Overall, we are the first to construct a stable and repeatable repair model of severe tooth germ injury, and our results reveal that mTORC1 signaling plays a crucial role during tooth germ repair, providing a potential target for clinical treatment of tooth germ injury.

Comprehensive evaluation of mitochondrial redox profile, calcium dynamics, membrane integrity and apoptosis markers in a preclinical model of severe penetrating traumatic brain injury.

Traumatic Brain Injury (TBI) is caused by the external physical assaults damages the brain. It is a heterogeneous disorder that remains a leading cause of death and disability in the military and civilian population of the United States. Preclinical investigations of mitochondrial responses in TBI have ascertained that mitochondrial dysfunction is an acute indicator of cellular damage and plays a pivotal role in long-term injury progression through cellular excitotoxicity. The current study was designed to provide an in-depth evaluation of mitochondrial endpoints with respect to redox and calcium homeostasis, and cell death responses following penetrating TBI (PTBI). To evaluate these pathological cascades, anesthetized adult male rats (N=6/group) were subjected to either 10% unilateral PTBI or Sham craniectomy. Animals were euthanized at 24h post-PTBI, and purified mitochondrial fractions were isolated from the brain injury core and perilesional areas. Overall, increased reactive oxygen and nitrogen species (ROS/RNS) production, and elevated oxidative stress markers such as 4-hydroxynonenal (4-HNE), 3-nitrotyrosine (3-NT), and protein carbonyls (PC) were observed in the PTBI group compared to Sham. Mitochondrial antioxidants such as glutathione, peroxiredoxin (PRX-3), thioredoxin (TRX), nicotinamide adenine dinucleotide phosphate (NADPH), superoxide dismutase (SOD), and catalase (CAT) levels were significantly decreased after PTBI. Likewise, PTBI mitochondria displayed significant loss of Ca2+ homeostasis, early opening of mitochondrial permeability transition pore (mPTP), and increased mitochondrial swelling. Both, outer and inner mitochondrial membrane integrity markers, such as voltage-dependent anion channels (VDAC) and cytochrome c (Cyt C) expression were significantly decreased following PTBI. The apoptotic cell death was evidenced by significantly decreased B-cell lymphoma-2 (Bcl-2) and increased glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression after PTBI. Collectively, current results highlight the comprehensive picture of mitochondria-centric acute pathophysiological responses following PTBI, which may be utilized as novel prognostic indicators of disease progression and theragnostic indicators for evaluating neuroprotection therapeutics following TBI.

Biliary epithelial cells are facultative liver stem cells during liver regeneration in adult zebrafish.

The liver is a highly regenerative organ, yet the presence of a dedicated stem cell population remains controversial. Here, we interrogate a severe hepatocyte injury model in adult zebrafish to define that regeneration involves a stem cell population. After near-total hepatocyte ablation, single-cell transcriptomic and high-resolution imaging analyses throughout the entire regenerative timeline reveal that biliary epithelial cells undergo transcriptional and morphological changes to become hepatocytes. As a population, biliary epithelial cells give rise to both hepatocytes and biliary epithelial cells. Biliary epithelial cells proliferate and dedifferentiate to express hepatoblast transcription factors prior to hepatocyte differentiation. This process is characterized by increased MAPK, PI3K, and mTOR signaling, and chemical inhibition of these pathways impairs biliary epithelial cell proliferation and fate conversion. We conclude that, upon severe hepatocyte ablation in the adult liver, biliary epithelial cells act as facultative liver stem cells in an EGFR-PI3K-mTOR-dependent manner.

Restricted physical activity after volumetric muscle loss alters whole-body and local muscle metabolism.

Volumetric muscle loss (VML) is the traumatic loss of skeletal muscle, resulting in chronic functional deficits and pathological comorbidities, including altered whole-body metabolic rate and respiratory exchange ratio (RER), despite no change in physical activity in animal models. In other injury models, treatment with β2 receptor agonists (e.g. formoterol) improves metabolic and skeletal muscle function. We aimed first to examine if restricting physical activity following injury affects metabolic and skeletal muscle function, and second, to enhance the metabolic and contractile function of the muscle remaining following VML injury through treatment with formoterol. Adult male C57Bl/6J mice (n=32) underwent VML injury to the posterior hindlimb compartment and were randomly assigned to unrestricted or restricted activity and formoterol treatment or no treatment; age-matched injury naïve mice (n=4) were controls for biochemical analyses. Longitudinal 24h evaluations of physical activity and whole-body metabolism were conducted following VML. In vivo muscle function was assessed terminally, and muscles were biochemically evaluated for protein expression, mitochondrial enzyme activity and untargeted metabolomics. Restricting activity chronically after VML had the greatest effect on physical activity and RER, reflected in reduced lipid oxidation, although changes were attenuated by formoterol treatment. Formoterol enhanced injured muscle mass, while mitigating functional deficits. These novel findings indicate physical activity restriction may recapitulate following VML clinically, and adjunctive oxidative treatment may create a metabolically beneficial intramuscular environment while enhancing the injured muscle's mass and force-producing capacity. Further investigation is needed to evaluate adjunctive oxidative treatment with rehabilitation, which may augment the muscle's regenerative and functional capacity following VML. KEY POINTS: The natural ability of skeletal muscle to regenerate and recover function is lost following complex traumatic musculoskeletal injury, such as volumetric muscle loss (VML), and physical inactivity following VML may incur additional deleterious consequences for muscle and metabolic health. Modelling VML injury-induced physical activity restriction altered whole-body metabolism, primarily by decreasing lipid oxidation, while preserving local skeletal muscle metabolic activity. The β2 adrenergic receptor agonist formoterol has shown promise in other severe injury models to improve regeneration, recover function and enhance metabolism. Treatment with formoterol enhanced mass of the injured muscle and whole-body metabolism while mitigating functional deficits resulting from injury. Understanding of chronic effects of the clinically available and FDA-approved pharmaceutical formoterol could be a translational option to support muscle function after VML injury.

Delayed viral vector mediated delivery of neurotrophin-3 improves skilled hindlimb function and stability after thoracic contusion

Intramuscular injection of an Adeno-associated viral vector serotype 1 (AAV1) encoding Neurotrophin-3 (NT3) into hindlimb muscles 24 h after a severe T9 spinal level contusion in rats has been shown to induce lumbar spinal neuroplasticity, partially restore locomotive function and reduce spasms during swimming. Here we investigate whether a targeted delivery of NT3 to lumbar and thoracic motor neurons 48 h following a severe contusive injury aids locomotive recovery in rats. AAV1-NT3 was injected bilaterally into the tibialis anterior, gastrocnemius and rectus abdominus muscles 48-h following trauma, persistently elevating serum levels of the neurotrophin. NT3 modestly improved trunk stability, accuracy of stepping during skilled locomotion, and alternation of the hindlimbs during swimming, but it had no effect on gross locomotor function in the open field. The number of vGlut1+ boutons, likely arising from proprioceptive afferents, on gastrocnemius α-motor neurons was increased after injury but normalised following NT3 treatment, suggestive of a mechanism in which functional benefits may be mediated through proprioceptive feedback. Ex vivo MRI revealed substantial loss of grey and white matter at the lesion epicentre but no effect of delayed NT3 treatment to induce neuroprotection. Lower body spasms and hyperreflexia of an intrinsic paw muscle were not reliably induced in this severe injury model suggesting a more complex anatomical or physiological cause to their induction. We have shown that delayed intramuscular AAV-NT3 treatment can promote recovery in skilled stepping and coordinated swimming, supporting a role for NT3 as a therapeutic strategy for spinal injuries potentially through modulation of somatosensory feedback.

Distinct stimulus-dependent neutrophil dynamics revealed by real-time imaging of intestinal mucosa after acute injury.

Clinical symptoms in many inflammatory diseases of the intestine are directly related to neutrophil (PMN) migration across colonic mucosa and into the intestinal lumen, yet in-vivo studies detailing this process are lacking. Using real-time intravital microscopy and a new distal colon loop model, we report distinct PMN migratory dynamics in response to several models of acute colonic injury. PMNs exhibited rapid swarming responses after mechanically induced intestinal wounds. Similar numbers of PMNs infiltrated colonic mucosa after wounding in germ-free mice, suggesting microbiota-independent mechanisms. By contrast, acute mucosal injury secondary to either a treatment of mice with dextran sodium sulfate or an IL-10 receptor blockade model of colitis resulted in lamina propria infiltration with PMNs that were largely immotile. Biopsy wounding of colonic mucosa in DSS-treated mice did not result in enhanced PMN swarming however, intraluminal application of the neutrophil chemoattractant LTB4 under such conditions resulted in enhanced transepithelial migration of PMNs. Analyses of PMNs that had migrated into the colonic lumen revealed that the majority of PMNs were directly recruited from the circulation and not from the immotile pool in the mucosa. Decreased PMN motility parallels upregulation of the receptor CXCR4 and apoptosis. Similarly, increased expression of CXCR4 on human PMNs was observed in colonic biopsies from people with active ulcerative colitis. This new approach adds an important tool to investigate mechanisms regulating PMN migration across mucosa within the distal intestine and will provide new insights for developing future anti-inflammatory and pro-repair therapies.

Study on injury parameters of severe blast injury in mice at plain and plateau based on equivalent trauma

To explore the establishment of the interconvertible injury parameters of same severe blast injury in mice at plain and plateau. A total of 157 C57BL/6 male mice were randomly divided into plain control group (8 mice), plain injury group (77 mice), plateau control group (8 mice) and plateau injury group (64 mice) according to random number table method. The mice in plateau control group and plateau blast injury group had been placed in animal experimental low-pressure oxygen chamber to simulate 4 000 meters plateau environment for 5 days in advance. Then the mice in plain blast injury group and plateau blast injury group were put into biological shock tube, respectively. Different pressures of the driving section were selected to establish the severe blast injury models in mice at plain and 4 000 meters plateau to reach approximately 70% mortality within 72 hours. The equivalent traumatic condition at 24 hours after blast injury in different groups was verified by the series of experiments including gross autopsy, lung wet/dry weight ratio (W/D), hematoxylin-eosin (HE) staining and histological scoring. The mice mortality were basically consistent between the plain injury group (65%) and plateau injury group (75%) when 5.4 MPa and 4.0 MPa of the driving section pressures were chosen, respectively. Compared with the corresponding control groups, the lungs showed massive hemorrhage (patchy and diffuse) with significant pulmonary edema in both plain 5.4 MPa-injured group and the plateau 4.0 MPa-injured group at 24 hours after blast injury. Compared with the plateau control group, the pulmonary W/D ratio were significantly increased in the plateau injury group (5.579±0.646 vs. 4.476±0.076, P < 0.05), while the difference between plateau injury group and the plain control group was not statistically significant (5.303±1.020 vs. 4.015±0.144, P > 0.05). Also, compared with the corresponding control groups, the analysis of lung histopathological sections showed that there were several pathological changes including large alveolar rupture and fusion, thickened alveolar walls, and a small amount of inflammatory cell infiltration in the alveolar lumen in the groups of plain 5.4 MPa and plateau 4.0 MPa. In addition, the histopathological scores of lung in the groups of plain 5.4 MPa and plateau 4.0 MPa were significantly higher than that in corresponding control group (8.67±0.82 vs. 1.67±0.52, 9.00±1.10 vs. 2.17±0.41, both P < 0.05), however, there was no statistical difference for the above score between plain blast injury group and plateau blast injury group. The pressures of driving section 5.4 MPa and 4.0 MPa are injury parameters to establish equivalent severe blast injury in mice at plain and plateau, respectively, which can be converted to each other. This study provides support for the application and evaluation of prevention and treatment technology for severe blast injury in special environment.

The mechanism of action of a novel neuroprotective low molecular weight dextran sulphate: New platform therapy for neurodegenerative diseases like Amyotrophic Lateral Sclerosis.

Background: Acute and chronic neurodegenerative diseases represent an immense socioeconomic burden that drives the need for new disease modifying drugs. Common pathogenic mechanisms in these diseases are evident, suggesting that a platform neuroprotective therapy may offer effective treatments. Here we present evidence for the mode of pharmacological action of a novel neuroprotective low molecular weight dextran sulphate drug called ILB®. The working hypothesis was that ILB® acts via the activation of heparin-binding growth factors (HBGF). Methods: Pre-clinical and clinical (healthy people and patients with ALS) in vitro and in vivo studies evaluated the mode of action of ILB®. In vitro binding studies, functional assays and gene expression analyses were followed by the assessment of the drug effects in an animal model of severe traumatic brain injury (sTBI) using gene expression studies followed by functional analysis. Clinical data, to assess the hypothesized mode of action, are also presented from early phase clinical trials. Results: ILB® lengthened APTT time, acted as a competitive inhibitor for HGF-Glypican-3 binding, effected pulse release of heparin-binding growth factors (HBGF) into the circulation and modulated growth factor signaling pathways. Gene expression analysis demonstrated substantial similarities in the functional dysregulation induced by sTBI and various human neurodegenerative conditions and supported a cascading effect of ILB® on growth factor activation, followed by gene expression changes with profound beneficial effect on molecular and cellular functions affected by these diseases. The transcriptional signature of ILB® relevant to cell survival, inflammation, glutamate signaling, metabolism and synaptogenesis, are consistent with the activation of neuroprotective growth factors as was the ability of ILB® to elevate circulating levels of HGF in animal models and humans. Conclusion: ILB® releases, redistributes and modulates the bioactivity of HBGF that target disease compromised nervous tissues to initiate a cascade of transcriptional, metabolic and immunological effects that control glutamate toxicity, normalize tissue bioenergetics, and resolve inflammation to improve tissue function. This unique mechanism of action mobilizes and modulates naturally occurring tissue repair mechanisms to restore cellular homeostasis and function. The identified pharmacological impact of ILB® supports the potential to treat various acute and chronic neurodegenerative disease, including sTBI and ALS.

ILB®, a Low Molecular Weight Dextran Sulphate, Restores Glutamate Homeostasis, Amino Acid Metabolism and Neurocognitive Functions in a Rat Model of Severe Traumatic Brain Injury.

In a previous study, we found that administration of ILB®, a new low molecular weight dextran sulphate, significantly improved mitochondrial functions and energy metabolism, as well as decreased oxidative/nitrosative stress, of brain tissue of rats exposed to severe traumatic brain injury (sTBI), induced by the closed-head weight-drop model of diffused TBI. Using aliquots of deproteinized brain tissue of the same animals of this former study, we here determined the concentrations of 24 amino acids of control rats, untreated sTBI rats (sacrificed at 2 and 7 days post-injury) and sTBI rats receiving a subcutaneous ILB® administration (at the dose levels of 1, 5 and 15 mg/kg b.w.) 30 min post-impact (sacrificed at 2 and 7 days post-injury). Additionally, in a different set of experiments, new groups of control rats, untreated sTBI rats and ILB®-treated rats (administered 30 min after sTBI at the dose levels of 1 or 5 mg/kg b.w.) were studied for their neurocognitive functions (anxiety, locomotor capacities, short- and long-term memory) at 7 days after the induction of sTBI. Compared to untreated sTBI animals, ILB® significantly decreased whole brain glutamate (normalizing the glutamate/glutamine ratio), glycine, serine and γ-aminobutyric acid. Furthermore, ILB® administration restored arginine metabolism (preventing nitrosative stress), levels of amino acids involved in methylation reactions (methionine, L-cystathionine, S-adenosylhomocysteine), and N-acetylaspartate homeostasis. The macroscopic evidences of the beneficial effects on brain metabolism induced by ILB® were the relevant improvement in neurocognitive functions of the group of animals treated with ILB® 5 mg/kg b.w., compared to the marked cognitive decline measured in untreated sTBI animals. These results demonstrate that ILB® administration 30 min after sTBI prevents glutamate excitotoxicity and normalizes levels of amino acids involved in crucial brain metabolic functions. The ameliorations of amino acid metabolism, mitochondrial functions and energy metabolism in ILB®-treated rats exposed to sTBI produced significant improvement in neurocognitive functions, reinforcing the concept that ILB® is a new effective therapeutic tool for the treatment of sTBI, worth being tested in the clinical setting.

Heat-killed probiotic Lactobacillus plantarum affects the function of neutrophils but does not improve survival in murine burn injury

Probiotics have become of interest as therapeutics in trauma or sepsis-induced inflammation due to their ability to affects the immune response. However, their use is still under debate due to the potential risk of septicemia. Therefore, heat-killed probiotics offer a potential alternative, with recent research suggesting a comparable immunomodulating potential and increased safety. In a previous study, we demonstrated decreased mortality by administration of live Lactobacillus plantarum in a mouse burn-sepsis model. Neutrophils are an essential innate defense against pathogens. Therefore, our present study aims to understand the impact of heat-killed probiotic L. plantarum (HKLP) on neutrophil function. Utilizing an in vitro stimulation with HKLP and a burn-infection in vivo model, we determined that administration of HKLP induced significant release of granulocyte-colony stimulating factor (G-CSF) and stimulated the release of pro-and anti-inflammatory cytokines. HKLP had no impact on neutrophil function, such as phagocytosis, oxidative burst, and NETosis, but increased apoptosis and activated neutrophils. HKLP did not improve survival. Together, contrary to our hypothesis, heat-killed probiotics did not improve neutrophil function and survival outcome in a murine severe burn injury model.

Targeting HDAC6 in the Dorsal Root Ganglia Attenuates Peripheral Nerve Injury‐induced Hypersensitivity

Histone deacetylase 6 (HDAC6) is a Class IIb histone deacetylase, which is primarily localized to the cytoplasm and plays an important role in cell structure and dynamism, transcriptional repression, exocytosis and endocytocis. Preclinical evidence has suggested that HDAC6 inhibitors alleviate signs of chemotherapy‐induced peripheral neuropathy (CIPN), such as mechanical allodynia. However, no group to our knowledge has investigated the mechanism of action of HDAC6 inhibitors in a severe nerve injury model, which has a different pathophysiology than milder models such as CIPN. In this study we use genetically modified mice and the tibial spared nerve injury (SNI) model to determine the impact of dorsal root ganglion (DRG) specific HDAC6 knockout in the induction and maintenance of sensory hypersensitivity. Downregulation of HDAC6 in the DRG was achieved by injection of the left sciatic nerve of adult male HDAC6fl\fl mice with AAV8‐CMV‐Cre‐EGFP or AAV8‐CMV‐EGFP vectors. DRG‐knockdown of HDAC6 prevented the development of mechanical allodynia after SNI. Using in situ hybridization (RNAScope), we also demonstrate that HDAC6 is upregulated at three weeks after the induction of nerve injury in L3‐6 DRG neurons. Furthermore, treatment with the peripherally acting HDAC6 inhibitor ACY1215 (Regenacy Pharmaceuticals, Waltham, MA) after SNI leads to full recovery from mechanical allodynia. We are continuing this work by performing bulk RNA sequencing on L3‐6 DRGs from mice who received 21 days of treatment with ACY1215 or vehicle starting at three weeks after SNI or Sham operation, in order to understand the transcriptomic events and upstream pathways associated with recovery from prolonged neuropathic states.Our findings highlight a promising therapeutic role of HDAC6 inhibitors for the prevention or recovery from sensory hypersensitivity behaviors associated with prolonged peripheral nerve injury. Our future work aims to define the mechanisms underlying HDAC6 action in the DRG and identify new pathways associated with recovery from sensory hypersensitivity to influence novel treatment strategies for pain.

The Role and Mechanism of Lysine Methyltransferase and Arginine Methyltransferase in Kidney Diseases.

Methylation can occur in both histones and non-histones. Key lysine and arginine methyltransferases under investigation for renal disease treatment include enhancer of zeste homolog 2 (EZH2), G9a, disruptor of telomeric silencing 1-like protein (DOT1L), and protein arginine methyltransferases (PRMT) 1 and 5. Recent studies have shown that methyltransferases expression and activity are also increased in several animal models of kidney injury, such as acute kidney injury(AKI), obstructive nephropathy, diabetic nephropathy and lupus nephritis. The inhibition of most methyltransferases can attenuate kidney injury, while the role of methyltransferase in different animal models remains controversial. In this article, we summarize the role and mechanism of lysine methyltransferase and arginine methyltransferase in various kidney diseases and highlight methyltransferase as a potential therapeutic target for kidney diseases.

Human placental hematopoietic stem cell derived natural killer cells (CYNK-001) mediate protection against influenza a viral infection

ABSTRACT Influenza A virus (IAV) infections are associated with a high healthcare burden around the world and there is an urgent need to develop more effective therapies. Natural killer (NK) cells have been shown to play a pivotal role in reducing IAV-induced pulmonary infections in preclinical models; however, little is known about the therapeutic potential of adoptively transferred NK cells for IAV infections. Here, we investigated the effects of CYNK-001, human placental hematopoietic stem cell derived NK cells that exhibited strong cytolytic activity against a range of malignant cells and expressed high levels of activating receptors, against IAV infections. In a severe IAV-induced acute lung injury model, mice treated with CYNK-001 showed a milder body weight loss and clinical symptoms, which led to a delayed onset of mortality, thus demonstrating their antiviral protection in vivo. Analysis of bronchoalveolar lavage fluid (BALF) revealed that CYNK-001 reduced proinflammatory cytokines and chemokines highlighting CYNK-001’s anti-inflammatory actions in viral induced-lung injury. Furthermore, CYNK-001-treated mice had altered immune responses to IAV with reduced number of neutrophils in BALF yet increased number of CD8+ T cells in the BALF and lung compared to vehicle-treated mice. Our results demonstrate that CYNK-001 displays protective functions against IAV via its anti-inflammatory and immunomodulating activities, which leads to alleviation of disease burden and progression in a severe IAV-infected mice model. The potential of adoptive NK therapy for IAV infections warrants clinical investigation.

Implication of autophagy in the antifibrogenic effect of Rilpivirine: when more is less

As the main extracellular matrix-producing cells, activated hepatic stellate cells (HSC) are fundamental mediators of liver fibrosis (LF), and understanding their activation/inactivation mechanisms is paramount to the search for novel therapeutics. The antiretroviral drug Rilpivirine (RPV) has demonstrated a hepatoprotective effect in several animal models of chronic liver injury that is related to its antifibrogenic and apoptotic action in HSC. In the present study, we evaluated whether autophagy is implicated in the hepatoprotective action of RPV, as autophagy plays an important role in HSC transdifferentiation. We employed two standard mouse models of chronic liver injury - fatty liver disease and carbon tetrachloride (CCl4)-induced hepatotoxicity -and cultured HSC activated with the profibrotic cytokine TGF-β. RPV enhanced autophagy in the whole liver of both mouse models and in activated HSC, evident in the protein expression of autophagy markers, increased autophagosome content and lysosomal mass. Moreover, increased autophagic flux was observed in RPV-exposed HSC as revealed by tandem fluorescence-tagged LC3 and p62 and analysis of LC3-II accumulation in cells exposed to the lysosomal inhibitor chloroquine. Importantly, autophagy was involved in the cytotoxic effect of RPV on HSC, though in a differential manner. Pharmacological inhibition of autophagy by 3-methyladenine (3-MA) did not affect the diminishing effect of RPV on viability, while treatment with wortmannin or depletion of specific autophagy proteins (ATG5, Beclin-1 and SQSTM1/p62) rescued the detrimental effect of high concentrations of RPV on the viability of activated HSC. Finally, we also provide evidence that RPV compromises the viability of TGF-β-induced HSC independently of its antifibrogenic effect, observed as reduced collagen 1A1 synthesis, and that this effect does not include RPV´s modulation of autophagy. In summary, as a contributor to the mechanisms involved in the hepatoprotective action of RPV, autophagy may be a good candidate to explore when developing novel therapeutics for LF.

Dysfunctional Endoplasmic Reticulum-Mitochondrion Coupling Is Associated with Endoplasmic Reticulum Stress-Induced Apoptosis and Neurological Deficits in a Rodent Model of Severe Head Injury.

Cellular homeostasis requires critical communications between the endoplasmic reticulum (ER) and mitochondria to maintain the viability of cells. This communication is mediated and maintained by the mitochondria-associated membranes and may be disrupted during acute traumatic brain injury (TBI), leading to structural and functional damage of neurons and supporting cells. To test this hypothesis, we subjected male C57BL/6 mice to severe TBI (sTBI) using a controlled cortical impact device. We analyzed the physical ER-mitochondrion contacts in the perilesional cortex using transmission electron microscopy, Western blot, and immunofluorescence. We specifically measured changes in the production of reactive oxygen species (ROS) in mitochondria, the unfolded protein response (UPR), the neuroinflammatory response, and ER stress-mediated apoptosis in the traumatic injured cerebral tissue. A modified neurological severity score was used to evaluate neurological function in the sTBI mice. We found that sTBI induced significant reorganizations of mitochondria-associated ER membranes (MAMs) in the cerebral cortex within the first 24 h post-injury. This ER-mitochondrion coupling was enhanced, reaching its peak level at 6 h post-sTBI. This enhanced coupling correlated closely with increases in the expression of the Ca2+ regulatory proteins (inositol 1,4,5-trisphosphate receptor type 1 [IP3R1], voltage-dependent anion channel 1 [VDAC1], glucose-regulated protein 75 [GRP75], Sigma 1 receptor [Sigma-1R]), production of ROS, degree of ER stress, levels of UPR, and release of proinflammatory cytokines. Further, the neurological function of sTBI mice was significantly improved by silencing the gene for the ER-mitochondrion tethering factor PACS2, restoring the IP3R1-GRP75-VDAC1 axis of Ca2+ regulation, alleviating mitochondria-derived oxidative stress, suppressing inflammatory response through the PERK/eIF2α/ATF4/CHOP pathway, and inhibiting ER stress and associated apoptosis. These results indicate that dysfunctional ER-mitochondrion coupling might be primarily involved in the neuronal apoptosis and neurological deficits, and modulating the ER-mitochondrion crosstalk might be a novel therapeutic strategy for sTBI.