NLRX1 Inhibits LPS-Induced Microglial Death via Inducing p62-Dependent HO-1 Expression, Inhibiting MLKL and Activating PARP-1.

The activation of microglia and the production of cytokines are key factors contributing to progressive neurodegeneration. Despite the well-recognized neuronal programmed cell death regulated by microglial activation, the death of microglia themselves is less investigated. Nucleotide-binding oligomerization domain, leucine-rich repeat-containing X1 (NLRX1) functions as a scaffolding protein and is involved in various central nervous system diseases. In this study, we used the SM826 microglial cells to understand the role of NLRX1 in lipopolysaccharide (LPS)-induced cell death. We found LPS-induced cell death is blocked by necrostatin-1 and zVAD. Meanwhile, LPS can activate poly (ADP-ribose) polymerase-1 (PARP-1) to reduce DNA damage and induce heme oxygenase (HO)-1 expression to counteract cell death. NLRX1 silencing and PARP-1 inhibition by olaparib enhance LPS-induced SM826 microglial cell death in an additive manner. Less PARylation and higher DNA damage are observed in NLRX1-silencing cells. Moreover, LPS-induced HO-1 gene and protein expression through the p62-Keap1-Nrf2 axis are attenuated by NLRX1 silencing. In addition, the Nrf2-mediated positive feedback regulation of p62 is accordingly reduced by NLRX1 silencing. Of note, NLRX1 silencing does not affect LPS-induced cellular reactive oxygen species (ROS) production but increases mixed lineage kinase domain-like pseudokinase (MLKL) activation and cell necroptosis. In addition, NLRX1 silencing blocks bafilomycin A1-induced PARP-1 activation. Taken together, for the first time, we demonstrate the role of NLRX1 in protecting microglia from LPS-induced cell death. The underlying protective mechanisms of NLRX1 include upregulating LPS-induced HO-1 expression via Nrf2-dependent p62 expression and downstream Keap1-Nrf2 axis, mediating PARP-1 activation for DNA repair via ROS- and autophagy-independent pathway, and reducing MLKL activation.

CASK Mediates Oxidative Stress-Induced Microglial Apoptosis-Inducing Factor-Independent Parthanatos Cell Death via Promoting PARP-1 Hyperactivation and Mitochondrial Dysfunction.

Calcium/calmodulin-dependent serine protein kinase (CASK) is a scaffold protein and plays critical roles in neuronal synaptic formation and brain development. Previously, CASK was shown to associate with EGFR to maintain the vulval cell differentiation in C. elegans. In this study, we explored the role of CASK in CHME3 microglial cells. We found that CASK silencing protects cells from H2O2-induced cell death by attenuating PARP-1 activation, mitochondrial membrane potential loss, reactive oxygen species production, and mitochondrial fission, but it increases oxidative phosphorylation. The PARP-1 inhibitor olaparib blocks H2O2-induced cell death, suggesting the death mode of parthanatos. CASK silencing also increases AKT activation but decreases AMPK activation under H2O2 treatment. Pharmacological data further indicate that both signaling changes contribute to cell protection. Different from the canonical parthanatos pathway, we did not observe the AIF translocation from mitochondria into the nucleus, suggesting a non-canonical AIF-independent parthanatos in H2O2-treated CHME3 cells. Moreover, we found that CASK silencing upregulates the EGFR gene and protein expression and increases H2O2-induced EGFR phosphorylation in CHME3 microglia. However, EGFR activation does not contribute to cell protection caused by CASK silencing. In conclusion, CASK plays a crucial role in microglial parthanatos upon H2O2 treatment via stimulation of PARP-1 and AMPK but the inhibition of AKT. These findings suggest that CASK might be an ideal therapeutic target for CNS disorders.

Epigenetic upregulation of spleen tyrosine kinase in cancer cells through p53-dependent downregulation of DNA methyltransferase.

Syk is a tumor suppressor gene in some solid tumors. Currently, it remains unknown how Syk gene hypermethylation is controlled by DNA methyltransferase (DNMT) and p53. In colorectal cancer HCT116 cells, we found that protein and mRNA levels of Syk were much higher in WT than in p53-/- cells. Both p53 inhibitor PFT-α and p53 silencing can reduce the protein and mRNA expression of Syk in WT cells, while DNMT inhibitor 5-Aza-2'-dC can increase Syk expression in p53-/- cells. Interestingly, the DNMT expression in p53-/- HCT116 cells was higher than that in WT cells. PFT-α can not only enhance Syk gene methylation but also increase DNMT1 protein and mRNA levels in WT HCT116 cells. In metastatic lung cancer cell lines A549 and PC9, which express WT p53 and gain function of p53, respectively, PFT-α can also downregulate Syk mRNA and protein expression. However, the Syk methylation level was increased by PFT-α in A549 but not in PC9 cells. Likewise, 5-Aza-2'-dC transcriptionally increased Syk gene expression in A549 cells, but not in PC9 cells. In summary methylation of Syk promoter requires DNMT1, and p53 can upregulate Syk expression via downregulation of DNMT1 at the transcriptional level.

P2X7 Is Involved in the Mouse Retinal Degeneration via the Coordinated Actions in Different Retinal Cell Types.

Adenosine triphosphate (ATP) released from dying cells with high concentrations is sensed as a danger signal by the P2X7 receptor. Sodium iodate (NaIO3) is an oxidative toxic agent, and its retinal toxicity has been used as the model of dry age-related macular degeneration (AMD). In this study, we used NaIO3-treated mice and cultured retinal cells, including BV-2 microglia, 661W photoreceptors, rMC1 Müller cells and ARPE-19 retinal epithelial cells, to understand the pathological action of P2X7 in retinal degeneration. We found that NaIO3 can significantly decrease the photoreceptor function by reducing a-wave and b-wave amplitudes in electroretinogram (ERG) analysis. Optical coherence tomography (OCT) analysis revealed the degeneration of retinal epithelium and ganglion cell layers. Interestingly, P2X7-/- mice were protected from the NaIO3-induced retinopathy and inflammatory NLRP3, IL-1ß and IL-6 gene expression in the retina. Hematoxylin and eosin staining indicated that the retinal epithelium was less deteriorated in P2X7-/- mice compared to the WT group. Although P2X7 was barely detected in 661W, rMC1 and ARPE-19 cells, its gene and protein levels can be increased after NaIO3 treatment, leading to a synergistic cytotoxicity of BzATP [2'(3')-O-(4-benzoylbenzoyl)adenosine-5'-triphosphate tri(triethyleneammonium)salt] and NaIO3 administration in ARPE-19 cells. In conclusion, the paracrine action of the ATP/P2X7 axis via cell-cell communication is involved in NaIO3-induced retinal injury. Our results show that P2X7 antagonist might be a potential therapy in inflammation-related retinal degeneration.

Pan-Caspase Inhibitor zVAD Induces Necroptotic and Autophagic Cell Death in TLR3/4-Stimulated Macrophages.

In addition to inducing apoptosis, caspase inhibition contributes to necroptosis and/or autophagy depending on the cell type and cellular context. In macrophages, necroptosis can be induced by co-treatment with Toll-like receptor (TLR) ligands (lipopolysaccharide [LPS] for TLR4 and polyinosinic-polycytidylic acid [poly I:C] for TLR3) and a cell-permeable pan-caspase inhibitor zVAD. Here, we elucidated the signaling pathways and molecular mechanisms of cell death. We showed that LPS/zVAD- and poly I:C/zVAD-induced cell death in bone marrow-derived macrophages (BMDMs) was inhibited by receptor-interacting protein kinase 1 (RIP1) inhibitor necrostatin-1 and autophagy inhibitor 3-methyladenine. Electron microscopic images displayed autophagosome/autolysosomes, and immunoblotting data revealed increased LC3II expression. Although zVAD did not affect LPS- or poly I:C-induced activation of IKK, JNK, and p38, it enhanced IRF3 and STAT1 activation as well as type I interferon (IFN) expression. In addition, zVAD inhibited ERK and Akt phosphorylation induced by LPS and poly I:C. Of note, zVAD-induced enhancement of the IRF3/IFN/STAT1 axis was abolished by necrostatin-1, while zVAD-induced inhibition of ERK and Akt was not. Our data further support the involvement of autocrine IFNs action in reactive oxygen species (ROS)-dependent necroptosis, LPS/zVAD-elicited ROS production was inhibited by necrostatin-1, neutralizing antibody of IFN receptor (IFNR) and JAK inhibitor AZD1480. Accordingly, both cell death and ROS production induced by TLR ligands plus zVAD were abrogated in STAT1 knockout macrophages. We conclude that enhanced TRIF-RIP1-dependent autocrine action of IFNß, rather than inhibition of ERK or Akt, is involved in TLRs/zVAD-induced autophagic and necroptotic cell death via the JAK/STAT1/ROS pathway.

Percutaneous pedicle screw placement under single dimensional fluoroscopy with a designed pedicle finder-a technical note and case series.

BACKGROUND CONTEXT: Minimally invasive spine surgery has become increasingly popular in clinical practice, and it offers patients the potential benefits of reduced blood loss, wound pain, and infection risk, and it also diminishes the loss of working time and length of hospital stay. However, surgeons require more intraoperative fluoroscopy and ionizing radiation exposure during minimally invasive spine surgery for localization, especially for guidance in instrumentation placement. In addition, computer navigation is not accessible in some facility-limited institutions. PURPOSE: This study aimed to demonstrate a method for percutaneous screws placement using only the anterior-posterior (AP) trajectory of intraoperative fluoroscopy. STUDY DESIGN: A technical report (a retrospective and prospective case series) was carried out. PATIENT SAMPLE: Patients who received posterior fixation with percutaneous pedicle screws for thoracolumbar degenerative disease or trauma comprised the patient sample. METHOD: We retrospectively reviewed the charts of consecutive 670 patients who received 4,072 pedicle screws between December 2010 and August 2015. Another case series study was conducted prospectively in three additional hospitals, and 88 consecutive patients with 413 pedicle screws were enrolled from February 2014 to July 2016. The fluoroscopy shot number and radiation dose were recorded. In the prospective study, 78 patients with 371 screws received computed tomography at 3 months postoperatively to evaluate the fusion condition and screw positions. RESULTS: In the retrospective series, the placement of a percutaneous screw required 5.1 shots (2-14, standard deviation [SD]=2.366) of AP fluoroscopy. One screw was revised because of a medialwall breach of the pedicle. In the prospective series, 5.8 shots (2-16, SD=2.669) were required forone percutaneous pedicle screw placement. There were two screws with a Grade 1 breach (8.6%), both at the lateral wall of the pedicle, out of 23 screws placed at the thoracic spine at T9-T12. Forthe lumbar and sacral areas, there were 15 Grade 1 breaches (4.3%), 1 Grade 2 breach (0.3%), and 1 Grade 3 breach (0.3%). No revision surgery was necessary. CONCLUSION: This method avoids lateral shots of fluoroscopy during screw placement and thus decreases the operation time and exposes surgeons to less radiation. At the same time, compared with the computer-navigated procedure, it is less facility-demanding, and provides satisfactory reliability and accuracy.

Clinical application of perfusion computed tomography in neurosurgery.

OBJECT: Currently, perfusion CT (PCT) is a valuable imaging technique that has been successfully applied to the clinical management of patients with ischemic stroke and aneurysmal subarachnoid hemorrhage (SAH). However, recent literature and the authors' experience have shown that PCT has many more important clinical applications in a variety of neurosurgical conditions. Therefore, the authors share their experiences of its application in various diseases of the cerebrovascular, neurotraumatology, and neurooncology fields and review the pertinent literature regarding expanding PCT applications for neurosurgical conditions, including pitfalls and future developments. METHODS: A pertinent literature search was conducted of English-language articles describing original research, case series, and case reports from 1990 to 2011 involving PCT and with relevance and applicability to neurosurgical disorders. RESULTS: In the cerebrovascular field, PCT is already in use as a diagnostic tool for patients suspected of having an ischemic stroke. Perfusion CT can be used to identify and define the extent of the infarct core and ischemic penumbra core, and thus aid patient selection for acute reperfusion therapy. For patients with aneurysmal SAH, PCT provides assessment of early brain injury, cerebral ischemia, and infarction, in addition to vasospasm. It may also be used to aid case selection for aggressive treatment of patients with poor SAH grade. In terms of oncological applications, PCT can be used as an imaging biomarker to assess angiogenesis and response to antiangiogenetic treatments, differentiate between glioma grades, and distinguish recurrent tumor from radiation necrosis. In the setting of traumatic brain injury, PCT can detect and delineate contusions at an early stage. In patients with mild head injury, PCT results have been shown to correlate with the severity and duration of postconcussion syndrome. In patients with moderate or severe head injury, PCT results have been shown to correlate with patients' functional outcome. CONCLUSIONS: Perfusion CT provides quantitative and qualitative data that can add diagnostic and prognostic value in a number of neurosurgical disorders, and also help with clinical decision making. With emerging new technical developments in PCT, such as characterization of blood-brain barrier permeability and whole-brain PCT, this technique is expected to provide more and more insight into the pathophysiology of many neurosurgical conditions.

The neuronal protective effects of local brain cooling at the craniectomy site after lateral fluid percussion injury in a rat model.

BACKGROUND: The aim of the present study is to investigate whether local brain cooling at the craniectomy site causes attenuation of traumatic brain injury (TBI) induced by fluid percussion injury (FPI). METHODS: Anesthetized male Sprague-Dawley rats were divided into two major treatment groups. Immediately after the onset of fluid percussion TBI, a craniectomy window of 6 × 8 mm was made at the right parietal, and a cold water bag (0°C-1°C or 5°C-6°C) was applied locally for 30 min. Additional groups of rats were used as craniectomy and craniectomy + FPI controls. Physiological parameters, such as brain and colonic temperature, mean arterial pressure, and heart rate, were monitored during FPI. Functional motor outcomes were evaluated using the inclined plane test (maximal grasp angle). Cellular infarction volume was calculated using triphenyltetrazolium chloride staining. Apoptosis and neuronal marker-positive cells in the cortex were measured by immunofluorescence staining. All functional and morphologic parameters were assessed 72 h after injury. RESULTS: Compared with the craniectomy + FPI control groups, the groups treated with 5°C-6°C local cold water therapy showed significant attenuation of the FPI-induced motor deficits, weight loss, and cerebral infarction but no effect on colonic temperature. The FPI-induced apoptosis and neuronal loss were also significantly reduced by local cooling. CONCLUSIONS: Our results suggest that local cooling with 5°C-6°C cold water therapy may ameliorate TBI in rats by reducing infarction volume, neuronal cell loss, and apoptosis, resulting in improved functional outcome. We propose that the use of local cooling at the craniectomy site after FPI might have clinical benefits in the future.

Early parenchymal contrast extravasation predicts subsequent hemorrhage progression, clinical deterioration, and need for surgery in patients with traumatic cerebral contusion.

BACKGROUND: This study aimed to identify early radiologic signs that are predictive of hemorrhage progression and clinical deterioration in patients with traumatic cerebral contusion. We hypothesized that contrast extravasation (CE) and blood-brain barrier disruption might be associated with hemorrhage progression, brain edema, and clinical deterioration in these patients. METHODS: Twenty-two patients with traumatic cerebral contusion (diagnosed on initial noncontrast head computed tomography [CT]) who initially did not require surgical intervention were enrolled in this study. Contrast-enhanced and perfusion CT scans were performed within 6 hours of injury, and follow-up noncontrast CT scans were performed at 24 hours and 72 hours. RESULTS: In each noncontrast CT scan, the volumes of the contusion hemorrhage and edema were calculated using computerized planimetric techniques. The initial Glasgow Coma Scale, hemorrhage progression, clinical deterioration, and the need for subsequent surgery were recorded. The early radiologic findings were compared with these parameters and functional outcome at 6 months to identify predictive radiologic signs. CE was present in 9 of 22 patients (41%) and was highly associated with hemorrhage progression (p < 0.05), clinical deterioration (p < 0.01), and need for subsequent surgery (p < 0.01). In addition, patients with CE had a greater volume of edema at 24 hours (p < 0.01) and 72 hours (p < 0.01) than those who did not have CE. However, CE was not found to be associated with poor outcome. CONCLUSIONS: Early parenchymal CE is associated with hemorrhage progression, cerebral edema, clinical deterioration, and need for subsequent surgery. These patients should be monitored closely, and early surgery may be needed if deterioration occurs. Further elucidation of the pathophysiology is needed to formulate effective treatment for these high-risk patients.

Metastatic gallbladder cancer presenting as a gingival tumor and deep neck infection.

Gallbladder cancer has an extremely poor prognosis because it is often diagnosed at an advanced stage. We describe a 63-year-old woman who was treated 4 years previously for gallbladder cancer, with laparoscopic cholecystectomy and secondary hepatectomy after presenting with acute cholecystitis and gallbladder rupture. At her second presentation, she had a left lower gingival tumor and deep neck infection. Incision and drainage and tumor biopsies were performed, and pathology at both sites revealed adenocarcinoma. Positron emission tomography revealed other tumors in the left breast and left lower lung field, which were both proven to be adenocarcinoma by biopsy. The patient's presentation with a metastatic oral tumor was rare. Although the incidence is very low, physicians should consider the possibility of metastatic cancer in a patient with a history of cancer, who presents with new oral tumor or deep neck infection.

Decompressive craniectomy as the primary surgical intervention for hemorrhagic contusion.

The standard surgical treatment of hemorrhagic cerebral contusion is craniotomy with evacuation of the focal lesion. We assessed the safety and feasibility of performing decompressive craniectomy and duraplasty as the primary surgical intervention in this group of patients. Fifty-four consecutive patients with Glasgow Coma Scale (GCS) scores of less than or equal to 8, a frontal or temporal hemorrhagic contusion greater than 20 cm(3) in volume, and a midline shift of at least 5 mm or cisternal compression on computer tomography (CT) scan were studied. Sixteen (29.7%) underwent traditional craniotomy with hematoma evacuation, and 38 (70.4%) underwent craniectomy as the primary surgical treatment. Mortality, reoperation rate, Glasgow Outcome Scale-Extended (GOSE) scores, and length of stay in both the acute care and rehabilitation phase were compared between these two groups. Mortality (13.2% vs. 25.0%) and reoperation rate (7.9% vs. 37.5%) were lower in the craniectomy group, whereas the length of stay in both the acute care setting and the rehabilitation phase were similar between these two groups. The craniectomy group also had better GOSE score (5.55 vs. 3.56) at 6 months. Decompressive craniectomy is safe and effective as the primary surgical intervention for treatment of hemorrhagic contusion. This study also suggests that patient with hemorrhagic contusion can possibly have better outcome after craniectomy than other subgroup of patients with severe traumatic brain injury.

Clinical experience of hydroxyethyl starch (10% HES 200/0.5) in cerebral perfusion pressure protocol for severe head injury.

BACKGROUND: The present study was undertaken to evaluate 10% hydroxyethyl starch (HES 200/0.5) with regard to its clinical outcome and safety in the treatment of severe head injury. METHODS: Retrospective review of patient data from a prospectively designed standard treatment protocol for severe head injury. The standard protocol included (1) cerebral perfusion pressure higher than 60 mm Hg, (2) colloid solution (10% HES 200/0.5) 1000 mL/d in combination with crystalloid solution, (3) stepwise management of intracranial hypertension. Renal function, coagulation function, and electrolytes were evaluated every other day. The data of intracranial pressure, mean arterial pressure, cerebral perfusion pressure, intake, output, mannitol, complications, and outcome were recorded and analyzed. RESULTS: There were 78 patients, aged 45.61 +/- 21.80 years, in this study. The initial Glasgow Coma Scale score was 6.35 +/- 1.38. Seventy-three patients received operations with intracranial pressure monitoring. Blood transfusion was surgery related (days 1 and 2); otherwise, it was rarely used (P<.05). Prolonged prothrombin time was shown only 7 (2.65%) times of 234 of blood sampling. There was no anaphylactic reaction, pulmonary complications, or renal function deterioration in the course of our observation. The chart review of the patients at 6 months revealed the following: favorable outcome, 55.1%; unfavorable outcome, 33.3%; and mortality, 11.6%. CONCLUSIONS: The 10% HES (200/0.5) can be used in the treatment protocol of severe head injury. There is no definite bleeding complications documented by current dosage of HES. Besides, balanced fluid management can be achieved without causing serious pulmonary complications. However, a further randomized, prospective study is needed to define the actual benefit of HES in fluid management and clinical outcome.

Subdural intracranial pressure monitoring in severe head injury: clinical experience with the Codman MicroSensor.

BACKGROUND: Our main objective was to study the clinical outcome and complications of the subdural ICP monitoring with the CMS (Johnson and Johnson Medical Ltd, Raynhan, MA) in severe head injury. METHODS: A retrospective analysis of patients with head injury with a GCS score of 8 or less was performed. Patients with severe systemic injury with hypotension (systolic blood pressure of <90 mm Hg on admission), a GCS score of 3 with fixed and dilated pupils after resuscitation, a GCS score of 3 to 4 whose family refused aggressive treatment, and those who were dead on arrival were excluded from this study. During the period from January 1997 to April 2004, 120 patients with severe head injuries were included and met criteria for insertion of a subdural ICP monitoring device (CMS). RESULTS: A total of 120 patients (84 males and 36 females), aged 16 to 80 years old (mean, 43.8 +/- 14.4), were enrolled in the study. The average duration of ICP monitoring device use was 7.6 +/- 0.4 days (range, 2-14 days). The overall clinical outcomes of these patients were as follows: mortality rate, 13.5%; percentage of unfavorable outcomes, 17.3%; percentage of favorable outcomes, 69.2%. There were no complications such as CNS infection or hemorrhage in this study. CONCLUSION: A subdural transducer-tipped catheter (CMS) can be used as the first-line equipment for monitoring ICP in patients with severe head injury. The clinical results are similar with other recent studies, but no complication such as infection or hemorrhage occurred in this study.