Recovery after human bone marrow mesenchymal stem cells (hBM-MSCs)-derived extracellular vesicles (EVs) treatment in post-MCAO rats requires repeated handling.

Rehabilitation is the only current intervention that improves sensorimotor function in ischemic stroke patients, similar to task-specific intensive training in animal models of stroke. Bone marrow mesenchymal stem cells (BM-MSCs)-derived extracellular vesicles (EVs) are promising in restoring brain damage and function in stroke models. Additionally, the non-invasive intranasal route allows EVs to reach the brain and target specific ischemic regions. Yet unclear is how handling might enhance recovery or influence other therapies such as EVs after stroke. We used the transient middle cerebral artery occlusion (MCAO) model of stroke in rats to assess how intensive handling alone, in the form of sensorimotor behavioral tests, or in combination with an intranasal treatment of EVs restored neurological function and ischemic damage. Handled rats were exposed to a battery of sensorimotor tests, including the modified Neurological Severity Score (mNSS), beam balance, corner, grid walking, forelimb placement, and cylinder tests, together with Magnetic Resonance Imaging (MRI) at 2, 7, 14, 21, and 28 days post-stroke (dps). Handled MCAO rats were also exposed to an intranasal multidose or single dose of EVs. Non-handled rats were evaluated only by mNSS and MRI at 2, 28, and 56 dps and were treated with a single intranasal dose of EVs. Our results showed that handling animals after MCAO is necessary for EVs to work at the tested dose and frequency, and that a single cumulative dose of EVs further improves the neurological function recovered during handling. These results show the importance of rehabilitation in combination with other treatments such as EVs, and highlight how extensive behavioral testing might influence functional recovery after stroke.

Interleukin-1ß orchestrates underlying inflammatory responses in microglia via Krüppel-like factor 4.

Microglia are the resident macrophages of the CNS, which secrete several pro- and anti-inflammatory cyto-chemokines including interleukin-1ß (IL-1ß), in response to pathogenic stimuli. Once secreted, IL-1ß binds to IL-1 receptor present on microglia and initiates the production of inflammatory cytokines in microglia. However, the detailed information regarding the molecular mechanisms of IL-1ß triggered inflammatory pathways in microglia is lacking. Our studies focused on the role of Krüppel-like factor 4 (Klf4) in mediating the regulation of pro-inflammatory gene expression upon IL-1ß stimulation in microglia. Our studies show that stimulation of microglia with IL-1ß robustly induces Klf4 via PI3K/Akt pathway which positively regulates the production of endogenous IL-1ß as well as other pro-inflammatory markers, cyclooxygenase-2, monocyte chemoattractant protein-1 and interleukin-6 (IL-6). In addition, we report that Klf4 negatively regulates the expression of inducible nitric oxide synthase, thereby playing a key role in regulating the immunomodulatory activities of microglia.

Ultrasound-guided anterior suprascapular nerve block versus interscalene brachial plexus block for arthroscopic shoulder surgery: A randomised controlled study.

Background and Aims: The interscalene brachial plexus block (ISB) affects the phrenic nerve, resulting in hemi-diaphragmatic paresis (HDP) and, possibly, respiratory distress. Suprascapular nerve block via an anterior approach (SSB-A) is performed more distally at the level of the trunk of the brachial plexus and, thus, may spare the phrenic nerve. This study compares the analgesic efficacy and decline of hemi-diaphragmatic excursion (HDE) following ultrasound (US)-guided SSB-A versus ISB for arthroscopic shoulder surgery. Methods: This study was conducted on 60 adult participants undergoing arthroscopic shoulder surgery under general anaesthesia. Both US-guided SSB-A (n = 30) and ISB (n = 30) were performed with a combination of 10 ml bupivacaine (0.5%) and 4 mg dexamethasone. The primary objective was to compare the duration of analgesia (time to first rescue analgesia), and secondary objectives were to compare 24-h postoperative numerical rating scale (NRS) scores, 24-h morphine consumption and post block change in HDE, and pulmonary function tests (PFTs) between the two groups. For analysing intergroup differences of NRS, HDE and PFT; Pearson's Chi-squared test or Fisher's exact test, unpaired t test, and Mann-Whitney U test were used. For intragroup differences, paired t test was used. A P value <0.05 was considered significant. Results: The duration of analgesia (mean ± Standard Deviation) was similar in two groups (SSB-A = 1,345 ± 182 min, ISB = 1,375 ± 156 min; P = 0.8). The reduction in HDE was significantly greater in the ISB group (44%) than in the SSB-A group (10%). Pulmonary function was better preserved in the SSB-A group. Conclusion: Compared to ISB, SSB-A has a similar analgesic efficacy for arthroscopic shoulder surgeries, but it is superior in preserving diaphragmatic function and pulmonary function.

Therapeutic targeting of Krüppel-like factor 4 abrogates microglial activation.

BACKGROUND: Neuroinflammation occurs as a result of microglial activation in response to invading micro-organisms or other inflammatory stimuli within the central nervous system. According to our earlier findings, Krüppel-like factor 4 (Klf4), a zinc finger transcription factor, is involved in microglial activation and subsequent release of proinflammatory cytokines, tumor necrosis factor alpha, macrophage chemoattractant protein-1 and interleukin-6 as well as proinflammatory enzymes, inducible nitric oxide synthase and cyclooxygenase-2 in lipopolysaccharide-treated microglial cells. Our current study focuses on finding the molecular mechanism of the anti-inflammatory activities of honokiol in lipopolysaccharide-treated microglia with emphasis on the regulation of Klf4. METHODS: For in vitro studies, mouse microglial BV-2 cell lines as well as primary microglia were treated with 500 ng/mL lipopolysaccharide as well as 1 µM and 10 µM of honokiol. We cloned full-length Klf4 cDNA in pcDNA3.1 expression vector and transfected BV-2 cells with this construct using lipofectamine for overexpression studies. For in vivo studies, brain tissues were isolated from BALB/c mice treated with 5 mg/kg body weight of lipopolysaccharide either with or without 2.5 or 5 mg/kg body weight of honokiol. Expression of Klf4, cyclooxygenase-2, inducible nitric oxide synthase and phospho-nuclear factor-kappa B was measured using immunoblotting. We also measured the levels of cytokines, reactive oxygen species and nitric oxide in different conditions. RESULTS: Our findings suggest that honokiol can substantially downregulate the production of proinflammatory cytokines and inflammatory enzymes in lipopolysaccharide-stimulated microglia. In addition, honokiol downregulates lipopolysaccharide-induced upregulation of both Klf4 and phospho-nuclear factor-kappa B in these cells. We also found that overexpression of Klf4 in BV-2 cells suppresses the anti-inflammatory action of honokiol. CONCLUSIONS: Honokiol potentially reduces inflammation in activated microglia in a Klf4-dependent manner.

Krüppel-like factor 4, a novel transcription factor regulates microglial activation and subsequent neuroinflammation.

BACKGROUND: Activation of microglia, the resident macrophages of the central nervous system (CNS), is the hallmark of neuroinflammation in neurodegenerative diseases and other pathological conditions associated with CNS infection. The activation of microglia is often associated with bystander neuronal death. Nuclear factor-κB (NF-κB) is one of the important transcription factors known to be associated with microglial activation which upregulates the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (Cox-2) and other pro-inflammatory cytokines. Recent studies have focused on the role of Krüppel-like factor 4 (Klf4), one of the zinc-finger transcription factors, in mediating inflammation. However, these studies were limited to peripheral system and its role in CNS is not understood. Our studies focused on the possible role of Klf4 in mediating CNS inflammation. METHODS: For in vitro studies, mouse microglial BV-2 cell lines were treated with 500 ng/ml Salmonella enterica lipopolysacchride (LPS). Brain tissues were isolated from BALB/c mice administered with 5 mg/kg body weight of LPS. Expressions of Klf4, Cox-2, iNOS and pNF-κB were evaluated using western blotting, quantitative real time PCR, and reverse transcriptase polymerase chain reactions (RT-PCRs). Klf4 knockdown was carried out using SiRNA specific for Klf4 mRNA and luciferase assays and electromobility shift assay (EMSA) were performed to study the interaction of Klf4 to iNOS promoter elements in vitro. Co-immunoprecipitation of Klf4 and pNF-κB was done in order to study a possible interaction between the two transcription factors. RESULTS: LPS stimulation increased Klf4 expression in microglial cells in a time- and dose-dependent manner. Knockdown of Klf4 resulted in decreased levels of the pro-inflammatory cytokines TNF-α, MCP-1 and IL-6, along with a significant decrease in iNOS and Cox-2 expression. NO production also decreased as a result of Klf4 knockdown. We found that Klf4 can potentially interact with pNF-κB and is important for iNOS and Cox-2 promoter activity in vitro. CONCLUSIONS: These studies demonstrate the role of Klf4 in microglia in mediating neuroinflammation in response to the bacterial endotoxin LPS.

Inflammasome signaling at the heart of central nervous system pathology.

Neuroinflammation is a complex innate response of neural tissue against harmful effects of diverse stimuli viz., pathogens, damaged cells and irritants within the Central Nervous System (CNS). Studies show that multiple inflammatory mediators including cytokines, chemokines and prostaglandins are elevated in the Cerebrospinal Fluid (CSF) and in post-mortem brain tissues of patients with history of neuroinflammatory conditions as well as neurodegenerative disorders like Alzheimer's disease, Parkinson's disease and Multiple Sclerosis. The innate immunity mediators in the brain, namely microglia and astrocytes, express certain Pattern Recognition Receptors (PRRs), which are always on 'high-alert' for pathogens or other inflammatory triggers and participate in the assembly and activation of the inflammasome. The inflammasome orchestrates the activation of the precursors of proinflammatory caspases, which in turn, cleave the precursor forms of interleukin-1beta, IL-18 and IL-33 into their active forms; the secretion of which leads to a potent inflammatory response, and/or influences the release of toxins from glial and endothelial cells. Altered expression of inflammasome mediators can either promote or inhibit neurodegenerative processes. Therefore, modulating the inflammasome machinery seems a better combat strategy than summarily suppressing all inflammation in most neuroinflammatory conditions. In the current review we have surveyed the identified triggers and pathways of inflammasome activation and the following events which ultimately accomplish the innate inflammatory response in the CNS, with a goal to provide an analytical insight into disease pathogenesis that might provide cues for devising novel therapeutic strategies.

Posterior migration of Ahmed glaucoma valve tube in a patient with Reiger anomaly: a case report.

BACKGROUND: To describe, a yet non-documented complication of GDI surgery (glaucoma drainage incision surgery)--anterior to posterior segment migration of Ahmed Glaucoma Valve (AGV) tube. CASE PRESENTATION: We report a young 9 year old boy, diagnosed with refractory glaucoma with Reiger anomaly. History included of poor vision in both eyes, left more than right with glare since childhood. He underwent GDI surgery with AGV implantation following which he developed posterior migration of AGV tube. The detailed ocular history, ophthalmic findings, clinical course, surgical management and development of the posterior tube migration is discussed. CONCLUSION: Posterior Migration of AGV tube has yet not been described. Also there is a role of expectant management of the complication in this case as evidenced by the benign course of events.

Human protein reference database--2006 update.

Human Protein Reference Database (HPRD) (http://www.hprd.org) was developed to serve as a comprehensive collection of protein features, post-translational modifications (PTMs) and protein-protein interactions. Since the original report, this database has increased to >20 000 proteins entries and has become the largest database for literature-derived protein-protein interactions (>30 000) and PTMs (>8000) for human proteins. We have also introduced several new features in HPRD including: (i) protein isoforms, (ii) enhanced search options, (iii) linking of pathway annotations and (iv) integration of a novel browser, GenProt Viewer (http://www.genprot.org), developed by us that allows integration of genomic and proteomic information. With the continued support and active participation by the biomedical community, we expect HPRD to become a unique source of curated information for the human proteome and spur biomedical discoveries based on integration of genomic, transcriptomic and proteomic data.

pH-responsive and enzymatically-responsive hydrogel microparticles for the oral delivery of therapeutic proteins: Effects of protein size, crosslinking density, and hydrogel degradation on protein delivery.

Two potential platform technologies for the oral delivery of protein therapeutics were synthesized and tested. pH-responsive poly(itaconic acid-co-N-vinyl-2-pyrrolidone) (P(IA-co-NVP)) hydrogel microparticles were tested in vitro with model proteins salmon calcitonin, urokinase, and rituximab to determine the effects of particle size, protein size, and crosslinking density on oral delivery capability. Particle size showed no significant effect on overall delivery potential but did improve percent release of encapsulated protein over the micro-scale particle size range studied. Protein size was shown to have a significant impact on the delivery capability of the P(IA-co-NVP) hydrogel. We show that when using P(IA-co-NVP) hydrogel microparticles with 3 mol% tetra(ethylene glycol) dimethacrylate crosslinker, a small polypeptide (salmon calcitonin) loads and releases up to 45 µg/mg hydrogel while the mid-sized protein urokinase and large monoclonal antibody rituximab load and release only 19 and 24 µg/mg hydrogel, respectively. We further demonstrate that crosslinking density offers a simple method for tuning hydrogel properties to variously sized proteins. Using 5 mol% TEGDMA crosslinker offers optimal performance for the small peptide, salmon calcitonin, whereas lower crosslinking density of 1 mol% offers optimal performance for the much larger protein rituximab. Finally, an enzymatically-degradable hydrogels of P(MAA-co-NVP) crosslinked with the peptide sequence MMRRRKK were synthesized and tested in simulated gastric and intestinal conditions. These hydrogels offer ideal loading and release behavior, showing no degradative release of encapsulated salmon calcitonin in gastric conditions while yielding rapid and complete release of encapsulated protein within 1h in intestinal conditions.

Bilateral familial vertical Duane Syndrome with synergistic convergence, aberrant trigeminal innervation, and facial hypoplasia.

A 5-year-old girl presented with bilateral familial vertical Duane retraction syndrome with alternating esotropia, elevation deficit, Marcus gunn phenomenon, and facial hypoplasia. Abnormal adducting downshoots on attempting abduction suggestive of a synergistic convergence were noted. Hypothesis suggests aberrant innervations or peripheral anatomic connections between inferior and medial recti.

NLRP3 inflammasome: key mediator of neuroinflammation in murine Japanese encephalitis.

BACKGROUND: Japanese Encephalitis virus (JEV) is a common cause of acute and epidemic viral encephalitis. JEV infection is associated with microglial activation resulting in the production of pro-inflammatory cytokines including Interleukin-1 ß (IL-1ß) and Interleukin-18 (IL-18). The Pattern Recognition Receptors (PRRs) and the underlying mechanism by which microglia identify the viral particle leading to the production of these cytokines is unknown. METHODOLOGY/PRINCIPAL FINDINGS: For our studies, we have used murine model of JEV infection as well as BV-2 mouse microglia cell line. In this study, we have identified a signalling pathway which leads to the activation of caspase-1 as the key enzyme responsible for the maturation of both IL-1ß and IL-18 in NACHT, LRR and PYD domains-containing protein-3 (NLRP3) dependent manner. Depletion of NLRP3 results in the reduction of caspase-1 activity and subsequent production of these cytokines. CONCLUSION/SIGNIFICANCE: Our results identify a mechanism mediated by Reactive Oxygen Species (ROS) production and potassium efflux as the two danger signals that link JEV infection to caspase-1 activation resulting in subsequent IL-1ß and IL-18 maturation.

Microglial response to viral challenges: every silver lining comes with a cloud.

Microglia, the resident macrophages of the Central Nervous System (CNS) mediate key innate immune responses against foreign invasions within the CNS and clear the debris after any damage to the nearby tissue. Blood Brain Barrier (BBB) segregates the CNS from the rest of the lymphatic system and prevents the entry of foreign molecules into the brain. Pathogens still cross the BBB via different mechanisms and can cause severe infections of the CNS. Viral encephalitis is the most common form of brain infection and the causative agents include Japanese Encephalitis Virus (JEV), West Nile Virus (WNV), Murray Valley Encephalitis Virus (MVEV), Herpes Simplex Virus (HSV), Human Immunodeficiency Virus (HIV), Cytomegalovirus (CMV) and Hepatitis C Virus (HCV) among several others. Microglia expresses various Pattern Recognition Receptors (PRRs) to identify viral signatures called Pathogen Associated Molecular Patterns (PAMPs) to which microglia respond by releasing several pro and anti-inflammatory cytokines like MCP1, IL-1beta, Type I IFN, IFN-gamma, TNF-alpha etc. This review discusses the various viral infections of the brain and strategies employed by microglia to detect them.

Atypical Duane's retraction syndrome: congenital adduction palsy with synergistic divergence in association with aberrant trigeminal innervation and facial hypoplasia.

The authors describe the atypical and unique features of trigeminal-oculomotor synkinesis in a 6-year-old boy with left congenital adduction palsy and synergistic divergence with facial hypoplasia. Adducting movements of the left eye were also seen on mastication. To the best of the authors' knowledge, a case of this nature has not previously been reported. This case illustrates the absence of changes in palpebral aperture and globe retraction on attempted adduction and also the complete absence of any abduction deficit of the involved eye. Additionally, presence of adducting movements on mastication suggests a trigemino-oculomotor synkinesis. Hypothesis favors an anomalous innervation of the medial rectus muscle from the motor branch of the trigeminal nerve that innervates the external pterygoids.

Esophageal replacement in the neonatal period in infants with esophageal atresia and tracheoesophageal fistula.

AIM: The aim of the study was to assess the outcome after esophageal replacement using gastric pull-up performed in critically ill neonates with esophageal atresia (EA) and tracheoesophageal fistula. METHODS: During 1998 to 2005, gastric transposition was performed in 27 neonates (mean birth weight, 2.32 kg [1.86-3.0 kg]; mean age, 6.08 days) for post-EA and tracheoesophageal fistula leaks in 17, long gap in 6, and pure EA in 4, using transhiatal route in all. Pyloromyotomy as the drainage procedure was added for all 27 neonates. Patients were followed up at 3, 6, and 12 months for clinical evaluation, gastric clearance, duodenogastric reflux, and gastric pressure profile. RESULTS: Six neonates had ongoing serious chest infection, 3 had lung collapse, and 2 had associated congenital heart disease. Postoperative elective ventilation was provided to all neonates for 2 to 40 days (mean, 10.6 days). Nine neonates developed postoperative leaks in the neck; all healed spontaneously before discharge. Mean hospital stay was 32.6 days (range, 9-87 days). Four newborns died on postoperative days 9, 13, 15, and 29 because of existing severe sepsis in 3 and major congenital heart disease in 1. Functional evaluations were done at 3, 6, and 12 months postoperatively. Values at 6 months revealed normal gastric emptying in 16 of 23, presence of duodenal gastric reflux in 11 of 23, and mass contractions with significant rise in intragastric pressure after bolus feeds in 16 of 23 cases. Values at 12 months revealed normal gastric emptying in 14 of 20, presence of duodenal gastric reflux in 8 of 20, and mass contractions with significant rise in intragastric pressure after bolus feeds in 13 (65%) of 20 cases. CONCLUSION: Gastric transposition could be a lifesaving alternative to diversion, even in the critically ill newborns after major leaks. However, it requires technical surgical expertise and an effective pain relief and neonatal intensive care.