Nociceptor-immune interactomes reveal insult-specific immune signatures of pain.

Inflammatory pain results from the heightened sensitivity and reduced threshold of nociceptor sensory neurons due to exposure to inflammatory mediators. However, the cellular and transcriptional diversity of immune cell and sensory neuron types makes it challenging to decipher the immune mechanisms underlying pain. Here we used single-cell transcriptomics to determine the immune gene signatures associated with pain development in three skin inflammatory pain models in mice: zymosan injection, skin incision and ultraviolet burn. We found that macrophage and neutrophil recruitment closely mirrored the kinetics of pain development and identified cell-type-specific transcriptional programs associated with pain and its resolution. Using a comprehensive list of potential interactions mediated by receptors, ligands, ion channels and metabolites to generate injury-specific neuroimmune interactomes, we also uncovered that thrombospondin-1 upregulated by immune cells upon injury inhibited nociceptor sensitization. This study lays the groundwork for identifying the neuroimmune axes that modulate pain in diverse disease contexts.

Immune drivers of physiological and pathological pain.

Physiological pain serves as a warning of exposure to danger and prompts us to withdraw from noxious stimuli to prevent tissue damage. Pain can also alert us of an infection or organ dysfunction and aids in locating such malfunction. However, there are instances where pain is purely pathological, such as unresolved pain following an inflammation or injury to the nervous system, and this can be debilitating and persistent. We now appreciate that immune cells are integral to both physiological and pathological pain, and that pain, in consequence, is not strictly a neuronal phenomenon. Here, we discuss recent findings on how immune cells in the skin, nerve, dorsal root ganglia, and spinal cord interact with somatosensory neurons to mediate pain. We also discuss how both innate and adaptive immune cells, by releasing various ligands and mediators, contribute to the initiation, modulation, persistence, or resolution of various modalities of pain. Finally, we propose that the neuroimmune axis is an attractive target for pain treatment, but the challenges in objectively quantifying pain preclinically, variable sex differences in pain presentation, as well as adverse outcomes associated with immune system modulation, all need to be considered in the development of immunotherapies against pain.

Macrophages protect against sensory axon degeneration in diabetic neuropathy.

Diabetic peripheral neuropathy (DPN) is a common complication of diabetes, causing sensory loss and debilitating neuropathic pain 1,2 . Although the onset and progression of DPN have been linked with dyslipidemia and hyperglycemia 3 , the contribution of inflammation in the pathogenesis of DPN has not been investigated. Here, we use a High Fat High Fructose Diet (HFHFD) to model DPN and the diabetic metabolic syndrome in mice. Diabetic mice develop persistent heat hypoalgesia after three months, but a reduction in epidermal skin innervation only manifests at 6 months. Using single-cell sequencing, we find that CCR2+ macrophages infiltrate the sciatic nerves of diabetic mice well before axonal degeneration is detectable. We show that these infiltrating macrophages share gene expression similarities with nerve crush-induced macrophages 4 and express neurodegeneration-associated microglia marker genes 5 although there is no axon loss or demyelination. Inhibiting this macrophage recruitment in diabetic mice by genetically or pharmacologically blocking CCR2 signaling results in a more severe heat hypoalgesia and accelerated skin denervation. These findings reveal a novel neuroprotective recruitment of macrophages into peripheral nerves of diabetic mice that delays the onset of terminal axonal degeneration, thereby reducing sensory loss. Potentiating and sustaining this early neuroprotective immune response in patients represents, therefore, a potential means to reduce or prevent DPN.

Nav1.7 gain-of-function mutation I228M triggers age-dependent nociceptive insensitivity and C-LTMR dysregulation.

Gain-of-function mutations in Scn9a, which encodes the peripheral sensory neuron-enriched voltage-gated sodium channel Nav1.7, cause paroxysmal extreme pain disorder (PEPD), inherited erythromelalgia (IEM), and small fiber neuropathy (SFN). Conversely, loss-of-function mutations in the gene are linked to congenital insensitivity to pain (CIP). These mutations are evidence for a link between altered sodium conductance and neuronal excitability leading to somatosensory aberrations, pain, or its loss. Our previous work in young adult mice with the Nav1.7 gain-of-function mutation, I228M, showed the expected DRG neuron hyperexcitability, but unexpectedly the mice had normal mechanical and thermal behavioral sensitivity. We now show that with aging both male and female mice with this mutation unexpectedly develop a profound insensitivity to noxious heat and cold, as well skin lesions that span the body. Electrophysiology demonstrates that, in contrast to young mice, aged I228M mouse DRGs have a profound loss of sodium conductance and changes in activation and slow inactivation dynamics, representing a loss-of-function. Through RNA sequencing we explored how these age-related changes may produce the phenotypic changes and found a striking and specific decrease in C-low threshold mechanoreceptor- (cLTMR) associated gene expression, suggesting a potential contribution of this DRG neuron subtype to Nav1.7 dysfunction phenotypes. A GOF mutation in a voltage-gated channel can therefore produce over a prolonged time, highly complex and unexpected alterations in the nervous system beyond excitability changes.

Nociceptor neuroimmune interactomes reveal cell type- and injury-specific inflammatory pain pathways.

Inflammatory pain associated with tissue injury and infections, results from the heightened sensitivity of the peripheral terminals of nociceptor sensory neurons in response to exposure to inflammatory mediators. Targeting immune-derived inflammatory ligands, like prostaglandin E2, has been effective in alleviating inflammatory pain. However, the diversity of immune cells and the vast array of ligands they produce make it challenging to systematically map all neuroimmune pathways that contribute to inflammatory pain. Here, we constructed a comprehensive and updatable database of receptor-ligand pairs and complemented it with single-cell transcriptomics of immune cells and sensory neurons in three distinct inflammatory pain conditions, to generate injury-specific neuroimmune interactomes. We identified cell-type-specific neuroimmune axes that are common, as well as unique, to different injury types. This approach successfully predicts neuroimmune pathways with established roles in inflammatory pain as well as ones not previously described. We found that thrombospondin-1 produced by myeloid cells in all three conditions, is a negative regulator of nociceptor sensitization, revealing a non-canonical role of immune ligands as an endogenous reducer of peripheral sensitization. This computational platform lays the groundwork to identify novel mechanisms of immune-mediated peripheral sensitization and the specific disease contexts in which they act.

Severe Traumatic Brain Injury in children-paradigm of decompressive craniectomy compared to a historic cohort.

PURPOSE: Traumatic brain injury (TBI) is one of the leading causes of death and disability in children. Medical therapy remains limited, and decompressive craniectomy (DC) is an established rescue therapy in case of elevated intracranial pressure (ICP). Much discussion deals with clinical outcome after severe TBI treated with DC, while data on the pediatric population is rare. We report our experience of treating severe TBI in two different treatment setups at the same academic institution. METHODS: Forty-eight patients (≤ 16 years) were hospitalized with severe TBI (GCS ≤ 8 points) between 2008 and 2018 in a pediatric intensive care unit (ICU) at a specialized tertiary pediatric care center. Data on treatment, clinical status, and outcome was retrospectively analyzed. Outcome data included Glasgow Outcome Scale (GOS) at 3-, 12-, and 36-month follow-up. Data was compared to a historic cohort with 53 pediatric severe TBI patients treated at the same institution in a neurointensive care unit between 1996 and 2007. Ethical approval was granted (EA2/076/21). RESULTS: Between 2008 and 2018, 11 patients were treated with DC. Compared to the historic cohort, patients were younger and GCS was worse, while in-hospital mortality and clinical outcome remained similar. A trend towards more aggressive EVD placement and the internal paradigm change for treatment in a specialized pediatric ICU was observed. CONCLUSIONS: In children with severe TBI treated over two decades, clinical outcome was comparable and mostly favorable in two different treatment setups. Consequent therapy is warranted to maintain the positive potential for favorable outcome in children with severe TBI.

Sonocatalytic degradation of EDTA in the presence of Ti and Ti@TiO2 nanoparticles.

The sonocatalytic degradation of EDTA (C0 = 5 10-3 M) in aqueous solutions was studied under 345 kHz (Pac = 0.25 W mL-1) ultrasound at 22-51 °C, Ar/20%O2, Ar or air, and in the presence of metallic titanium (Ti0) or core-shell Ti@TiO2 nanoparticles (NPs). Ti@TiO2 NPs have been obtained using simultaneous action of hydrothermal conditions (100-214 °C, autogenic pressure P = 1.0-19.0 bar) and 20 kHz ultrasound, called sonohydrothermal (SHT) treatment, on Ti0 NPs in pure water. Ti0 is composed of quasi-spherical particles (30-150 nm) of metallic titanium coated with a metastable titanium suboxide Ti3O. SHT treatment at 150-214 °C leads to the oxidation of Ti3O and partial oxidation of Ti0 and formation of nanocrystalline shell (10-20 nm) composed of TiO2 anatase. It was found that Ti0 NPs do not exhibit catalytic activity in the absence of ultrasound. Moreover, Ti0 NPs remain inactive under ultrasound in the absence of oxygen. However, significant acceleration of EDTA degradation was achieved during sonication in the presence of Ti0 NPs and Ar/20%O2 gas mixture. Coating of Ti0 with TiO2 nanocrystalline shell reduces sonocatalytic activity. Pristine TiO2 anatase nanoparticles do not show a sonocatalytic activity in studied system. Suggested mechanism of EDTA sonocatalytic degradation involves two reaction pathways: (i) sonochemical oxidation of EDTA by OH/HO2 radicals in solution and (ii) EDTA oxidation at the surface of Ti0 NPs in the presence of oxygen activated by cavitation event. Ultrasonic activation most probably occurs due to the local heating of Ti0/O2 species at cavitation bubble/solution interface.

Mechanisms of Efficacy of the FGFR1-3 Inhibitor AZD4547 in Pediatric Solid Tumor Models.

Children with aggressive pediatric solid tumors have poor outcomes and novel treatments are needed. Pediatric solid tumors demonstrate aberrant expression and activity of the fibroblast growth factor receptor (FGFR) family, suggesting FGFR inhibitors may be effective therapeutic agents. AZD4547 is a multikinase inhibitor of the FGFR1-3 kinases, and we hypothesized that AZD4547 would be effective in pediatric solid tumor preclinical models. We evaluated the effects of AZD4547 on neuroblastoma, rhabdomyosarcoma, and Ewing sarcoma cells alone and in combination with STAT3 inhibition. Continuous live cell imaging was used to measure induction of apoptosis and effects on migration. Receptor inhibition and intracellular signaling were examined by western blotting. AZD4547 treatment resulted in decreased cell confluence, increased apoptosis and reduced cell migration in all tested cell lines. AZD4547 treatment led to decreased phosphorylation of signaling proteins involved in cell survival and apoptotic pathways and increased phosphorylation of STAT3, and treatment of cell lines with AZD4547 combined with STAT3 inhibition demonstrated increased efficacy. Sensitivity to AZD4547 appears to be mediated by effects on the Ras/MAPK and JAK/STAT pathways, and AZD4547 represents a potential novel therapeutic agent for children with solid tumors.

Quantification of 18F-fluorodeoxyglucose uptake to detect residual nodal disease in locally advanced head and neck squamous cell carcinoma after chemoradiotherapy: results from the ECLYPS study.

BACKGROUND: The Hopkins criteria were introduced for nodal response evaluation after therapy in head and neck cancer, but its superiority over quantification is not yet confirmed. METHODS: SUVbody weight thresholds and lesion-to-background ratios were explored in a prospective multicenter study of standardized FDG-PET/CT 12 weeks after CRT in newly diagnosed locally advanced head and neck squamous cell carcinoma (LAHNSCC) patients (ECLYPS). Reference standard was histology, negative FDG-PET/CT at 12 months after treatment or ≥ 2 years of negative follow-up. Area under the receiver operator characteristics curves (AUROC) were estimated and obtained thresholds were validated in an independent cohort of HNSCC patients (n = 127). RESULTS: In ECLYPS, 124 patients were available for quantification. With a median follow-up of 20.4 months, 23 (18.5%) nodal neck recurrences were observed. A SUV70 threshold of 2.2 (AUROC = 0.89; sensitivity = 79.7%; specificity = 80.8%) was identified as optimal metric to identify nodal recurrence within 1 year after therapy. For lesion-to-background ratios, an SUV50/SUVliver threshold of 0.96 (AUROC = 0.89; sensitivity = 79.7%; specificity = 82.8%) had the best performance. Compared with Hopkins criteria (AUROC = 0.81), SUV70 and SUV50/SUVliver provided a borderline significant (p = 0.040 and p = 0.094, respectively) improvement. Validation of thresholds yielded similar AUROC values (SUV70 = 0.93, SUV50/SUVliver = 0.95), and were comparable to the Hopkins score (AUROC = 0.91; not statistically significant). CONCLUSION: FDG quantification detects nodal relapse in LAHNSCC patients. When using EARL standardized PET acquisitions and reconstruction, absolute SUV metrics (SUV70 threshold 2.2) prove robust, yet ratios (SUV50/SUVliver, threshold 0.96) may be more useful in routine clinical care. In this setting, the diagnostic value of quantification is comparable to the Hopkins criteria. TRIAL REGISTRATION: US National Library for Medicine, NCT01179360. Registered 11 August 2010, https://clinicaltrials.gov/ct2/show/NCT01179360.

Unraveling the Plastic Peripheral Neuroimmune Interactome.

Sensory neurons and immune cells share a common microenvironmental niche for surveying tissue integrity. The immune and nervous systems both sense deviations in homeostasis and initiate protective responses and, upon malfunction, also jointly contribute to disease. Barrier tissues are heavily innervated by nociceptors, the sensory neurons that detect noxious stimuli, leading to pain and itch. The same tissues are also home to diverse immune cells that respond to infections and injury. The physical proximity of nociceptors and immune cells allows for direct local interactions between the two, independent of the CNS. We discuss in this study their ligand-receptor-based interactions and propose the need to shift from studying individual neuroimmune interactions to exploring the reciprocal neuroimmune interaction network in its entirety: the "neuroimmune interactome." Identification of the nature of the interactome in health and its plasticity in disease will unravel the functional consequences of interactions between nociceptors and immune cells.

TROPHY registry study design: a prospective, international multicenter study for the surgical treatment of posthemorrhagic hydrocephalus in neonates.

INTRODUCTION: Among children with hydrocephalus, neonates with intraventricular hemorrhage (IVH) and posthemorrhagic hydrocephalus (PH) are considered a group with one of the highest complication rates of treatment. Despite continued progress in neonatal care, a standardized and reliable guideline for surgical management is missing for this challenging condition. Thus, further research is warranted to compare common methods of surgical treatment. The introduction of neuroendoscopic lavage has precipitated the establishment of an international registry aimed at elaborating key elements of a standardized surgical treatment. METHODS: The registry is designed as a multicenter, international, prospective data collection for neonates aged 41 weeks gestation, with an indication for surgical treatment for IVH with ventricular dilatation and progressive hydrocephalus. The following initial temporizing surgical interventions, each used as standard treatment at participating centers, will be compared: external ventricular drainage (EVD), ventricular access device (VAD), ventricular subgaleal shunt (VSGS), and neuroendoscopic lavage (NEL). Type of surgery, perioperative data including complications and mortality, subsequent shunt surgeries, ventricular size, and neurological outcome will be recorded at 6, 12, 36, and 60 months. RESULTS: An online, password-protected website will be used to collect the prospective data in a synchronized manner. As a prospective registry, data collection will be ongoing, with no prespecified endpoint. A prespecified analysis will take place after a total of 100 patients in the NEL group have been entered. Analyses will be performed for safety (6 months), shunt dependency (12, 24 months), and neurological outcome (60 months). CONCLUSION: The design and online platform of the TROPHY registry will enable the collection of prospective data on different surgical procedures for investigation of safety, efficacy, and neurodevelopmental outcome of neonates with IVH and hydrocephalus. The long-term goal is to provide valid data on NEL that is prospective, international, and multicenter. With the comparison of different surgical treatment modalities, we hope to develop better therapy guidelines for this complex neurosurgical condition.

Treatment of Frontal Secondary Headache Attributed to Supratrochlear and Supraorbital Nerve Entrapment With Oral Medication or Botulinum Toxin Type A vs Endoscopic Decompression Surgery.

IMPORTANCE: Endoscopic surgical decompression of the supratrochlear nerve (STN) and supraorbital nerve (SON) is a new treatment for patients with frontal chronic headache who are refractory to standard treatment options. OBJECTIVE: To evaluate and compare treatment outcomes of oral medication, botulinum toxin type A (BoNT/A) injections, and endoscopic decompression surgery in frontal secondary headache attributed to STN and supraorbital SON entrapment. DESIGN, SETTING, AND PARTICIPANTS: Prospective cohort study of 22 patients from a single institution (Diakonessen Hospital Utrecht) with frontal headache of moderate-to-severe intensity (visual analog scale [VAS] score, 7-10), frontally located, experienced more than 15 days per month, and described as pressure or tension that intensifies with pressure on the area of STN and SON. A screening algorithm was used that included examination, questionnaire, computed tomography of the sinus, injections of local anesthetic, and BoNT/A in the corrugator muscle. INTERVENTIONS: Different oral medication therapy for headache encountered in the study cohort, as well as BoNT/A injections (15 IU) into the corrugator muscle. Surgical procedures were performed by a single surgeon using an endoscopic surgical approach to release the supraorbital ridge periosteum and to bluntly dissect the glabellar muscle group. MAIN OUTCOMES AND MEASURES: Headache VAS intensity after oral medication and BoNT/A injections. Additionally, early postoperative follow-up consisted of a daily headache questionnaire that was evaluated after 1 year. RESULTS: In total, 22 patients (mean [SD] age, 42.0 [15.3] years; 7 men and 15 women) were included in this cohort study. Oral medication therapy reduced the headache intensity significantly (mean [standard error of the mean {SEM}] VAS score, 6.45 [0.20] [95% CI, 0.34-3.02; P < .001] compared with mean [SEM] pretreatment VAS score, 8.13 [0.22]). Botulinum toxin type A decreased the mean (SEM) headache intensity VAS scores significantly as well (pretreatment, 8.1 [0.22] vs posttreatment, 2.9 [0.42]; 95% CI, 3.89-6.56; P < .001). The mean (SEM) pretreatment headache intensity VAS score (8.10 [0.22]) decreased significantly after surgery at 3 months (1.30 [0.55]; 95% CI, 5.48-8.16; P < .001) and 12 months (1.09 [0.50]; 95% CI, 5.71-8.38; P < .001). There was a significant decrease of headache intensity VAS score in the surgical group over the BoNT/A group (mean [SEM] VAS score, 2.90 [0.42]) after 3 months (mean [SEM] VAS score, 1.30 [0.55]; 95% CI, 0.25-2.93; P < .001) and 12 months (mean [SEM] VAS score, 1.09 [0.50]; 95% CI, 0.48-3.16; P < .001) after surgery. CONCLUSIONS AND RELEVANCE: Endoscopic decompression surgery had a long-lasting successful outcome in this type of frontal secondary headache. Even though BoNT/A had a positive effect, the effect of surgery was significantly higher. LEVEL OF EVIDENCE: 3.

Experience in shunt management on revision free survival in infants with myelomeningocele.

OBJECTIVE: Depending on the etiology of hydrocephalus in childhood, the shunt therapy still remains challenging due to frequent shunt complications leading to possible revisions such as shunt infection or shunt malfunction. In myelomeningocele (MMC) patients who often require shunt therapy, higher revisions rates were reported. In a single-center retrospective study, experiences on shunt regimen on hydrocephalus associated with MMC are presented. METHODS: Data of 160 infant hydrocephalus cases younger than 1 year of age at the time of implantation were retrospectively reviewed from the hospital database. These patients received an adjustable differential pressure valve with gravitational unit and antibiotic impregnated catheters as a primary or secondary implant during the time period of April 2007 to July 2015. The subgroup of infants cases with MMC (n = 44; age 50.6 ± 80.6 days) were compared to the remaining cohort of other hydrocephalus etiology (control group). The shunt and valve revision free survival rates were recorded until July 2017. RESULTS: During the mean follow-up of 48.7 ± 19.2 (7-114) months, the shunt revision free survival was 87% at 1 year and 49% at 60 months in the MMC cohort. The control group showed a shunt survival rate of 68% at 1 year and 39% at 60 months. Similarly, the valve revision free survival rate showed a significant higher rate of 92% at 1 year and 69% at 60 months in the MMC group compared to the control group (75% at 1 year and 51% at 60 months; p < 0.05). During the entire follow-up period, 37% of the MMC infants underwent a revision operation in contrast to the control group of 40%. CONCLUSION: The presented shunt strategy showed improved revision free survival rates in infants with a MMC-related hydrocephalus in comparison to other etiologies of hydrocephalus in infants, which might relate to infection prophylaxis and high drainage resistance integrated in the shunt system.

Fluorodeoxyglucose-Positron Emission Tomography/Computed Tomography After Concurrent Chemoradiotherapy in Locally Advanced Head-and-Neck Squamous Cell Cancer: The ECLYPS Study.

Purpose To assess the standardized implementation and reporting of surveillance [18F]fluorodeoxyglucose-positron emission tomography/computed tomography (FDG-PET/CT) scan of the neck in locoregionally advanced head-and-neck squamous cell carcinoma (LAHNSCC) after concurrent chemoradiotherapy (CCRT). Patients and Methods We performed a prospective multicenter study of FDG-PET/CT scanning 12 weeks after CCRT in newly diagnosed patients with LAHNSCC (stage IVa/b) that used standardized reconstruction and Hopkins reporting criteria. The reference standard was histology or > 12 months of clinical follow-up. The primary outcome measure was the negative predictive value (NPV) of FDG-PET/CT scans and other supporting diagnostic test characteristics, including time dependency with increasing follow-up time. Results Of 152 patients, 125 had adequate primary tumor control after CCRT and entered follow-up (median, 20.4 months). Twenty-three (18.4%) had residual neck disease. Overall, NPV was 92.1% (95% CI, 86.9% to 95.3%; null hypothesis: NPV = 85%; P = .012) with sensitivity of 65.2% (95% CI, 44.9% to 81.2%), specificity of 91.2% (95% CI, 84.1% to 95.3%), positive predictive value of 62.5% (95% CI, 45.5% to 76.9%), and accuracy of 86.4% (95% CI, 79.3% to 91.3%). Sensitivity was time dependent and high for residual disease manifesting up to 9 months after imaging but lower (59.7%) for disease detected up to 12 months after imaging. Standardized reporting criteria reduced the number of equivocal reports (95% CI for the difference, 2.6% to 15.0%; P = .003). Test characteristics were not improved with the addition of lymph node CT morphology criteria. Conclusion FDG-PET/CT surveillance using Hopkins criteria 12 weeks after CCRT is reliable in LAHNSCC except for late manifesting residual disease, which may require an additional surveillance scan at 1 year after CCRT to be detected.

Interaction of quantitative (18)F-FDG-PET-CT imaging parameters and human papillomavirus status in oropharyngeal squamous cell carcinoma.

BACKGROUND: Patients with human papillomavirus (HPV)-positive oropharyngeal squamous cell carcinoma (SCC) have a better survival than with HPV-negative oropharyngeal SCC. An (18) F-fluorodeoxyglucose positron emission tomography-CT ((18) F-FDG-PET-CT) may also provide prognostic information. We evaluated glycolytic characteristics in HPV-negative and HPV-positive oropharyngeal SCC. METHODS: Forty-four patients underwent pretreatment (18) F-FDG-PET-CT. Standardized uptake values (SUVs) and metabolic active tumor volumes (MATVs) were determined for primary tumors. HPV status was determined with p16 immunostaining, followed by high-risk HPV DNA detection on the positive cases. RESULTS: Twenty-seven patients were HPV-positive (61.4%). Median MATV was 2.8 mL (range = 1.6-5.1 mL) for HPV-positive and 6.0 mL (range = 4.4-18.7 mL) for HPV-negative tumors (p < .001). SUV values are volume dependent (partial volume effect), therefore, MATV was included as covariate in multivariate analysis. In this multivariate analysis, the maximum SUV in HPV-positive tumors was 3.9 units lower than in HPV-negative tumors (p = .01). CONCLUSION: The (18) F-FDG-PET-CT parameters are lower in HPV-positive than in HPV-negative patients. Low pretreatment SUV values in HPV-positive oropharyngeal SCC may be at least partly explained by HPV-induced tumor changes.

Benefits of a short, practical questionnaire to measure subjective perception of nasal appearance after aesthetic rhinoplasty.

BACKGROUND: The authors tested a short, practically designed questionnaire to assess changes in subjective perception of nasal appearance in patients before and after aesthetic rhinoplasty. METHODS: A prospective cohort study was conducted in a group of 121 patients who desired aesthetic rhinoplasty and were operated on by one surgeon. The questionnaire contained five questions (E1-E5) based on a five-point Likert scale and a visual analogue scale (range, 0 to 10). Two questions were designed as trick questions to help the surgeon screen for signs of body dysmorphic disorder. RESULTS: All patients rated the appearance of their nose as improved after surgery. The visual analogue scale revealed a Gaussian curve of normal distribution (range, 0.5 to 10) around a significant improvement (mean, 4.36 points, p = 0.018). Also, question E1, question E2, and the sum of questions E1 through E5 showed a statistically significant improvement after surgery (p = 1.74 × 10, p = 4.29 × 10, and p = 9.23 × 10, respectively). The authors found a linear relationship between preoperative score on the trick questions and postoperative increase in visual analogue scale score. Test-retest reliability could be investigated in 74 of 121 patients (61 percent) and showed a positive correlation between postoperative (1 year after surgery) and repostoperative response (2 to 4 years after surgery). CONCLUSIONS: The authors concluded that a surgeon performing aesthetic rhinoplasty can benefit from using this questionnaire. It is simple, takes no more than 2 minutes to complete, and provides helpful subjective information regarding patients' preoperative nasal appearance and postoperative surgical outcome. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, IV.

Reduced plakoglobin immunoreactivity in arrhythmogenic cardiomyopathy: methodological considerations.

BACKGROUND: Arrhythmogenic cardiomyopathy (AC) primarily is considered to be a desmosomal disease with a predominant right ventricular phenotype. Reduced signal intensity for junctional plakoglobin (JUP) at the intercalated disks has been proposed as a marker that contributes to diagnosis of the disease. In this technical study, we investigated how methodology-related differences caused by tissue preservation and antibody dilutions affect an appropriate diagnosis. METHODS: Autopsy and biopsy material was available from a total of 7 control and 25 AC patients that fulfilled the diagnostic Task Force Criteria as proposed in 2010. Immunohistochemical analysis was performed on cryosections and formalin-fixed material using antibodies against JUP and N-Cadherin. RESULTS: Immunohistochemistry (1:1000 antibody dilution) on formalin-fixed material showed a reduced signal for JUP in 7/10 AC patients in a bidirectional, double-blinded exchange experiment in which 77% of individuals were correctly classified. Unmasking this disturbed JUP pattern was highly dependent on tissue preservation and antibody dilution since on cryosections the disturbed pattern in patients could only be unmasked at a very strong antibody dilution of 1:100000. CONCLUSIONS: Reduced immunoreactive signal of JUP at the intercalated disks can be observed in a majority of AC patients. These changes can comparably be detected on both cryo- (74%) and formalin-fixed material (70%) but demand a different, highly defined, and uniformly used approach.

Remodeling of the cardiac sodium channel, connexin43, and plakoglobin at the intercalated disk in patients with arrhythmogenic cardiomyopathy.

BACKGROUND: Arrhythmogenic cardiomyopathy (AC) is closely associated with desmosomal mutations in a majority of patients. Arrhythmogenesis in patients with AC is likely related to remodeling of cardiac gap junctions and increased levels of fibrosis. Recently, using experimental models, we also identified sodium channel dysfunction secondary to desmosomal dysfunction. OBJECTIVE: To assess the immunoreactive signal levels of the sodium channel protein NaV1.5, as well as connexin43 (Cx43) and plakoglobin (PKG), in myocardial specimens obtained from patients with AC. METHODS: Left and right ventricular free wall postmortem material was obtained from 5 patients with AC and 5 controls matched for age and sex. Right ventricular septal biopsies were taken from another 15 patients with AC. All patients fulfilled the 2010 revised Task Force Criteria for the diagnosis of AC. Immunohistochemical analyses were performed using antibodies against Cx43, PKG, NaV1.5, plakophilin-2, and N-cadherin. RESULTS: N-cadherin and desmoplakin immunoreactive signals and distribution were normal in patients with AC compared to controls. Plakophilin-2 signals were unaffected unless a plakophilin-2 mutation predicting haploinsufficiency was present. Distribution was unchanged compared to that in controls. Immunoreactive signal levels of PKG, Cx43, and NaV1.5 were disturbed in 74%, 70%, and 65% of the patients, respectively. CONCLUSIONS: A reduced immunoreactive signal of PKG, Cx43, and NaV1.5 at the intercalated disks can be observed in a large majority of the patients. Decreased levels of Nav1.5 might contribute to arrhythmia vulnerability and, in the future, potentially could serve as a new clinically relevant tool for risk assessment strategies.