Optogenetic Spreading Depolarizations Do Not Worsen Acute Ischemic Stroke Outcome.

BACKGROUND: Spreading depolarizations (SDs) are believed to contribute to injury progression and worsen outcomes in focal cerebral ischemia because exogenously induced SDs have been associated with enlarged infarct volumes. However, previous studies used highly invasive methods to trigger SDs that can directly cause tissue injury (eg, topical KCl) and confound the interpretation. Here, we tested whether SDs indeed enlarge infarcts when induced via a novel, noninjurious method using optogenetics. METHODS: Using transgenic mice expressing channelrhodopsin-2 in neurons (Thy1-ChR2-YFP), we induced 8 optogenetic SDs to trigger SDs noninvasively at a remote cortical location in a noninjurious manner during 1-hour distal microvascular clip or proximal an endovascular filament occlusion of the middle cerebral artery. Laser speckle imaging was used to monitor cerebral blood flow. Infarct volumes were then quantified at 24 or 48 hours. RESULTS: Infarct volumes in the optogenetic SD arm did not differ from the control arm in either distal or proximal middle cerebral artery occlusion, despite a 6-fold and 4-fold higher number of SDs, respectively. Identical optogenetic illumination in wild-type mice did not affect the infarct volume. Full-field laser speckle imaging showed that optogenetic stimulation did not affect the perfusion in the peri-infarct cortex. CONCLUSIONS: Altogether, these data show that SDs induced noninvasively using optogenetics do not worsen tissue outcomes. Our findings compel a careful reexamination of the notion that SDs are causally linked to infarct expansion.

Efficacy profile of noninvasive vagus nerve stimulation on cortical spreading depression susceptibility and the tissue response in a rat model.

BACKGROUND: Noninvasive vagus nerve stimulation (nVNS) has recently emerged as a promising therapy for migraine. We previously demonstrated that vagus nerve stimulation inhibits cortical spreading depression (CSD), the electrophysiological event underlying migraine aura and triggering headache; however, the optimal nVNS paradigm has not been defined. METHODS: Various intensities and doses of nVNS were tested to improve efficacy on KCl-evoked CSD frequency and electrical threshold of CSD in a validated rat model. Chronic efficacy was evaluated by daily nVNS delivery for four weeks. We also examined the effects of nVNS on neuroinflammation and trigeminovascular activation by western blot and immunohistochemistry. RESULTS: nVNS suppressed susceptibility to CSD in an intensity-dependent manner. Two 2-minute nVNS 5 min apart afforded the highest efficacy on electrical CSD threshold and frequency of KCl-evoked CSD. Daily nVNS for four weeks did not further enhance efficacy over a single nVNS 20 min prior to CSD. The optimal nVNS also attenuated CSD-induced upregulation of cortical cyclooxygenase-2, calcitonin gene-related peptide in trigeminal ganglia, and c-Fos expression in trigeminal nucleus caudalis. CONCLUSIONS: Our study provides insight on optimal nVNS parameters to suppress CSD and suggests its benefit on CSD-induced neuroinflammation and trigeminovascular activation in migraine treatment.

Anti-migraine Calcitonin Gene-Related Peptide Receptor Antagonists Worsen Cerebral Ischemic Outcome in Mice.

OBJECTIVE: Calcitonin gene-related peptide (CGRP) pathway inhibitors are emerging treatments for migraine. CGRP-mediated vasodilation is, however, a critical rescue mechanism in ischemia. We, therefore, investigated whether gepants, small molecule CGRP receptor antagonists, worsen cerebral ischemia. METHODS: Middle cerebral artery was occluded for 12 to 60 minutes in mice. We compared infarct risk and volumes, collateral flow, and neurological deficits after pretreatment with olcegepant (single or 10 daily doses of 0.1-1mg/kg) or rimegepant (single doses of 10-100mg/kg) versus vehicle. We also determined their potency on CGRP-induced relaxations in mouse and human vessels, in vitro. RESULTS: Olcegepant (1mg/kg, single dose) increased infarct risk after 12- to 20-minute occlusions mimicking transient ischemic attacks (14/19 vs 6/18 with vehicle, relative risk = 2.21, p < 0.022), and doubled infarct volumes (p < 0.001) and worsened neurological deficits (median score = 9 vs 5 with vehicle, p = 0.008) after 60-minute occlusion. Ten daily doses of 0.1 to 1mg/kg olcegepant yielded similar results. Rimegepant 10mg/kg increased infarct volumes by 60% after 20-minute ischemia (p = 0.03); 100mg/kg caused 75% mortality after 60-minute occlusion. In familial hemiplegic migraine type 1 mice, olcegepant 1mg/kg increased infarct size after 30-minute occlusion (1.6-fold, p = 0.017). Both gepants consistently diminished collateral flow and reduced reperfusion success. Olcegepant was 10-fold more potent than rimegepant on CGRP-induced relaxations in mouse aorta. INTERPRETATION: Gepants worsened ischemic stroke in mice via collateral dysfunction. CGRP pathway blockers might thus aggravate coincidental cerebral ischemic events. The cerebrovascular safety of these agents must therefore be better delineated, especially in patients at increased risk of ischemic events or on prophylactic CGRP inhibition. ANN NEUROL 2020;88:771-784.

Adipokines as Immune Cell Modulators in Multiple Sclerosis.

Multiple sclerosis (MS), a chronic inflammatory and demyelinating disease of the central nervous system (CNS), is a major clinical and societal problem, which has a tremendous impact on the life of patients and their proxies. Current immunomodulatory and anti-inflammatory therapies prove to be relatively effective; however, they fail to concomitantly stop ongoing neurological deterioration and do not reverse acquired disability. The proportion to which genetic and environmental factors contribute to the etiology of MS is still incompletely understood; however, a recent association between MS etiology and obesity was shown, with obesity greatly increasing the risk of developing MS. An altered balance of adipokines, which are white adipose tissue (WAT) hormones, plays an important role in the low-grade chronic inflammation during obesity by their pervasive modification of local and systemic inflammation. Vice versa, inflammatory factors secreted by immune cells affect adipokine function. To explore the role of adipokines in MS pathology, we will here review the reciprocal effects of adipokines and immune cells and summarize alterations in adipokine levels in MS patient cohorts. Finally, we will discuss proof-of-concept studies demonstrating the therapeutic potential of adipokines to target both neuroinflammation and neurodegeneration processes in MS.

Increased Mortality and Vascular Phenotype in a Knock-In Mouse Model of Retinal Vasculopathy With Cerebral Leukoencephalopathy and Systemic Manifestations.

Background and Purpose- Retinal vasculopathy with cerebral leukoencephalopathy and systemic manifestations (RVCL-S) is an autosomal dominant small vessel disease caused by C-terminal frameshift mutations in the TREX1 gene that encodes the major mammalian 3' to 5' DNA exonuclease. RVCL-S is characterized by vasculopathy, especially in densely vascularized organs, progressive retinopathy, cerebral microvascular disease, white matter lesions, and migraine, but the underlying mechanisms are unknown. Methods- Homozygous transgenic RVCL-S knock-in mice expressing a truncated Trex1 (three prime repair exonuclease 1) protein (similar to what is seen in patients) and wild-type littermates, of various age groups, were subjected to (1) a survival analysis, (2) in vivo postocclusive reactive hyperemia and ex vivo Mulvany myograph studies to characterize the microvascular and macrovascular reactivity, and (3) experimental stroke after transient middle cerebral artery occlusion with neurological deficit assessment. Results- The mutant mice show increased mortality starting at midlife (P=0.03 with hazard ratio, 3.14 [95% CI, 1.05-9.39]). The mutants also show a vascular phenotype as evidenced by attenuated postocclusive reactive hyperemia responses (across all age groups; F[1, 65]=5.7, P=0.02) and lower acetylcholine-induced relaxations in aortae (in 20- to 24-month-old mice; RVCL-S knock-in: Emax: 37±8% versus WT: Emax: 65±6%, P=0.01). A vascular phenotype is also suggested by the increased infarct volume seen in 12- to 14-month-old mutant mice at 24 hours after infarct onset (RVCL-S knock-in: 75.4±2.7 mm3 versus WT: 52.9±5.6 mm3, P=0.01). Conclusions- Homozygous RVCL-S knock-in mice show increased mortality, signs of abnormal vascular function, and increased sensitivity to experimental stroke and can be instrumental to investigate the pathology seen in patients with RVCL-S.

Migraine and Cerebrovascular Atherosclerosis in Patients With Ischemic Stroke.

BACKGROUND AND PURPOSE: Migraine is a well-established risk factor for ischemic stroke, but migraine is also related to other vascular diseases. This study aims to investigate the association between migraine and cerebrovascular atherosclerosis in patients with acute ischemic stroke. METHODS: We retrieved data on patients with ischemic stroke from the DUST (Dutch Acute Stroke Study). Migraine history was assessed with a migraine screener and confirmed by telephone interview based on the ICHD criteria (International Classification of Headache Disorders). We assessed intra- and extracranial atherosclerotic changes and quantified intracranial internal carotid artery calcifications as measure of atherosclerotic burden on noncontrast computed tomography and computed tomographic angiography. We calculated risk ratios with adjustments for possible confounders with multivariable Poisson regression analyses. RESULTS: We included 656 patients, aged 18 to 99 years, of whom 53 had a history of migraine (29 with aura). Patients with migraine did not have more frequent atherosclerotic changes in intracranial (51% versus 74%; adjusted risk ratio, 0.82; 95% confidence interval, 0.64-1.05) or extracranial vessels (62% versus 79%; adjusted risk ratio, 0.93; 95% confidence interval, 0.77-1.12) than patients without migraine and had comparable internal carotid artery calcification volumes (largest versus medium and smallest volume tertile, 23% versus 35%; adjusted risk ratio, 0.93; 95% confidence interval, 0.57-1.52). CONCLUSIONS: Migraine is not associated with excess atherosclerosis in large vessels in patients with acute ischemic stroke. Our findings suggest that the biological mechanisms by which migraine results in ischemic stroke are not related to macrovascular cerebral atherosclerosis.

Funnel-freezing versus heat-stabilization for the visualization of metabolites by mass spectrometry imaging in a mouse stroke model.

Tissue preparation is the key to a successful matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) experiment. Rapid post-mortem changes contribute a significant challenge to the use of MSI approaches for the analysis of peptides and metabolites. In this technical note we aimed to compare the tissue fixation method ex-vivo heat-stabilization with in-situ funnel-freezing in a middle cerebral artery occlusion (MCAo) mouse model of stroke, which causes profound alterations in metabolite concentrations. The influence of the duration of the thaw-mounting of the tissue sections on metabolite stability was also determined. We demonstrate improved stability and biomolecule visualization when funnel-freezing was used to sacrifice the mouse compared with heat-stabilization. Results were further improved when funnel-freezing was combined with fast thaw-mounting of the brain sections.

The novel ROCK2 selective inhibitor NRL-1049 preserves the blood-brain barrier after acute injury.

Endothelial blood-brain barrier (BBB) dysfunction is critical in the pathophysiology of brain injury. Rho-associated protein kinase (ROCK) activation disrupts BBB integrity in the injured brain. We aimed to test the efficacy of a novel ROCK2 inhibitor in preserving the BBB after acute brain injury. We characterized the molecular structure and pharmacodynamic and pharmacokinetic properties of a novel selective ROCK2 inhibitor, NRL-1049, and its first metabolite, 1-hydroxy-NRL-1049 (referred to as NRL-2017 hereon) and tested the efficacy of NRL-1049 on the BBB integrity in rodent models of acute brain injury. Our data show that NRL-1049 and NRL-2017 both inhibit ROCK activity and are 44-fold and 17-fold more selective towards ROCK2 than ROCK1, respectively. When tested in a mouse model of cortical cryoinjury, NRL-1049 significantly attenuated the increase in water content. Interestingly, 60% of the mice in the vehicle arm developed seizures within 2 hours after cryoinjury versus none in the NRL-1049 arm. In spontaneously hypertensive rats, NRL-1049 attenuated the dramatic surge in Evans Blue extravasation compared with the vehicle arm after transient middle cerebral artery occlusion. Hemorrhagic transformation was also reduced. We show that NRL-1049, a selective ROCK2 inhibitor, is a promising drug candidate to preserve the BBB after brain injury.

Non-invasively triggered spreading depolarizations induce a rapid pro-inflammatory response in cerebral cortex.

Cortical spreading depolarization (CSD) induces pro-inflammatory gene expression in brain tissue. However, previous studies assessing the relationship between CSD and inflammation have used invasive methods that directly trigger inflammation. To eliminate the injury confounder, we induced CSDs non-invasively through intact skull using optogenetics in Thy1-channelrhodopsin-2 transgenic mice. We corroborated our findings by minimally invasive KCl-induced CSDs through thinned skull. Six CSDs induced over 1 h dramatically increased cortical interleukin-1ß (IL-1ß), chemokine (C-C motif) ligand 2 (CCL2), and tumor necrosis factor-α (TNF-α) mRNA expression peaking around 1, 2 and 4 h, respectively. Interleukin-6 (IL-6) and intercellular adhesion molecule-1 (ICAM-1) were only modestly elevated. A single CSD also increased IL-1ß, CCL2, and TNF-α, and revealed an ultra-early IL-1ß response within 10 min. The response was blunted in IL-1 receptor-1 knockout mice, implicating IL-1ß as an upstream mediator, and suppressed by dexamethasone, but not ibuprofen. CSD did not alter systemic inflammatory indices. In summary, this is the first report of pro-inflammatory gene expression after non-invasively induced CSDs. Altogether, our data provide novel insights into the role of CSD-induced neuroinflammation in migraine headache pathogenesis and have implications for the inflammatory processes in acute brain injury where numerous CSDs occur for days.

A clinical decision support system to improve adequate dosing of gentamicin and vancomycin.

OBJECTIVE: Gentamicin and vancomycin meet the criteria for measuring plasma concentrations during therapy. However, compliance with the accompanying guidelines remains low. The primary objective of this study was to determine whether the implementation of a clinical decision support system, which displays an alert if a plasma concentration should be measured and a daily reviewed patient list resulted in improved compliance. MATERIALS AND METHODS: This intervention study was performed at the Spaarne Gasthuis, Haarlem/Hoofddorp, the Netherlands. The authors included 261 treatments with either gentamicin or vancomycin intravenously for at least 48 h in the year before and after implementation of the clinical decision support system in May 2015. The authors analyzed whether plasma concentrations were measured sooner and more frequently after the implementation, and determined whether the time until the correct dosage, with adequate drug concentrations, was reduced after implementation. RESULTS: Before implementation, plasma concentrations were measured within 72 h in 47% of the treatments. After implementation, this percentage increased to 80% (p < 0.01). After implementation, the time was significantly shorter until the correct dosage was given. CONCLUSION: The implementation of a clinical decision support system and a patient list resulted in improved compliance with the guidelines and optimized the treatment with gentamicin and vancomycin.

Circle of Willis variations in migraine patients with ischemic stroke.

OBJECTIVES: Migraine is a risk factor for stroke, which might be explained by a higher prevalence in anatomical variants in the circle of Willis (CoW). Here, we compared the presence of CoW variants in patients with stroke with and without migraine. MATERIALS AND METHODS: Participants were recruited from the prospective Dutch acute Stroke Study. All participants underwent CT angiography on admission. Lifetime migraine history was assessed with a screening questionnaire and confirmed by an interview based on International Classification of Headache Disorders criteria. The CoW was assessed for incompleteness/hypoplasia (any segment <1 mm), for anterior cerebral artery asymmetry (difference > 1/3), and for posterior communicating artery (Pcom) dominance (Pcom-P1 difference > 1/3). Odds ratios with adjustments for age and sex (aOR) were calculated with logistic regression. RESULTS: We included 646 participants with stroke, of whom 52 had a history of migraine. Of these, 45 (87%) had an incomplete or hypoplastic CoW versus 506 (85%) of the 594 participants without migraine (aOR: 1.47; 95% CI: 0.63-3.44). There were no differences between participants with and without migraine in variations of the anterior or posterior CoW, anterior cerebral artery asymmetry (aOR: 0.86; 95% CI: 0.43-1.74), or Pcom dominance (aOR: 0.64; 95% CI: 0.32-1.30). There were no differences in CoW variations between migraine patients with or without aura. CONCLUSION: We found no significant difference in the completeness of the CoW in acute stroke patients with migraine compared to those without.

Stroke progression and clinical outcome in ischemic stroke patients with a history of migraine.

BACKGROUND: Patients with migraine might be more susceptible of spreading depolarizations, which are known to affect vascular and neuronal function and penumbra recovery after stroke. We investigated whether these patients have more severe stroke progression and less favorable outcomes after recanalization therapy. METHODS: We included patients from a prospective multicenter ischemic stroke cohort. Lifetime migraine history was based on the International Classification of Headache Disorders II criteria. Patients without confirmed migraine diagnosis were excluded. Patients underwent CT angiography and CT perfusion <9 h of onset and follow-up CT after three days. On admission, presence of a perfusion deficit, infarct core and penumbra volume, and blood brain barrier permeability (BBBP) were assessed. At follow-up we assessed malignant edema, hemorrhagic transformation, and final infarct volume. Outcome at three months was evaluated with the modified Rankin Scale (mRS). We calculated adjusted relative risks (aRR) or difference of means (aB) with regression analyses. RESULTS: We included 600 patients of whom 43 had migraine. There were no differences between patients with or without migraine in presence of a perfusion deficit on admission (aRR: 0.98, 95%CI: 0.77-1.25), infarct core volume (aB: -10.8, 95%CI: -27.04-5.51), penumbra volume (aB: -11.6, 95%CI: -26.52-3.38), mean blood brain barrier permeability (aB: 0.08, 95%CI: -3.11-2.96), malignant edema (0% vs. 5%), hemorrhagic transformation (aRR: 0.26, 95%CI: 0.04-1.73), final infarct volume (aB: -14.8, 95%CI: 29.9-0.2) or outcome after recanalization therapy (mRS > 2, aRR: 0.50, 95%CI: 0.21-1.22). CONCLUSION: Elderly patients with a history of migraine do not seem to have more severe stroke progression and have similar treatment outcomes compared with patients without migraine.

Automated Ischemic Lesion Segmentation in MRI Mouse Brain Data after Transient Middle Cerebral Artery Occlusion.

Magnetic resonance imaging (MRI) has become increasingly important in ischemic stroke experiments in mice, especially because it enables longitudinal studies. Still, quantitative analysis of MRI data remains challenging mainly because segmentation of mouse brain lesions in MRI data heavily relies on time-consuming manual tracing and thresholding techniques. Therefore, in the present study, a fully automated approach was developed to analyze longitudinal MRI data for quantification of ischemic lesion volume progression in the mouse brain. We present a level-set-based lesion segmentation algorithm that is built using a minimal set of assumptions and requires only one MRI sequence (T2) as input. To validate our algorithm we used a heterogeneous data set consisting of 121 mouse brain scans of various age groups and time points after infarct induction and obtained using different MRI hardware and acquisition parameters. We evaluated the volumetric accuracy and regional overlap of ischemic lesions segmented by our automated method against the ground truth obtained in a semi-automated fashion that includes a highly time-consuming manual correction step. Our method shows good agreement with human observations and is accurate on heterogeneous data, whilst requiring much shorter average execution time. The algorithm developed here was compiled into a toolbox and made publically available, as well as all the data sets.

A DGGE system for comprehensive mutation screening of BRCA1 and BRCA2: application in a Dutch cancer clinic setting.

Rapid and reliable identification of deleterious changes in the breast cancer genes BRCA1 and BRCA2 has become one of the major issues in most DNA services laboratories. To rapidly detect all possible changes within the coding and splice site determining sequences of the breast cancer genes, we established a semiautomated denaturing gradient gel electrophoresis (DGGE) mutation scanning system. All exons of both genes are covered by the DGGE scan, comprising 120 amplicons. We use a semiautomated approach, amplifying all individual amplicons with the same PCR program, after which the amplicons are pooled. DGGE is performed using three slightly different gel conditions. Validation was performed using DNA samples with known sequence variants in 107 of the 120 amplicons; all variants were detected. This DGGE mutation scanning, in combination with a PCR test for two Dutch founder deletions in BRCA1 was then applied in 431 families in which 52 deleterious changes and 70 unclassified variants were found. Fifteen unclassified variants were not reported before. The system was easily adopted by five other laboratories, where in another 3,593 families both exons 11 were analyzed by the protein truncation test (PTT) and the remaining exons by DGGE. In total, a deleterious change (nonsense, frameshift, splice-site mutation, or large deletion) was found in 661 families (16.4%), 462 in BRCA1 (11.5%), 197 in BRCA2 (4.9%), and in two index cases a deleterious change in both BRCA1 and BRCA2 was identified. Eleven deleterious changes in BRCA1 and 36 in BRCA2 had not been reported before. In conclusion, this DGGE mutation screening method for BRCA1 and BRCA2 is proven to be highly sensitive and is easy to adopt, which makes screening of large numbers of patients feasible. The results of screening of BRCA1 and BRCA2 in more than 4,000 families present a valuable overview of mutations in the Dutch population.

Role of atherosclerosis, clot extent, and penumbra volume in headache during ischemic stroke.

OBJECTIVE: To investigate the role of large vessel atherosclerosis, blood clot extent, and penumbra volume in relation to headache in ischemic stroke patients. METHODS: In this cross-sectional study, we performed noncontrast CT, CT angiography (CTA), and CT perfusion (CTP) in 284 participants from the Dutch Acute Stroke Study and Leiden Stroke Cohort within 9 hours after ischemic stroke onset. We collected headache characteristics prospectively using a semi-structured questionnaire. Atherosclerosis was assessed by evaluating presence of plaques in extracranial and intracranial vessels and by quantifying intracranial carotid artery calcifications. Clot extent was estimated by the clot burden score on CTA and penumbra volume by CTP. We calculated risk ratios (RRs) with adjustments (aRR) for possible confounders using multivariable Poisson regression. RESULTS: Headache during stroke was reported in 109/284 (38%) participants. Headache was less prevalent in patients with than in patients without atherosclerosis in the extracranial anterior circulation (35% vs 48%; RR 0.72; 95% confidence interval [CI] 0.54-0.97). Atherosclerosis in the intracranial arteries was also associated with less headache, but this association was not statistically significant. Penumbra volume (aRR 1.08; 95% CI 0.63-1.85) and clot extent (aRR 1.02; 95% CI 0.86-1.20) were not related with headache. CONCLUSIONS: Headache in the early phase of ischemic stroke tends to occur less often in patients with atherosclerosis than in patients without atherosclerosis in the large cerebral arteries. This finding lends support to the hypothesis that vessel wall elasticity is a necessary contributing factor in the occurrence of headache during acute ischemic stroke.

Spreading depolarization-modulating drugs and delayed cerebral ischemia after subarachnoid hemorrhage: A hypothesis-generating retrospective clinical study.

BACKGROUND: Delayed cerebral ischemia (DCI) occurs in approximately one-third of patients with aneurysmal subarachnoid hemorrhage (aSAH). A proposed underlying mechanism for DCI is spreading depolarization (SD). Our aim was to, retrospectively, investigate the influence of the use of SD-modulating drugs on the occurrence of DCI. METHODS: We, retrospectively, combined data from four cohorts of aSAH patients with data on the use of home medication prior to hospital admission, occurrence of DCI, and clinical outcome. Home medication was classified as "SD-inhibiting", "SD-facilitating", or "SD-neutral based" on a comprehensive literature review. We defined subgroups "likely", "possibly" and "weak" concerning the amount of evidence in literature. We performed Cox and Poisson regression analysis and calculated hazard ratios (HR) and risk ratios (RR) for the influence of "SD-modulating" drugs on primary outcome measure DCI and secondary outcome measure poor clinical outcome (modified Rankin Scale ≥3) three months after aSAH. We adjusted for age, sex and clinical condition on admission (aHR/aRR). RESULTS: DCI occurred in 343 (29%) of 1194 patients. Patients using SD-inhibiting home medication had an aHR for DCI of 0.66 (95% CI: 0.42-1.06) and an aRR for poor outcome of 1.13 (95% CI: 0.90-1.41). Patients using SD-facilitating drugs had an aHR for DCI of 1.24 (95% CI: 0.83-1.87) and an aRR for poor outcome of 1.19 (95% CI: 0.95-1.50). When comparing patients using SD-inhibiting drugs with patients using SD-facilitating drugs, the aHR was 0.54 (95% CI: 0.29-0.99) for DCI and the aRR 0.97 (95% CI: 0.71-1.32) for outcome. CONCLUSIONS: In this exploratory study chronic use of SD-inhibiting drugs tended to reduce DCI but did not result in a better clinical outcome. Additional research is needed to investigate the specific effects of SD-modulation on DCI and outcome and to further explore its effectiveness in preventing DCI after aSAH.

Stability assessment of repackaged bevacizumab for intravitreal administration.

Intravitreal bevacizumab is frequently used off-label for the treatment of neovascular age-related macular degeneration, but there are concerns about the safety of intravitreal administered bevacizumab. It is suggested that repackaging bevacizumab in plastic syringes could affect the safety due to the unknown shelf life of the syringes. In this study, we analyzed the shelf life of the repackaged bevacizumab syringes, stored at 4 degrees C, at certain time intervals. Over the 32 days tested, bevacizumab concentration and the pH were stable. However, the number of particles in the repackaged bevacizumab syringes increased during storage at 4 degrees C and had exceeded the limits for intravitreal injections after 7 days. Since the number of particles seems to be the limitation of the shelf life of repackaged bevacizumab, it is necessary to quantify the number of particles in repackaged bevacizumab. Based on our results the maximum shelf life of repackaged bevacizumab should be 3 days.

DGGE-based whole-gene mutation scanning of the dystrophin gene in Duchenne and Becker muscular dystrophy patients.

Duchenne and Becker muscular dystrophy (DMD and BMD) are caused by mutations in the dystrophin gene. Large rearrangements in the gene are found in about two-thirds of DMD patients, with approximately 60% carrying deletions and 5-10% carrying duplications. Most of the remaining 30-35% of patients are expected to have small nucleotide substitutions, insertions, or deletions. To detect these subtle changes within the coding and splice site determining sequences of the dystrophin gene, we established a semiautomated denaturing gradient gel electrophoresis (DGGE) mutation scanning system. The DGGE scan covers the dystrophin gene with 95 amplicons, PCRed either individually or in a multiplex setup. PCR and pooling were performed semiautomatically, using a pipetting robot and 384-well plates, enabling concurrent amplification of DNA of four patients in one run. Amplification of individual fragments was performed using one PCR program. The products were pooled just before gel loading; DGGE requires only a single gel condition. Validation was performed using DNA samples harboring 39 known DMD variants, all of which could be readily detected. DGGE mutation scanning was applied to analyze 135 DMD/BMD patients and potential DMD carriers without large deletions or duplications. In DNA from 25 out of 44 DMD patients (57%) and from 5 out of 39 BMD patients (13%), we identified clear pathogenic changes. All mutations were different, with the exception of one DMD mutation, which occurred twice. In DNA from 10 out of 44 potential DMD carriers, including four obligate carriers, we detected causative changes, including one pathogenic change in every obligate carrier. In addition to these pathogenic changes, we detected 15 unique unclassified variants, i.e., changes for which a pathogenic nature is uncertain.

Keep moving forward: a new energy returning prosthetic device with low installation height after Syme or Pirogoff amputation.

BACKGROUND: The incidence of foot amputations increased in the Netherlands to 3.3/100,000 people up to 1994. Despite these numbers, only a few basic prosthetic and orthotic devices are available, and all lack functionality to restore ankle and foot mobility. OBJECTIVES: The aim of this explorative study was to design and test a unique prosthesis for Syme or Pirogoff amputees with the necessary low installation height but restoring ankle and foot mobility. STUDY DESIGN: A case study was performed. METHODS: The new prosthesis was designed and numerically analyzed on aspects concerning strength and deformation. A prototype was tested in a case study to assess the biomechanical behavior of the new foot. As a reference, six Syme/Pirogoff amputees were measured. Additionally, all volunteers filled out a questionnaire to evaluate their prosthetic feet. RESULTS: The self-selected and maximum walking speed of the case subject at 0° and 5° slopes was higher using the new foot (0.36 m/s and 0.53 m/s, respectively) comparing to the Low Rider (Otto Bock HealthCare) (0.31 m/s and 0.31 m/s, respectively). Using the new foot, a more symmetrical walking pattern was achieved. CONCLUSION: The case study shows that this new prosthetic foot could be an improvement compared to existing prosthetic feet. CLINICAL RELEVANCE: Foot amputees with low available installation height still experience daily the inconvenience of missing ankle and foot mobility. Their low velocity and cosmetically poor walking pattern influence on their sound leg and overall walking functionality. A more functional prosthesis would have a great impact on their daily activities.