Evaluation of a luminometric cell counting system in context of antimicrobial photodynamic inactivation

Antimicrobial resistance belongs to the most demanding medical challenges, and antimicrobial photodynamic inactivation (aPDI) is considered a promising alternative to classical antibiotics. However, the pharmacologic characterization of novel compounds suitable for aPDI is a tedious and time-consuming task that usually requires preparation of bacterial cultures and counting of bacterial colonies. In this study, we established and utilized a luminescence-based microbial cell viability assay to analyze the aPDI effects of two porphyrin-based photosensitizers (TMPyP and THPTS) on several bacterial strains with antimicrobial resistance. We demonstrate that after adaptation of the protocol and initial calibration to every specific bacterial strain and photosensitizer, the luminometric method can be used to reliably quantify aPDI effects in most of the analyzed bacterial strains. The interference of photosensitizers with the luminometric readout and the bioluminescence of some bacterial strains were identified as possible confounders. Using this method, we could confirm the susceptibility of several bacterial strains to photodynamic treatment, including extensively drug-resistant pathogens (XDR). In contrast to the conventional culture-based determination of bacterial density, the luminometric assay allowed for a much more time-effective analysis of various treatment conditions. We recommend this luminometric method for high-throughput tasks requiring measurements of bacterial viability in the context of photodynamic treatment approaches

Photodynamic inactivation of SARS-CoV-2 infectivity and antiviral treatment effects in vitro

Despite available vaccines, antibodies and antiviral agents, the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic still continues to cause severe disease and death. Current treatment options are limited, and emerging new mutations are a challenge. Thus, novel treatments and measures for prevention of viral infections are urgently required. Photodynamic inactivation (PDI) is a potential treatment for infections by a broad variety of critical pathogens, including viruses. We explored the infectiousness of clinical SARS-CoV-2 isolates in Vero cell cultures after PDI-treatment, using the photosensitizer Tetrahydroporphyrin-tetratosylate (THPTS) and near-infrared light. Replication of viral RNA (qPCR), viral cytopathic effects (microscopy) and mitochondrial activity were assessed. PDI of virus suspension with 1 µM THPTS before infection resulted in a reduction of detectable viral RNA by 3 log levels at day 3 and 6 after infection to similar levels as in previously heat-inactivated virions (<99.9%; p < 0.05). Mitochondrial activity, which was significantly reduced by viral infection, was markedly increased by PDI to levels similar to uninfected cell cultures. When applying THPTS-based PDI after infection, a single treatment had a virus load-reducing effect only at a higher concentration (3 µM) and reduced cell viability in terms of PDI-induced toxicity. Repeated PDI with 0.3 µM THPTS every 4 h for 3 d after infection reduced the viral load by more than 99.9% (p < 0.05), while cell viability was maintained. Our data demonstrate that THPTS-based antiviral PDI might constitute a promising approach for inactivation of SARS-CoV-2. Further testing will demonstrate if THPTS is also suitable to reduce the viral load in vivo

The meta-substituted isomer of TMPyP enables more effective photodynamic bacterial inactivation than para-TMPyP in vitro

Porphyrinoid-based photodynamic inactivation (PDI) provides a promising approach to treating multidrug-resistant infections. However, available agents for PDI still have optimization potential with regard to effectiveness, toxicology, chemical stability, and solubility. The currently available photosensitizer TMPyP is provided with a para substitution pattern (para-TMPyP) of the pyridinium groups and has been demonstrated to be effective for PDI of multidrug-resistant bacteria. To further improve its properties, we synthetized a structural variant of TMPyP with an isomeric substitution pattern in a meta configuration (meta-TMPyP), confirmed the correct structure by crystallographic analysis and performed a characterization with NMR-, UV/Vis-, and IR spectroscopy, photostability, and singlet oxygen generation assay. Meta-TMPyP had a hypochromic shift in absorbance (4 nm) with a 55% higher extinction coefficient and slightly improved photostability (+6.9%) compared to para-TMPyP. Despite these superior molecular properties, singlet oxygen generation was increased by only 5.4%. In contrast, PDI, based on meta-TMPyP, reduced the density of extended spectrum β-lactamase-producing and fluoroquinolone-resistant Escherichia coli by several orders of magnitude, whereby a sterilizing effect was observed after 48 min of illumination, while para-TMPyP was less effective (p < 0.01). These findings demonstrate that structural modification with meta substitution increases antibacterial properties of TMPyP in PDI

Nimodipine vs. Milrinone – Equal or Complementary Use? A Retrospective Analysis

Background: Cerebral vasospasm (CVS) continues to account for high morbidity and mortality in patients surviving the initial aneurysmal subarachnoid hemorrhage (SAH). Nimodipine is the only drug known to reduce delayed cerebral ischemia (DCI), but it is believed not to affect large vessel CVS. Milrinone has emerged as a promising option. Our retrospective study focused on the effectiveness of the intra-arterial application of both drugs in monotherapy and combined therapy. Methods: We searched for patients with aneurysmal SAH, angiographically confirmed CVS, and at least one intra-arterial pharmacological angioplasty. Ten defined vessel sections on angiograms were assessed before and after vasodilator infusion. The improvement in vessel diameters was compared to the frequency of DCI-related cerebral infarction before hospital discharge and functional outcome reported as the modified Rankin Scale (mRS) score after 6 months. Results: Between 2014 and 2021, 132 intra-arterial interventions (144 vascular territories, 12 bilaterally) in 30 patients were analyzed for this study. The vasodilating effect of nimodipine was superior to milrinone in all intradural segments. There was no significant intergroup difference concerning outcome in mRS (p = 0.217). Only nimodipine or the combined approach could prevent DCI-related infarction (both 57.1%), not milrinone alone (87.5%). Both drugs induced a doubled vasopressor demand due to blood pressure decrease, but milrinone alone induced tachycardia. Conclusions: The monotherapy with intra-arterial nimodipine was superior to milrinone. Nimodipine and milrinone may be used complementary in an escalation scheme with the administration of nimodipine first, complemented by milrinone in cases of severe CVS. Milrinone monotherapy is not recommended

Delayed Stroke after Aneurysm Treatment with Flow Diverters in Small Cerebral Vessels: A Potentially Critical Complication Caused by Subacute Vasospasm

Flow diversion (FD) is a novel endovascular technique based on the profound alteration of cerebrovascular hemodynamics, which emerged as a promising minimally invasive therapy for intracranial aneurysms. However, delayed post-procedural stroke remains an unexplained concern. A consistent follow-up-regimen has not yet been defined, but is required urgently to clarify the underlying cause of delayed ischemia. In the last two years, 223 patients were treated with six different FD devices in our center. We identified subacute, FD-induced segmental vasospasm (SV) in 36 patients as a yet unknown, delayed-type reaction potentially compromising brain perfusion to a critical level. Furthermore, 86% of all patients revealed significant SV approximately four weeks after treatment. In addition, 56% had SV with 25% stenosis, and 80% had additional neointimal hyperplasia. Only 13% exhibited SV-related high-grade stenosis. One of those suffered stroke due to prolonged SV, requiring neurocritical care and repeated intra-arterial (i.a.) biochemical angioplasty for seven days to prevent territorial infarction. Five patients suffered newly manifested, transient hemicrania accompanying a compensatorily increased ipsilateral leptomeningeal perfusion. One treated vessel obliterated permanently. Hence, FD-induced SV is a frequent vascular reaction after FD treatment, potentially causing symptomatic ischemia or even stroke, approximately one month post procedure. A specifically early follow-up-strategy must be applied to identify patients at risk for ischemia, requiring intensified monitoring and potentially anti-vasospastic treatment

Segment Occlusion vs. Reconstruction—A Single Center Experience With Endovascular Strategies for Ruptured Vertebrobasilar Dissecting Aneurysms

Objective: Ruptured dissecting aneurysms of the intracranial vertebral arteries exhibit an extraordinarily high risk for morbidity and mortality and are prone to re-rupture. Therefore, early treatment is mandatory to induce stagnation of the critical dynamic mural process. Appropriate endovascular approaches are segment sacrifice and reconstruction, however, both carry specific risks and benefits. To date most studies discuss only one of these approaches and focus on one specific device or technique. Therefore, our study aimed to present our experiences with both techniques, providing a considered approach on when to perform endovascular reconstruction or sacrifice.Materials and Methods: We retrospectively reviewed patients with subarachnoid hemorrhage in our database, suffering from dissecting aneurysms of the intradural vertebral arteries and treated endovascularly in the acute setting. A total of 16 cases were included. Clinical history, radiologic findings and outcomes were analyzed.Results: In 7 patients a reconstructive approach was chosen with 4 of them receiving stent-assisted coiling as primary strategy. One of the 7 patients suffered early re-bleeding due to progression of the dissection and therefore treatment was augmented with implantation of 2 flow diverters. The remaining 2 patients were primarily treated with flow diverters in telescoping technique. In 9 patients a deconstructive approach was followed: 6 patients were treated with proximal coil-occlusion of the V4 segment, 3 patients received distal coiling of the V4 segment. Two patients died (GOS 1) in the subacute stage due to sequelae of recurrent episodes of raised intracranial pressure and parenchymal hemorrhage. Two patients kept severe disability (GOS 3), six patients had moderate disability (GOS 4) and seven patients showed full recovery (GOS 5). None of the patients suffered from a procedural or postprocedural ischemic stroke.Conclusions: In patients with good collateral vascularization, proximal, or distal partial segment sacrifice via with endovascular coil occlusion seems to yield the best risk-benefit ratio for treatment of ruptured dissecting V4 aneurysms, especially since no continued anticoagulation is required and possibly essential surgery remains feasible in this scenario. If possible, PICA occlusion should be avoided—although even proximal PICA occlusion can become necessary, when weighing against the risk of an otherwise untreated ruptured V4 dissecting aneurysm. Contrarily, if the dominant V4 segment is affected, the hemodynamic asymmetry prohibits occlusion and necessitates reconstruction of the respective segment. For this, implants with high metal coverage treating the entire affected segment appear to be the most promising approach

Efficient Photodynamic Therapy against Gram-Positive and Gram-Negative Bacteria Using THPTS, a Cationic Photosensitizer Excited by Infrared Wavelength

The worldwide rise in the rates of antibiotic resistance of bacteria underlines the need for alternative antibacterial agents. A promising approach to kill antibiotic-resistant bacteria uses light in combination with a photosensitizer to induce a phototoxic reaction. Concentrations of 1, 10 and 100µM of tetrahydroporphyrin-tetratosylat (THPTS) and different incubation times (30, 90 and 180min) were used to measure photodynamic efficiency against two Gram-positive strains of S.aureus (MSSA and MRSA), and two Gram-negative strains of E.coli and P.aeruginosa. We found that phototoxicity of the drug is independent of the antibiotic resistance pattern when incubated in PBS for the investigated strains. Also, an incubation with 100µM THPTS followed by illumination, yielded a 6lg (≥99.999%) decrease in the viable numbers of all bacteria strains tested, indicating that the THPTS drug has a high degree of photodynamic inactivation. We then modulated incubation time, photosensitizer concentration and monitored the effect of serum on the THPTS activity. In doing so, we established the conditions to obtain the strongest bactericidal effect. Our results suggest that this new and highly pure synthetic compound should improve the efficiency of photodynamic therapy against multiresistant bacteria and has a significant potential for clinical applications in the treatment of nosocomial infections

Reference Values of Cerebral Artery Diameters of the Anterior Circulation by Digital Subtraction Angiography: A Retrospective Study

A threshold-based classification of cerebral vasospasm needs reference values for intracranial vessel diameters on digital subtraction angiography (DSA). We aimed to generate adjusted reference values for this purpose by retrospectively analyzing angiograms and potential influencing factors on vessel diameters. Angiograms of the anterior circulation were evaluated in 278 patients aged 18&ndash;81 years. The vessel diameters of 453 angiograms (175 bilateral) were gathered from nine defined measuring sites. The effect sizes of physical characteristics (i.e., body weight and height, body mass index, gender, age, and cranial side) and anatomical variations were calculated with MANOVA. Segments bearing aneurysms were excluded for the calculation of reference values. Adjusted vessel diameters were calculated via linear regression analysis of the vessel diameter data. Vessel diameters increased with age and body height. Male and right-sided vessels were larger in diameter. Of the anatomical variations, only the hypoplastic/aplastic A1 segment had a significant influence (p &lt; 0.05) on values of the anterior cerebral artery and the internal carotid artery with a small effect size (|&omega;2| &gt; 0.01) being excluded from the reference values. We provide gender-, age-, and side-adjusted reference values and nomograms of arterial vessel diameters in the anterior circulation

The Supratrochlear Artery Sign

Background: Cerebral vasospasm (CVS) after subarachnoid hemorrhage (SAH) has been extensively investigated, but the impact of collateralization remains unclear. We investigated the predictive value of collateral activation for delayed cerebral ischemia (DCI)-related infarctions and functional outcome. Methods: Data from 43 patients with CVS (January 2014 to August 2021) were evaluated for the angiographic presence of leptomeningeal and ophthalmic collaterals (anterior falcine artery (AFA), supratrochlear artery (STA), dorsal nasal artery (DNA)) on internal carotid artery angiograms. Vasospasm-related infarction and the modified Rankin Scale (mRS) score after six months were chosen as the endpoints. Results: 77% of the patients suffered from DCI-related infarctions. In 233 angiograms (at hospitalization, before spasmolysis, after six months), positive vessel signs were observed in 31 patients for STA, 35 for DNA, and 31 for AFA. The STA sign had the highest positive (84.6%) and negative (85.7%) predictive value for unfavorable outcome (mRS 4–6) in patients aged 50 years. DNA and AFA signs were not meaningful predictors for either endpoint. Leptomeningeal collaterals showed a positive Pearson’s correlation with the STA sign in 87.5% (p = 0.038) without providing any prediction for either endpoint. Conclusions: The STA sign is associated with clinical outcome in patients with CVS after SAH aged 50 years, and was correlated with the occurrence of leptomeningeal collaterals

The Supratrochlear Artery Sign

Background: Cerebral vasospasm (CVS) after subarachnoid hemorrhage (SAH) has been extensively investigated, but the impact of collateralization remains unclear. We investigated the predictive value of collateral activation for delayed cerebral ischemia (DCI)-related infarctions and functional outcome. Methods: Data from 43 patients with CVS (January 2014 to August 2021) were evaluated for the angiographic presence of leptomeningeal and ophthalmic collaterals (anterior falcine artery (AFA), supratrochlear artery (STA), dorsal nasal artery (DNA)) on internal carotid artery angiograms. Vasospasm-related infarction and the modified Rankin Scale (mRS) score after six months were chosen as the endpoints. Results: 77% of the patients suffered from DCI-related infarctions. In 233 angiograms (at hospitalization, before spasmolysis, after six months), positive vessel signs were observed in 31 patients for STA, 35 for DNA, and 31 for AFA. The STA sign had the highest positive (84.6%) and negative (85.7%) predictive value for unfavorable outcome (mRS 4–6) in patients aged 50 years. DNA and AFA signs were not meaningful predictors for either endpoint. Leptomeningeal collaterals showed a positive Pearson’s correlation with the STA sign in 87.5% (p = 0.038) without providing any prediction for either endpoint. Conclusions: The STA sign is associated with clinical outcome in patients with CVS after SAH aged 50 years, and was correlated with the occurrence of leptomeningeal collaterals