Activation of NLRP3 inflammasome in a rat model of cerebral small vessel disease.

Cerebral small vessel disease (CSVD) is increasingly being recognized as a leading contributor to cognitive impairment in the elderly. However, there is a lack of effective preventative or therapeutic options for CSVD. In this exploratory study, we investigated the interplay between neuroinflammation and CSVD pathogenesis as well as the cognitive performance, focusing on NLRP3 signaling as a new therapeutic target. Spontaneously hypertensive stroke-prone (SHRSP) rats served as a CSVD model. We found that SHRSP rats showed decline in learning and memory abilities using morris water maze test. Activated NLRP3 signaling and an increased expression of the downstream pro-inflammatory factors, including IL (interleukin)-6 and tumor necrosis factor α were determined. We also observed a remarkable increase in the production of pyroptosis executive protein gasdermin D, and elevated astrocytic and microglial activation. In addition, we identify several neuropathological hallmarks of CSVD, including blood-brain barrier breakdown, white matter damage, and endothelial dysfunction. These results were in correlation with the activation of NLRP3 inflammasome. Thus, our findings reveal that the NLRP3-mediated inflammatory pathway could play a central role in the pathogenesis of CSVD, presenting a novel target for potential CSVD treatment.

GEOMETRIC DETERMINANTS OF CELL VIABILITY FOR 3D-PRINTED HOLLOW MICRONEEDLE ARRAY-MEDIATED DELIVERY.

A wide range of emerging biomedical applications and clinical interventions rely on the ability to deliver living cells via hollow, high-aspect-ratio microneedles. Recently, microneedle arrays (MNA) have gained increasing interest due to inherent benefits for drug delivery; however, studies exploring the potential to harness such advantages for cell delivery have been impeded due to the difficulties in manufacturing high-aspect-ratio MNAs suitable for delivering mammalian cells. To bypass these challenges, here we leverage and extend our previously reported hybrid additive manufacturing (or "three-dimensional (3D) printing) strategy-i.e., the combined the "Vat Photopolymerization (VPP)" technique, "Liquid Crystal Display (LCD)" 3D printing with "Two-Photon Direct Laser Writing (DLW)"-to 3D print hollow MNAs that are suitable for cell delivery investigations. Specifically, we 3D printed four sets of 650 µm-tall MNAs corresponding to needle-specific inner diameters (IDs) of 25 µm, 50 µm, 75 µm, and 100 µm, and then examined the effects of these MNAs on the post-delivery viability of both dendritic cells (DCs) and HEK293 cells. Experimental results revealed that the 25 µm-ID case led to a statistically significant reduction in post-MNA-delivery cell viability for both cell types; however, MNAs with needle-specific IDs ≥ 50 µm were statistically indistinguishable from one another as well as conventional 32G single needles, thereby providing an important benchmark for MNA-mediated cell delivery.

Unlocking the potential of ultra-high dose fractionated radiation for effective treatment of glioblastoma.

Background: Conventional radiation therapy for glioblastoma (GBM) has limited efficacy. Regenerative medicine brings hope for repairing damaged tissue, opening opportunities for elevating the maximum acceptable radiation dose. In this study, we explored the effect of ultra-high dose fractionated radiation on brain injury and tumor responses in immunocompetent mice. We also evaluated the role of the HIF-1α under radiation. Methods: Naïve and hypoxia-inducible factor-1 alpha (HIF-1α)+/- heterozygous mice received a fractionated daily dose of 20 Gy for three or five consecutive days. Magnetic resonance imaging (MRI) and histology were performed to assess brain injury post-radiation. The 2×105 human GBM1 luciferase-expressing cells were transplanted with tolerance induction protocol. Fractionated radiotherapy was performed during the exponential phase of tumor growth. BLI, MRI, and immunohistochemistry staining were performed to evaluate tumor growth dynamics and radiotherapy responses. Additionally, animal lifespan was recorded. Results: Fractionated radiation of 5×20 Gy induced severe brain damage, starting 3 weeks after radiation. All animals from this group died within 12 weeks. In contrast, later onset and less severe brain injury were observed starting 12 weeks after radiation of 3×20 Gy. It resulted in complete GBM eradication and survival of all treated animals. Furthermore, HIF-1α+/- mice exhibited more obvious vascular damage 63 weeks after fractionated radiation of 3×20 Gy. Conclusion: Ultra-high dose fractionated 3×20 Gy radiation can eradicate the GBM cells at the cost of only mild brain injury. The HIF-1α gene is a promising target for ameliorating vascular impairment post-radiation, encouraging the implementation of neurorestorative strategies.

3D printing-based frugal manufacturing of glass pipettes for minimally invasive delivery of therapeutics to the brain.

Objective: Intracerebral delivery of agents in liquid form is usually achieved through commercially available and durable metal needles. However, their size and texture may contribute to mechanical brain damage. Glass pipettes with a thin tip may significantly reduce injection-associated brain damage but require access to prohibitively expensive programmable pipette pullers. This study is to remove the economic barrier to the application of minimally invasive delivery of therapeutics to the brain, such as chemical compounds, viral vectors, and cells. Methods: We took advantage of the rapid development of free educational online resources and emerging low-cost 3D printers by designing an affordable pipette puller (APP) to remove the cost obstacle. Results: We showed that our APP could produce glass pipettes with a sharp tip opening down to 20 µm or less, which is sufficiently thin for the delivery of therapeutics into the brain. A pipeline from pipette pulling to brain injection using low-cost and open-source equipment was established to facilitate the application of the APP. Conclusion: In the spirit of frugal science, our device may democratize glass pipette-puling and substantially promote the application of minimally invasive and precisely controlled delivery of therapeutics to the brain for finding more effective therapies of brain diseases.

In vivo Mapping of Cellular Resolution Neuropathology in Brain Ischemia by Diffusion MRI.

Non-invasive mapping of cellular pathology can provide critical diagnostic and prognostic information. Recent developments in diffusion MRI have produced new tools for examining tissue microstructure at a level well below the imaging resolution. Here, we report the use of diffusion time ( t )-dependent diffusion kurtosis imaging ( t DKI) to simultaneously assess the morphology and transmembrane permeability of cells and their processes in the context of pathological changes in hypoxic-ischemic brain (HI) injury. Through Monte Carlo simulations and cell culture organoid imaging, we demonstrate feasibility in measuring effective size and permeability changes based on the peak and tail of t DKI curves. In a mouse model of HI, in vivo imaging at 11.7T detects a marked shift of the t DKI peak to longer t in brain edema, suggesting swelling and beading associated with the astrocytic processes and neuronal neurites. Furthermore, we observed a faster decrease of the t DKI tail in injured brain regions, reflecting increased membrane permeability that was associated with upregulated water exchange upon astrocyte activation at acute stage as well as necrosis with disrupted membrane integrity at subacute stage. Such information, unavailable with conventional diffusion MRI at a single t, can predict salvageable tissues. For a proof-of-concept, t DKI at 3T on an ischemic stroke patient suggested increased membrane permeability in the stroke region. This work therefore demonstrates the potential of t DKI for in vivo detection of the pathological changes in microstructural morphology and transmembrane permeability after ischemic injury using a clinically translatable protocol.

Cutting-edge advances in modeling the blood-brain barrier and tools for its reversible permeabilization for enhanced drug delivery into the brain.

The blood-brain barrier (BBB) is a sophisticated structure whose full functionality is required for maintaining the executive functions of the central nervous system (CNS). Tight control of transport across the barrier means that most drugs, particularly large size, which includes powerful biologicals, cannot reach their targets in the brain. Notwithstanding the remarkable advances in characterizing the cellular nature of the BBB and consequences of BBB dysfunction in pathology (brain metastasis, neurological diseases), it remains challenging to deliver drugs to the CNS. Herein, we outline the basic architecture and key molecular constituents of the BBB. In addition, we review the current status of approaches that are being explored to temporarily open the BBB in order to allow accumulation of therapeutics in the CNS. Undoubtedly, the major concern in field is whether it is possible to open the BBB in a meaningful way without causing negative consequences. In this context, we have also listed few other important key considerations that can improve our understanding about the dynamics of the BBB.

Real-Time MRI Monitoring of Liquid Embolic Agent (Onyx) Injection in a Swine Arteriovenous Malformation Model.

The paradigm is gradually shifting, with radiosurgery and endovascular embolization being increasingly chosen over surgical resection in the selected cases of brain arteriovenous malformations. Routinely used X-ray monitoring of liquid embolic infusion has very good spatial and temporal resolution but is not without significant drawbacks regarding poor visualization of the complex AVM angioarchitecture, especially after many embolizations in the past and therefore limiting the technical ability of the embocure-total occlusion of the feeding arteries, nidus, and draining veins. The purpose of this study was to evaluate the use of real-time MRI guidance in endovascular embolization with Onyx (instead of X-ray) in a single swine rete mirabile (RM) AVM model in order to provide the scaffolding for the real-time MRI guidance method. Onyx propagation was observed in real-time dynamic GE-EPI scan with initial ipsilateral RM filling followed by main cerebral arterial branch distribution. The relatively bright signal within RM and the brain prior to Onyx injection provided a good background for the dark, low signal of the embolic agent spreading in rete mirabile and brain arteries. X-ray picture confirmed Onyx cast distribution at the end of the procedure. In this initial experience, real-time MRI seems to be a promising method that may significantly improve liquid embolic agent infusion monitoring in the future, although requiring further development before clinical use.

Reconsidering the route of drug delivery in refractory multiple sclerosis: Toward a more effective drug accumulation in the central nervous system.

Multiple sclerosis is a chronic demyelinating disease with different disease phenotypes. The current FDA-approved disease-modifying therapeutics (DMTs) cannot cure the disease, but only alleviate the disease progression. While the majority of patients respond well to treatment, some of them are suffering from rapid progression. Current drug delivery strategies include the oral, intravenous, subdermal, and intramuscular routes, so these drugs are delivered systemically, which is appropriate when the therapeutic targets are peripheral. However, the potential benefits may be diminished when these targets sequester behind the barriers of the central nervous system. Moreover, systemic drug administration is plagued with adverse effects, sometimes severe. In this context, it is prudent to consider other drug delivery strategies improving their accumulation in the brain, thus providing better prospects for patients with rapidly progressing disease course. These targeted drug delivery strategies may also reduce the severity of systemic adverse effects. Here, we discuss the possibilities and indications for reconsideration of drug delivery routes (especially for those "non-responding" patients) and the search for alternative drug delivery strategies. More targeted drug delivery strategies sometimes require quite invasive procedures, but the potential therapeutic benefits and reduction of adverse effects could outweigh the risks. We characterized the major FDA-approved DMTs focusing on their therapeutic mechanism and the potential benefits of improving the accumulation of these drugs in the brain.

Mn-Based Methacrylated Gellan Gum Hydrogels for MRI-Guided Cell Delivery and Imaging.

This work aims to engineer a new stable injectable Mn-based methacrylated gellan gum (Mn/GG-MA) hydrogel for real-time monitored cell delivery into the central nervous system. To enable the hydrogel visualization under Magnetic Resonance Imaging (MRI), GG-MA solutions were supplemented with paramagnetic Mn2+ ions before its ionic crosslink with artificial cerebrospinal fluid (aCSF). The resulting formulations were stable, detectable by T1-weighted MRI scans and also injectable. Cell-laden hydrogels were prepared using the Mn/GG-MA formulations, extruded into aCSF for crosslink, and after 7 days of culture, the encapsulated human adipose-derived stem cells remained viable, as assessed by Live/Dead assay. In vivo tests, using double mutant MBPshi/shi/rag2 immunocompromised mice, showed that the injection of Mn/GG-MA solutions resulted in a continuous and traceable hydrogel, visible on MRI scans. Summing up, the developed formulations are suitable for both non-invasive cell delivery techniques and image-guided neurointerventions, paving the way for new therapeutic procedures.

3D-Printed Microinjection Needle Arrays via a Hybrid DLP-Direct Laser Writing Strategy.

Microinjection protocols are ubiquitous throughout biomedical fields, with hollow microneedle arrays (MNAs) offering distinctive benefits in both research and clinical settings. Unfortunately, manufacturing-associated barriers remain a critical impediment to emerging applications that demand high-density arrays of hollow, high-aspect-ratio microneedles. To address such challenges, here, a hybrid additive manufacturing approach that combines digital light processing (DLP) 3D printing with "ex situ direct laser writing (esDLW)" is presented to enable new classes of MNAs for fluidic microinjections. Experimental results for esDLW-based 3D printing of arrays of high-aspect-ratio microneedles-with 30 µm inner diameters, 50 µm outer diameters, and 550 µm heights, and arrayed with 100 µm needle-to-needle spacing-directly onto DLP-printed capillaries reveal uncompromised fluidic integrity at the MNA-capillary interface during microfluidic cyclic burst-pressure testing for input pressures in excess of 250 kPa (n = 100 cycles). Ex vivo experiments perform using excised mouse brains reveal that the MNAs not only physically withstand penetration into and retraction from brain tissue but also yield effective and distributed microinjection of surrogate fluids and nanoparticle suspensions directly into the brains. In combination, the results suggest that the presented strategy for fabricating high-aspect-ratio, high-density, hollow MNAs could hold unique promise for biomedical microinjection applications.

Childhood Brain Tumors: A Review of Strategies to Translate CNS Drug Delivery to Clinical Trials.

Brain and spinal tumors affect 1 in 1000 people by 25 years of age, and have diverse histological, biological, anatomical and dissemination characteristics. A mortality of 30-40% means the majority are cured, although two-thirds have life-long disability, linked to accumulated brain injury that is acquired prior to diagnosis, and after surgery or chemo-radiotherapy. Only four drugs have been licensed globally for brain tumors in 40 years and only one for children. Most new cancer drugs in clinical trials do not cross the blood-brain barrier (BBB). Techniques to enhance brain tumor drug delivery are explored in this review, and cover those that augment penetration of the BBB, and those that bypass the BBB. Developing appropriate delivery techniques could improve patient outcomes by ensuring efficacious drug exposure to tumors (including those that are drug-resistant), reducing systemic toxicities and targeting leptomeningeal metastases. Together, this drug delivery strategy seeks to enhance the efficacy of new drugs and enable re-evaluation of existing drugs that might have previously failed because of inadequate delivery. A literature review of repurposed drugs is reported, and a range of preclinical brain tumor models available for translational development are explored.

Application of optical coherence tomography in decision-making of post-thrombectomy adjunctive treatments.

An adult patient with acute basilar artery occlusion underwent mechanical thrombectomy. After complete reperfusion, a 70% residual stenosis of the proximal basilar artery was observed. Intravascular optical coherence tomography (OCT) identified lipid plaques with an intact fibrous cap and thrombus in the culprit lesion, indicating plaque erosion was the mechanism of in situ thrombosis. Adjunctive antiplatelet therapy rather than rescue interventions was pursued for its beneficial effects in acute coronary syndrome caused by plaque erosion. The patient had a 90-day modified Rankin Scale score of 0. OCT enables precise evaluation of vessel characteristics following thrombectomy, so may improve outcomes through subsequent tailored treatments.

What is the Accuracy of a Rapid Strip Test That Detects D-lactic Acid in Synovial Fluid for the Diagnosis of Periprosthetic Joint Infections?

BACKGROUND: D-lactic acid is a specific marker produced almost exclusively by bacterial species; thus, the appearance of this marker in synovial fluid may indicate periprosthetic joint infection (PJI). Recently, studies have investigated the accuracy of enzyme-linked laboratory tests that detect D-lactic acid in synovial fluid to diagnose PJI. However, to our knowledge, no studies have determined the usefulness of rapid strip tests that detect D-lactic acid in synovial fluid in the diagnosis of PJI. QUESTIONS/PURPOSES: (1) What is the best cutoff value for the rapid D-lactic acid strip test for diagnosing PJI? (2) What are the diagnostic accuracies (sensitivity, specificity, positive predictive value [PPV], and negative predictive value [NPV]) of the rapid D-lactic acid strip test and two different rapid leukocyte esterase (LE) strip tests? METHODS: This prospective study enrolled 157 patients who underwent revision THA or TKA from May 2021 to February 2022 at a single orthopaedic center. Seventy percent (110 of 157) were eligible for analysis; 10% of these patients (15 of 157) were excluded based on the exclusion criteria (causes of revisions and additional comorbidities that may interfere with the results), and 20% (32 of 157) of the synovial fluid samples could not be tested (dry taps and blood-contaminated samples that could not be centrifuged). We performed the following off-label diagnostic tests on synovial fluid samples collected from all patients: the D-lactic acid strip test (QuantiQuick TM , BioAssay System), two different LE strip tests (10 EA from ARKRAY and BM 10 from BioMaxima). Differently colored strips were marked with symbols (from [-] to [++++] for D-lactic acid and from [-] to [+++] for LE tests) according to the manufacturers' instructions. For the LE tests, results were different for (++), which corresponds to a minimal value of 250 leu/mL for 10 EA and 125 leu/mL for BM 10 tests. The diagnostic standard for the presence or absence of PJI in this study was the International Consensus Meeting (ICM) 2018 criteria; based on these criteria (without the application of an LE test as a minor criterion), all patients were assessed and divided into two groups. Patients who did not meet the criteria for PJI and underwent revision for aseptic loosening, implant malposition, instability, or implant damage were included in the aseptic revision total joint arthroplasty group (68 patients). Patients with a fistula penetrating the joint, those with two positive culture results of the same pathogen, or those with ≥ 6 points according to ICM 2018 minor criteria were enrolled in the PJI group (42 patients). To ascertain the best cutoff value for the rapid D-lactic acid and both LE strip tests for diagnosing PJI, we used collected results, generated a receiver operating characteristic curve, and calculated the Youden index. To determine the accuracies of the diagnostic tests, we calculated their sensitivities, specificities, PPVs, and NPVs against the diagnostic standard (the ICM 2018 criteria). RESULTS: The best cutoff value for D-lactic acid was 22.5 mg/L, which corresponded to a reading of (+) on the test strip. For D-lactic acid, in the diagnosis of PJI, the sensitivity was 83% (95% confidence interval [CI] 68% to 92%) and specificity was 100% (95% CI 93% to 100%). For both LE strip tests, the best cutoff value was the same as that proposed in the ICM 2018 criteria. For LE (10 EA), the sensitivity was 81% (95% CI 66% to 91%) and specificity was 99% (95% CI 91% to 100%); for LE (BM 10), sensitivity was 81% (95% CI 65% to 91%) and specificity was 97% (95% CI 89% to 100%). CONCLUSION: A rapid off-label D-lactic acid strip test is valuable for diagnosing PJI. The results of this study indicate very good accuracy with comparable sensitivity and specificity for both LE strip tests. The usefulness of the test in a group of patients with chronic inflammatory diseases and the reproducibility of the reading by different researchers were not analyzed in this study and require further investigations. Before a rapid D-lactic strip test is routinely used for diagnosing PJI, multicenter studies on a larger group of patients should be conducted.Level of Evidence Level II, diagnostic study.

Preserving cognitive function in patients with Alzheimer's disease: The Alzheimer's disease neuroprotection research initiative (ADNRI).

The global trend toward aging populations has resulted in an increase in the occurrence of Alzheimer's disease (AD) and associated socioeconomic burdens. Abnormal metabolism of amyloid-ß (Aß) has been proposed as a significant pathomechanism in AD, supported by results of recent clinical trials using anti-Aß antibodies. Nonetheless, the cognitive benefits of the current treatments are limited. The etiology of AD is multifactorial, encompassing Aß and tau accumulation, neuroinflammation, demyelination, vascular dysfunction, and comorbidities, which collectively lead to widespread neurodegeneration in the brain and cognitive impairment. Hence, solely removing Aß from the brain may be insufficient to combat neurodegeneration and preserve cognition. To attain effective treatment for AD, it is necessary to (1) conduct extensive research on various mechanisms that cause neurodegeneration, including advances in neuroimaging techniques for earlier detection and a more precise characterization of molecular events at scales ranging from cellular to the full system level; (2) identify neuroprotective intervention targets against different neurodegeneration mechanisms; and (3) discover novel and optimal combinations of neuroprotective intervention strategies to maintain cognitive function in AD patients. The Alzheimer's Disease Neuroprotection Research Initiative's objective is to facilitate coordinated, multidisciplinary efforts to develop systemic neuroprotective strategies to combat AD. The aim is to achieve mitigation of the full spectrum of pathological processes underlying AD, with the goal of halting or even reversing cognitive decline.

Label-Free Assessment of Mannitol Accumulation Following Osmotic Blood-Brain Barrier Opening Using Chemical Exchange Saturation Transfer Magnetic Resonance Imaging.

PURPOSE: Mannitol is a hyperosmolar agent for reducing intracranial pressure and inducing osmotic blood-brain barrier opening (OBBBO). There is a great clinical need for a non-invasive method to optimize the safety of mannitol dosing. The aim of this study was to develop a label-free Chemical Exchange Saturation Transfer (CEST)-based MRI approach for detecting intracranial accumulation of mannitol following OBBBO. METHODS: In vitro MRI was conducted to measure the CEST properties of D-mannitol of different concentrations and pH. In vivo MRI and MRS measurements were conducted on Sprague-Dawley rats using a Biospec 11.7T horizontal MRI scanner. Rats were catheterized at the internal carotid artery (ICA) and randomly grouped to receive either 1 mL or 3 mL D-mannitol. CEST MR images were acquired before and at 20 min after the infusion. RESULTS: In vitro MRI showed that mannitol has a strong, broad CEST contrast at around 0.8 ppm with a mM CEST MRI detectability. In vivo studies showed that CEST MRI could effectively detect mannitol in the brain. The low dose mannitol treatment led to OBBBO but no significant mannitol accumulation, whereas the high dose regimen resulted in both OBBBO and mannitol accumulation. The CEST MRI findings were consistent with 1H-MRS and Gd-enhanced MRI assessments. CONCLUSION: We demonstrated that CEST MRI can be used for non-invasive, label-free detection of mannitol accumulation in the brain following BBBO treatment. This method may be useful as a rapid imaging tool to optimize the dosing of mannitol-based OBBBO and improve its safety and efficacy.

Statin Use in Cancer Patients with Acute Myocardial Infarction and Its Impact on Long-Term Mortality.

Statin use and its impact on long-term clinical outcomes in active cancer patients following acute myocardial infarction (MI) remains insufficiently elucidated. Of the 1011 consecutive acute MI patients treated invasively between 2012 and 2017, cancer was identified in 134 (13.3%) subjects. All patients were observed within a median follow-up of 69.2 (37.8−79.9) months. On discharge, statins were prescribed less frequently in MI patients with cancer as compared to the non-cancer MI population (79.9% vs. 91.4%, p < 0.001). The most common statin in both groups was atorvastatin. The long-term mortality was higher in MI patients not treated vs. those treated with statins, both in non-cancer (29.5%/year vs. 6.7%/year, p < 0.001) and cancer groups (53.9%/year vs. 24.9%/year, p < 0.05), respectively. Patient's age (hazard ratio (HR) 1.04, 95% confidence interval (CI) 1.03−1.05, p < 0.001, per year), an active cancer (HR 2.42, 95% CI 1.89−3.11, p < 0.001), hemoglobin level (HR 1.14, 95% CI 1.09−1.20, p < 0.001, per 1 g/dL decrease), and no statin on discharge (HR 2.13, 95% CI 1.61−2.78, p < 0.001) independently increased long-term mortality. In MI patients, simultaneous diagnosis of an active cancer was associated with less frequently prescribed statins on discharge. Irrespective of cancer diagnosis, no statin use was found as an independent predictor of increased long-term mortality.