Caffeine Protects Against Retinal Inflammation

Caffeine, one of the most consumed central nervous system (CNS) stimulants, is an antagonist of A1 and A2A adenosine receptors. In this study, we investigated the potential protective effects of this methylxanthine in the retinal tissue. We tested caffeine by using in vitro and in vivo paradigms of retinal inflammation. Human retinal pigment epithelial cells (ARPE-19) were exposed to lipopolysaccharide (LPS) with or without caffeine. This latter was able to reduce the inflammatory response in ARPE-19 cells exposed to LPS, attenuating the release of IL-1β, IL-6, and TNF-α and the nuclear translocation of p-NFκB. Additionally, caffeine treatment restored the integrity of the ARPE-19 monolayer assessed by transepithelial electrical resistance (TEER) and the sodium fluorescein permeability test. Finally, the ischemia reperfusion (I/R) injury model was used in C57BL/6J mice to induce retinal inflammation and investigate the effects of caffeine treatment. Mouse eyes were treated topically with caffeine, and a pattern electroretinogram (PERG) was used to assess the retinal ganglion cell (RGC) function; furthermore, we evaluated the levels of IL-6 and BDNF in the retina. Retinal BDNF dropped significantly (p < 0.05) in the I/R group compared to the control group (normal mice); on the contrary, caffeine treatment maintained physiological levels of BDNF in the retina of I/R eyes. Caffeine was also able to reduce IL-6 mRNA levels in the retina of I/R eyes. In conclusion, these findings suggest that caffeine is a good candidate to counteract inflammation in retinal diseases.

Determining the Mechanism of Propofol-induced Neurotoxicity

Background and Hypothesis: Propofol is an IV anesthetic agent with many clinical applications, including general anesthesia, sedation, delirium, and palliative care. Despite its versatility and effectiveness, Propofol has been shown to have detrimental effects on the vascular interface between the brain and circulation. The Blood-Brain Barrier (BBB) acts as a selective interface that serves to protect the brain from its cerebrovascular network. BBB dysfunction can have lethal complications such as cerebral edema and stroke. Matrix Metalloproteinases (MMP’s), specifically MMP-2 and MMP-9 have been linked to BBB breakdown. Using human induced pluripotent stem cell (IPSC) derived brain microvascular endothelial cells (BMECs), we evaluated the impact of inhibiting MMP-2 on restoring BBB integrity following exposure to Propofol with the intent to reveal the mechanism by which Propofol disrupts the BBB. We hypothesized that inhibiting MMP-2 would lead to phenotype recovery after exposure to Propofol. Methods: IPSC-differentiated BMECs were treated with MMP inhibitors at varying time points and concentrations relative to exposure to Propofol for 3 hours. Trans-endothelial electrical resistance (TEER) and sodium fluorescein permeability was used to assess BBB structural integrity. A MTT assay was conducted to assess cell viability. Results: Inhibiting MMP-2 did not result in in recovery of BBB integrity following exposure to Propofol as no significant differences were observed in TEER and sodium fluorescein permeability between the Propofol control and Propofol + MMP-2 inhibitor groups. Conclusions & Future Directions: Propofol-induced disruption of BBB integrity does not appear to be through MMP-2 activity. High concentrations of MMP-inhibitor compounds result in increased disruption of BBB tightness and permeability. It is plausible to suspect that Propofol may act through other MMP’s to facilitate BBB break-down, thus future studies should investigate the effects of the other selective MMP inhibitors in their ability to achieve phenotype recovery following exposure to Propofol.

Inhibition of Neovascularization and Inflammation in a Mouse Model of Corneal Alkali Burns Using Cationic Liposomal Tacrolimus

Eye drops account for more than 90% of commercialized ophthalmic drugs. However, eye drops have certain shortcomings, such as short precorneal retention time and weak corneal penetration. The requirement of frequent instillation of eye drops also causes poor patient compliance, which may lead to further aggravation of the disease. We aimed to develop a cationic liposome formulation to increase the bioavailability of the therapeutic agent and solve the aforementioned problems. In the present study, we prepared cationic liposomal tacrolimus (FK506) with a surface potential of approximately +30 mV, which could bind to the negatively charged mucin layer of the ocular surface. Our results showed that the content of FK506 in the cornea was increased by 93.77, 120.30, 14.24, and 20.36 times at 5, 30, 60, and 90 min, respectively, in the FK506 liposome group (0.2 mg/ml) compared with the free drug group (0.2 mg/ml). Moreover, FITC-labeled FK506 liposomes significantly prolonged the ocular surface retention time to 50 min after a single dose. In addition, the results of the Cell Counting Kit-8 assay, live and dead cell assay, sodium fluorescein staining, and hematoxylin and eosin staining all indicated that FK506 liposomes had good biological compatibility in both human corneal epithelial cells and mouse eyeballs. Compared with the free drug at the same concentration, FK506 liposomes effectively inhibited vascular endothelial growth factor-induced green fluorescent protein-transduced human umbilical vein endothelial cell migration and tube formation in vitro. In a mouse corneal neovascularization model induced by alkali burns, FK506 liposomes (0.2 mg/ml) enhanced corneal epithelial recovery, inhibited corneal neovascularization, and reduced corneal inflammation, and its therapeutic effect was better than those of the commercial FK506 eye drops (1 mg/ml) and the free drug (0.2 mg/ml). Collectively, these results indicate that cationic FK506 liposomes could increase the efficacy of FK506 in the corneal neovascularization model. Therefore, cationic FK506 liposomes can be considered as a promising ocular drug delivery system.

Combined Application of Sodium Fluorescein and Neuronavigation Techniques in the Resection of Brain Gliomas

Objectives: To explore the effectiveness and safety of the combined application of sodium fluorescein and neuronavigation techniques in the resection of brain gliomas in different locations and patients of different ages.Methods: Fifty clinical cases of brain gliomas treated at the Department of Neurosurgery of Beijing Tiantan Hospital were collected from March 2014 to March 2019. These cases were divided into a supratentorial group (24 cases) and a brainstem group (26 cases) based on location and an adult group (28 cases) and a pediatric group (22 cases) based on age. Fluorescein-guided surgery was performed: the adult group received 5 mg/kg sodium fluorescein before opening the dura, while the pediatric group received 2.5 mg/kg during resection. Tumor visualization was evaluated by the enhancement of yellow fluorescein and considered “satisfactory” if the illumination demarcated the tumor boundary. Additionally, the consistency between fluorescein and neuronavigation was analyzed. The Karnofsky performance score (KPS) of all patients was recorded and assessed at admission, discharge, and the 6-month follow-up.Results: In the 28 adult cases, 4 were unsatisfactory, while in the 22 pediatric cases, 2 were unsatisfactory; in 7 cases, there was an inconsistency between yellow fluorescein enhancement and neuronavigation, 6 were in the supratentorial group, and 1 was in the brainstem group. Statistical analysis showed no significant differences in the satisfactory rate between the adult and pediatric groups (P = 0.575), whereas there were significant differences inconsistency between the supratentorial group and brainstem group (P = 0.031). The mean KPS at admission was between 70 and 100, which was not significantly different from that at discharge (P = 0.839), but the KPS at the 6-month follow-up was significantly higher than that at admission (P = 0.041).Conclusions: The consistency between sodium fluorescein and the neuronavigation system was higher in the brainstem group than in the supratentorial group; a half dose of sodium fluorescein (2.5 mg/kg) was sufficient for pediatric patients. The combined utilization of sodium fluorescein and neuronavigation techniques may confer glioma patients the opportunity to obtain better clinical outcomes after surgery.

Two-step versus 1-step subretinal injection to compare subretinal drug delivery: a randomised study protocol

IntroductionThere is increasing interest in subretinal injections as a surgical procedure, largely as a result of emerging treatments for ocular diseases which necessitate this manoeuvre. However, surgical variables in the efficacy of such treatments have to date been largely overlooked and the proportion of drug which reaches the intended compartment of the subretinal space remains unknown. Our aims are twofold: first, to determine the proportion of subretinally injected medication retained following surgical delivery and second, to compare two different techniques of injection (‘1-step’ vs ‘2-step’).MethodsWe outline a randomised controlled trial of subretinal injection of alteplase following vitrectomy for the management of submacular haemorrhage secondary to age-related macular degeneration. Patients will be randomised to receive either 1-step injection, where the therapeutic solution simultaneously defines the surgical plane or 2-step injection, where the surgical plane is first identified with balanced salt solution prior to injection of subretinal alteplase, as outlined below. Sodium fluorescein will be used as an optical label to track drug reflux into the vitreous cavity using quantitative protocols established in our laboratory. All patients will undergo fluid air exchange at the completion of surgery, with injection of bevacizumab 1.25 mg and 20% sulfahexafluoride gas as the vitreous substitute (both of which may help improve outcomes). Alteplase, sodium fluorescein and bevacizumab will all be used for off-label indications in the trial.Ethics and disseminationEthical approval has been obtained from the South Eastern Sydney Local Health District’s Human Research Ethics Committee (HREC 17/092). The results of this trial will be disseminated in peer-reviewed proceedings (associated with conference presentation) and in scholarly journals.Trial registration numberACTRN12619001121156.

Modeling ischemic stroke in a triculture neurovascular unit on-a-chip

Background In ischemic stroke, the function of the cerebral vasculature is impaired. This vascular structure is formed by the so-called neurovascular unit (NVU). A better understanding of the mechanisms involved in NVU dysfunction and recovery may lead to new insights for the development of highly sought therapeutic approaches. To date, there remains an unmet need for complex human in vitro models of the NVU to study ischemic events seen in the human brain. Methods We here describe the development of a human NVU on-a-chip model using a platform that allows culture of 40 chips in parallel. The model comprises a perfused vessel of primary human brain endothelial cells in co-culture with induced pluripotent stem cell derived astrocytes and neurons. Ischemic stroke was mimicked using a threefold approach that combines chemical hypoxia, hypoglycemia, and halted perfusion. Results Immunofluorescent staining confirmed expression of endothelial adherens and tight junction proteins, as well as astrocytic and neuronal markers. In addition, the model expresses relevant brain endothelial transporters and shows spontaneous neuronal firing. The NVU on-a-chip model demonstrates tight barrier function, evidenced by retention of small molecule sodium fluorescein in its lumen. Exposure to the toxic compound staurosporine disrupted the endothelial barrier, causing reduced transepithelial electrical resistance and increased permeability to sodium fluorescein. Under stroke mimicking conditions, brain endothelial cells showed strongly reduced barrier function (35-fold higher apparent permeability) and 7.3-fold decreased mitochondrial potential. Furthermore, levels of adenosine triphosphate were significantly reduced on both the blood- and the brain side of the model (4.8-fold and 11.7-fold reduction, respectively). Conclusions The NVU on-a-chip model presented here can be used for fundamental studies of NVU function in stroke and other neurological diseases and for investigation of potential restorative therapies to fight neurological disorders. Due to the platform’s relatively high throughput and compatibility with automation, the model holds potential for drug compound screening.

FLGS-03. Fluorescein sodium-guided biopsy or resection in patients with contrast enhancing brainstem lesion in MRI

BACKGROUND It is challenging to resect or just biopsy the lesions in the brainstem, due to the essential function of the surfaces and limited space. Neuro-navigation is not always reliable and stereotatic biopsy is infrequently inconclusive due to small or inadequate samples. We want to share our experiences in the application of fluorescein sodium in surgery on patients with brainstem lesion which is contrast enhancing in MRI. METHODS Between January 2017 and June 2021, 5 patients with brainstem lesion underwent fluorescein sodium-guided surgery in neurosurgery department of Sun Yat-sen University Cancer Center. After injection of low dose of sodium fluorescein (3 mg/kg), the lesions with strong fluorescence staining were identified as the target area for biopsy or resection. RESULTS 5 consecutive patients (aged 6–47 years) with brainstem lesions prospectively underwent fluorescein sodium-guided surgery. The lesions were located in pontine in 3 patients and in the medulla in 2 patients. Gross total resection was achieved in 2 patients, and partial resection in the other 3 patients. In all patients, a pathological diagnosis was obtained (4 gliomas and 1 metastasis from non-small cell lung carcinoma) without severe complications, including mild facial or abduct nerve palsy in 3 patients. And all the specimens with strong fluorescence staining sent for pathology were proved to be tumorous. CONCLUSION Fluorescein sodium-guided technique was helpful to locate the lesion in brainstem which was contrast-enhanced in MRI. It was effective and safe to figure out an ideal trajectory to avoid damage of the crucial structures and improve the diagnostic rate.

EXTH-36. ELECTROCONVULSIVE SEIZURE-INDUCED CHANGES IN THE TUMOR MICROENVIRONMENT PROMOTE SURVIVAL IN GLIOMA-BEARING MICE

PURPOSE Growing evidence indicates that the neurotransmitters dysregulated in psychiatric disorders are similarly dysregulated in glioblastoma (GBM) biology. GBM cells are dependent on bountiful neuronal glutamate, utilize elevated dopamine receptor expression to augment progression, and catabolize serotonin to drive proliferation and inhibit anti-tumor immunity. The clinical induction of seizure, known as electroconvulsive therapy (ECT), has been used by psychiatrists since the 1930s to correct these dysregulations and can additionally improve medication blood-brain barrier (BBB) penetrance. We hypothesized that seizure-induced changes in the glioma microenvironment occur with ECT, slowing tumor progression, increasing BBB permeability, and prolonging overall survival in glioma-bearing mice. METHODS C57BL6 mice were orthotopically injected with CT-2A-Luc mouse glioma cells. Mice were randomized to receive ECT via ear-clip electrodes or sham treatment daily up to five times per week. Intracranial progression was monitored via bioluminescent signal from CT-2A-Luc xenografts. BBB permeability was assessed by subjecting mice to ECT or sham treatment immediately following intravenous injection of sodium fluorescein. RESULTS Intracranial progression was maximally reduced in ECT-treated mice relative to sham-treated mice after 17 ECT treatments (ECT radiance 2.6 x 109 photons/second versus sham 4.7 x 109 photons/second, p=0.013), which was further confirmed by both decreased tumor weight and tumor size on histologic evaluation. This translated into an improvement in overall survival from median 29 days in sham-treated mice to 38 days in ECT-treated mice (p=0.0018). Mean seizure duration was 41.8 seconds and positively correlated with overall survival (Pearson coefficient r=0.63, p=0.028). Brain parenchymal uptake of sodium fluorescein was significantly higher in ECT-treated mice (mean relative increase in ECS to sham radiance of 1.47, p< 0.05). CONCLUSION Repeated ECT slows tumor progression and prolongs overall survival in C57BL6 mice bearing CT-2A-Luc xenografts. The BBB is compromised immediately following ECT. ECT merits further oncologic investigation as a potential therapeutic in GBM.

Effects of 1,2-Dimethylhydrazine on Barrier Properties of Rat Large Intestine and IPEC-J2 Cells

Colon cancer is accompanied by a decrease of epithelial barrier properties, which are determined by tight junction (TJ) proteins between adjacent epithelial cells. The aim of the current study was to analyze the expression of TJ proteins in a rat model of 1,2-dimethylhydrazine (DMH)-induced colorectal cancer, as well as the barrier properties and TJ protein expression of IPEC-J2 cell monolayers after incubation with DMH. Transepithelial electrical resistance and paracellular permeability for sodium fluorescein of IPEC-J2 were examined by an epithelial volt/ohm meter and spectrophotometry. The expression and localization of TJ proteins were analyzed by immunoblotting and immunohistochemistry. In the colonic tumors of rats with DMH-induced carcinogenesis, the expression of claudin-3 and -4 was significantly increased compared to controls. The transepithelial electrical resistance of IPEC-J2 cells increased, while paracellular permeability for sodium fluorescein decreased, accompanied by an increased expression of claudin-4. The increase of claudin-4 in rat colon after chronic DMH exposure was consistent with the acute effect of DMH on IPEC-J2 cells, which may indicate an essential role of this protein in colorectal cancer development.