Intracarotid Injection Research Articles

Abstract 3161: PCTK1 driven glutamine metabolic adaptation in brain metastasis

Abstract Brain metastasis (BrM) is the most common malignancy of the central nervous system and the median survival time of patients with BrM is less than 1 year. Although recent impressive advances in targeted therapy and immunotherapy enable more effective control of the systemic diseases, the incidence of BrM upon disease recurrence is steadily increasing. Unfortunately, there is no effective treatments for BrM. To surmount this challenge, we have performed an in vivo human kinome screening to uncover novel kinases that promote breast cancer (BC) BrMs in mice and may serve as therapeutic targets. We identified >20 different kinases enriched in BrM lesions of our mouse models and PCTK1 is one of the top “hits”. We transduced PCTK1 expression vector and the control vector into MDA-MB-231 human BC cells to generate 231.PCTK1 and 231.vector cells, respectively. Intra-carotid injection of the 231.PCTK1 cells into mice induced BrM significantly faster with shortened mouse survival than that of the control 231.vector cells, confirming PCTK1 as a BrM-promoting kinase. However, when 231.vector or 231.PCTK1 cells were injected into the mouse’ mammary glands, PCTK1 overexpression did not lead to enhanced primary tumor growth, suggesting that its pro-tumoral functions are BrM-specific. To uncover how PCTK1 promotes BrM but not primary tumor, we examined whether PCTK1 may confer growth advantage in BrM by utilizing the unique metabolic microenvironment of the brain. We analyzed the relationship between PCTK1 gene expression and gene expression of members of the glutamine metabolism pathway in TCGA database. We found that in the brain tumors, such as low-grade glioma, PCTK1 positively correlated with the expression of glutamine metabolizing genes, including glutaminase 1 and 2 (GLS and GLS2). To evaluate whether PCTK1-overexpressing cells can uptake glutamine from the media directly, we cultured 231.PCTK1 and 231.vector cells in low-nutrient BME media plus 5% d.FBS, with addition of different concentrations of glutamine. The PCTK1-overexpressing cells showed a clear growth advantage at glutamine concentrations ranging from 400 μM to 2 mM. Then, we knocked down PCTK1 in MDA-MB-231 cells by shRNAs and found that PCTK1 knocking down reduced the cell proliferation in 5% d.FBS.BME with 500 µM glutamine, a physiological glutamine level in the brain. We measured the expression of GLS, an enzyme that converts glutamine to glutamate, one of the key steps in the glutamine metabolism, and found that knockdown PCTK1 in MDA-MB-231 cells dramatically reduced the GLS expression, which indicates that PCTK1 can modulate glutamine metabolism in cancer cells. In summary, our data suggest that PCTK1 promotes BC BrM, at least partly, through increasing glutamine metabolism. Currently, we are exploring whether targeting PCTK1 or inhibiting glutamine metabolism can deter BrM progression. Our studies may reveal novel therapeutic targets for BrM inhibition. Citation Format: Lin Zhang, Frank Lowery, Chenyu Zhang, Yu-Wen Huang, Ya-Jing Jiang, Yimin Duan, Dihua Yu. PCTK1 driven glutamine metabolic adaptation in brain metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3161.

Interleukin 17A Contributes to Blood-Brain Barrier Disruption of Hypothalamic Paraventricular Nucleus in Rats With Myocardial Infarction.

Elevated inflammatory cytokines in the periphery have been identified as active contributors to neuroinflammation and sympathetic overactivity in heart failure (HF). Yet, the exact mechanisms by which these cytokines breach the blood-brain barrier (BBB) to exert their effects on the brain remain elusive. Interleukin 17A has been linked to BBB disruption in various neurologic disorders, and its levels were significantly augmented in circulation and the brain in HF. The present study aimed to determine whether the BBB integrity was compromised within the hypothalamic paraventricular nucleus (PVN), and if so, whether interleukin 17A contributes to BBB disruption in myocardial infarction-induced HF. Male Sprague-Dawley rats underwent coronary artery ligation to induce HF or sham surgery. Some HF rats received bilateral PVN microinjections of an interleukin 17 receptor A small interfering RNA or a scrambled small interfering RNA adeno-associated virus. Four weeks after coronary artery ligation, the permeability of the BBB was evaluated by intracarotid injection of fluorescent dyes (fluorescein isothiocyanate-dextran 10 kDa+rhodamine-dextran 70 kDa). Compared with sham-operated rats, HF rats exhibited an elevated extravasation of fluorescein isothiocyanate-dextran 10 kDa within the PVN but not in the brain cortex. The plasma interleukin 17A levels were positively correlated with fluorescein isothiocyanate 10 kDa extravasation in the PVN. The expression of caveolin-1, a transcytosis marker, was augmented, whereas the expression of tight junction proteins was diminished in HF rats. Interleukin 17 receptor A was identified within the endothelium of PVN microvessels. Treatment with interleukin 17 receptor A small interfering RNA led to a significant attenuation of fluorescein isothiocyanate 10 kDa extravasation in the PVN and reversed expression of caveolin-1 and tight junction-associated proteins in the PVN. Collectively, these data indicate that BBB permeability within the PVN is enhanced in HF and is likely attributable to increased interleukin 17A/interleukin 17 receptor A signaling in the BBB endothelium, by promoting caveolar transcytosis and degradation of tight junction complexes.

TRPV1 on the endings of thin fiber muscle afferents is not needed to evoke the exercise pressor reflex

Laura Anselmi, Victor Ruiz-Velasco, Sean D. Stocker, Shannon P. Higgins, Guillaume P. Ducrocq and Marc P. Kaufman. The role played by the transient receptor potential vallinoid-1 (TRPV1) on the endings of thin fiber muscle afferents in evoking the exercise pressor reflex is controversial. To shed light on this controversy, we used TRPV1-/- rats that were made by a CRISPR-Cas9 deletion to introduce a 26-bp frameshift deletion in exon 3. We compared the magnitude of the reflex between TRPV1 +/+ wild-type rats (WT), TRPV1+/- heterozygous rats (HET), and TRPV1-/- knockout rats (KO). The exercise pressor reflex was evoked by stimulating the tibial nerve (40Hz, 0.01ms, 1.5 times motor threshold) in precollicular decerebrated unanesthetized rats whose femoral arteries were either freely perfused or were occluded for 30 sec before the start of contraction. We found that there was no difference between the magnitude of the reflex in the three groups of rats with freely perfused hindlimbs (p=0.61). Specifically, the exercise pressor reflex averaged 16.4 ± 2.2 mmHg in WT rats (n=9), 21.1 ± 5 mmHg in HET rats (n=9), and 17.7 ± 2.3 mmHg in KO rats (n=9). No difference was also observed among the three groups of rats that received acute femoral artery occlusion (p=0.79). Specifically, the peak exercise pressor reflex averaged 15.3 ± 2.2 mmHg in WT rats (n=4), 19.8 ± 4.7 mmHg in HET rats (n=6), and 19 ± 8 mmHg in KO rats (n=3). Stimulation of the tibial nerve after paralysis of the rats with pancuronium (iv) had trivial effects on arterial pressure indicating that the pressor responses to contraction were not caused by electrical stimulation within the axon of the tibial nerve. Intra-carotid arterial injection of the TRPV1 agonist, capsaicin (0.5 mg), evoked a significant pressor response in the WT rats (34.7 ± 9.2 mmHg) and in the HET rats (36.3 ± 7.2 mmHg), but not in the KO rats (3.2 ± 0.4 mmHg). In electrophysiological studies of dorsal root ganglion cells innervating the gastrocnemius muscles, capsaicin evoked inward currents in the WT and HET rats, but not in the KO rats. Moreover, immunofluorescence revealed the presence of TRPV1 in the DRG of WT, but not in the DRG of KO rats. We conclude that TRPV1 is not needed to evoke the exercise pressor reflex either in rats whose femoral arteries were either freely perfused or were occluded. These results are consistent with our previous finding that pharmacological antagonism of TRPV1 had no effect on the exercise pressor reflex. Supported by HL156594 and HL 156513 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Proof of Principle for Intracarotid Injection of Leukemia As Model for Late Stage Central Nervous System Leukemia

Proof of Principle for Intracarotid Injection of Leukemia As Model for Late Stage Central Nervous System Leukemia

Unintentional intracarotid injections in the horse—15 cases (2010–2020)

SummaryBackgroundIntracarotid injections are a well‐recognised complication of jugular injections in the horse; however, little information is available about outcome and complications.ObjectivesThe aim of this study was to describe venipuncture techniques and short‐ and longer‐term complications related to intracarotid injections.Study designCase series.MethodsThe survey was distributed through the American College of Veterinary Internal Medicine (ACVIM) mailing list and a veterinary social media site. Data collected in the questionnaire included injection technique, drug administered, personnel performing the injection, clinical signs and short‐ and longer‐term complications.ResultsAccidental intracarotid injections were most commonly performed by veterinarians (n = 9/13). The majority of reported accidental intracarotid injections occurred during routine sedation in healthy horses (n = 8/13) and occurred when the jugular vein was accessed at the middle jugular groove (n = 6/13). Most events occurred while using longer needles (1.5 inches; n = 10/13) of 20G calibre (n = 9/13), attached to the syringe (n = 11/13) and against jugular blood flow (n = 11/13). Short‐term injuries included superficial abrasions (n = 13/13) and cardiac ailments (3/13). One of the 15 horses described in this report died. Longer‐term complications were not reported for the remaining 14 horses.Main limitationsPotential selection, recall and response bias and putative risk factors identified cannot be correlated with an increased risk of carotid injection without a control group.ConclusionAccidental intracarotid injections led to seizures but rarely resulted in death of the case. Premonitory clinical signs occurred while or soon after injection and can be used for early identification of this inadvertent injection and to establish safety measures for the horse and handlers.

Abstract 2675: Discovery of potent CNS-penetrant covalent KRAS G12C inhibitors

Abstract The RAS family of GTPases, which include KRAS, NRAS, and HRAS, are key regulators of cellular signaling, acting as molecular switches downstream of receptor tyrosine kinases (RTKs) and as key regulators of the RAS/MAPK pathway. RAS cycles between inactive (GDP) and active (GTP) states to regulate the activity of the RAS/MAPK pathway. In many cancers, activating KRAS mutations enable KRAS to persist in the active GTP-bound state, resulting in hyperactive RAS/MAPK pathway signaling that drives cell growth and survival. The KRAS G12C mutation occurs in 14% of lung adenocarcinoma, a cancer type that frequently metastasizes to the brain (40%). To address the high prevalence of KRAS G12C mutant CNS metastases in lung adenocarcinoma, we are developing CNS-penetrant covalent KRAS G12C inhibitors (“ERAS G12Ci’s”) to treat both CNS and systemic disease. ERAS G12Ci’s rapidly form covalent adducts with the KRAS G12C protein in its inactive GDP state and exhibit low nanomolar IC50’s in a nucleotide exchange assay. ERAS G12Ci’s demonstrate high selectivity in a cell-based proteome selectivity assay and inhibit the growth of RAS Initiative KRAS G12C mutant cells, and not RAS Initiative KRAS WT cells. ERAS G12Ci’s potently inhibit cell proliferation in 3-dimensional Cell-Titer Glo (3D-CTG) assays in KRAS G12C mutant lung and pancreatic cell lines (NCI-H1373, NCI-H2122, and MIA PaCa-2). Mechanistically, ERAS G12Ci’s blocked RAS-RAF complex formation and inhibited ERK1/2 phosphorylation. This in vitro activity translates in vivo where ERAS G12Ci’s induce pharmacodynamic modulation in the pancreatic cancer MIA PaCa-2 model. ERAS G12Ci’s significantly inhibit tumor growth in NCI-H1373 and NCI-H2122 CDX lung adenocarcinoma and MIA PaCa-2 PDAC models. No meaningful body weight loss or clinical adverse events were observed with any of these compounds. Human efflux transporter substrate assessments indicate that ERAS G12Ci’s are either not substrates or weak/modest substrates of P-gp, a crucial efflux transporter that can limit CNS penetration. In rat CNS studies, these inhibitors exhibit good CNS penetration performance as measured by brain-to-plasma partition coefficients, which are comparable to those of approved CNS-active small molecule inhibitors. Daily oral administration demonstrates dose-dependent tumor regression in intracranial and intra-carotid injection (ICA) KRAS G12C CDX models. We are optimizing multiple covalent CNS-penetrant KRAS G12C inhibitors that exhibit both CNS and systemic activity in vivo. Citation Format: Jae Hyun Bae, Erin D. Lew, Jun Feng, Marcos Gonzalez-Lopez, Joanne Oh, Patrick Fagan, Matt Salie, Nick Isley, Richard Lam, Adriana Irimia, Robin Nevarez, Bingzhen Lin, Taylor Congdon, Jingchuan Zhang, Dawei Xuan, Ping Chen, Jean-Michel Vernier, Robert Shoemaker. Discovery of potent CNS-penetrant covalent KRAS G12C inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2675.

Porcine model of neurocysticercosis by intracarotid injection of Taenia solium oncospheres: Dose assessment, infection outcomes and serological responses.

BackgroundNeurocysticercosis (NCC) is the infection of the human central nervous system (CNS) by Taenia solium larvae that cause significant neurological morbidity. Studies on NCC pathophysiology, host-parasite interactions or therapeutic agents are limited by the lack of suitable animal models. We have previously reported that carotid injection of activated T. solium oncospheres directs parasites into the CNS and consistently reproduces NCC. This study assessed the minimal dose required to consistently obtain NCC by intracarotid oncosphere injection and compared antigen and antibody response profiles by dose-group.Methods/Principal findingsThree groups of pigs were infected with either 2500 (n = 10), 5000 (n = 11), or 10000 (n = 10) oncospheres. Two pigs died during the study. Necropsy exam at day 150 post-infection (PI) demonstrated viable NCC in 21/29 pigs (72.4%), with higher NCC rates with increasing oncosphere doses (4/9 [44.4%], 9/11 [81.8%] and 8/9 [88.9%] for 2500, 5000, and 10000 oncospheres respectively, P for trend = 0.035). CNS cyst burden was also higher in pigs with increasing doses (P for trend = 0.008). Viable and degenerated muscle cysticerci were also found in all pigs, with degenerated cysticerci more frequent in the 2500 oncosphere dose-group. All pigs were positive for circulating parasite antigens on ELISA (Ag-ELISA) from day 14 PI; circulating antigens markedly increased at day 30 PI and remained high with plateau levels in pigs infected with either 5000 or 10000 oncospheres, but not in pigs infected with 2500 oncospheres. Specific antibodies appeared at day 30 PI and were not different between dose-groups.Conclusion/SignificanceIntracarotid injection of 5000 or more oncospheres produces high NCC rates in pigs with CNS cyst burdens like those usually found in human NCC, making this model appropriate for studies on the pathogenesis of NCC and the effects of antiparasitic treatment.

Pre-Clinical Proof of Concept: Intra-Carotid Injection of Autologous CD34-Positive Cells for Chronic Ischemic Stroke.

This study was conducted to evaluate the safety and efficacy of human peripheral blood CD34 positive (CD34+) cells transplanted into a murine chronic stroke model to obtain pre-clinical proof of concept, prior to clinical testing. Granulocyte colony stimulating factor (G-CSF) mobilized human CD34+ cells [1 × 104 cells in 50 μl phosphate-buffered saline (PBS)] were intravenously (iv) or intra-carotid arterially (ia) transplanted 4 weeks after the induction of stroke (chronic stage), and neurological function was evaluated. In this study, severe combined immune deficiency (SCID) mice were used to prevent excessive immune response after cell therapy. Two weeks post cell therapy, the ia CD34+ cells group demonstrated a significant improvement in neurological functions compared to the PBS control. The therapeutic effect was maintained 8 weeks after the treatment. Even after a single administration, ia transplantation of CD34+ cells had a significant therapeutic effect on chronic stroke. Based on the result of this pre-clinical proof of concept study, a future clinical trial of autologous peripheral blood CD34+ cells administration in the intra-carotid artery for chronic stroke patients is planned.

Dynamic Observation: Immune-Privileged Microenvironment Limited the Effectiveness of Immunotherapy in an Intraocular Metastasis Mouse Model

Introduction: Intraocular metastasis (IM) occurred in approximately 8–10% of patients with metastatic malignancy, for whom oncological immunotherapies showed poor visual potential. However, the mechanism for that inefficiency remains unclear and requires further exploration. Methods: We established a novel mouse model of IM by intracarotid injection of cutaneous melanoma cells. We investigated disease progression using ophthalmic and histological examinations. We used combined anti-PD-1 and anti-CTLA4 antibodies for immunotherapy and evaluated the therapeutic effects in the mouse model. In addition, we characterized the immune microenvironment of tumor-infiltrating CD8+ T by fluorescence staining and assessed their cytotoxicity by flow cytometry. Results: All mice presented IM in the left eye, while the right eye was healthy. Uveal tissues with rich vascularity (e.g., the iris, ciliary body, and choroid) initiated IM at an early stage, and IM development resulted in several secondary changes, including corneal swelling, retinal detachment, and intratumoral hemorrhage. Immunotherapy could inhibit IM and prolong the time to eye rupture but did not prevent rupture ending. This inefficiency might be attributed to ocular tissues specificities that inhibited CD8+ T-cell infiltration via PD-L1 expression. PD-L1low corneal tissue resisted tumor invasion with high levels of CD8+ T-cell infiltration, whereas CD8+ T cells were deficient in PD-L1high uveal metastasis. Furthermore, we found a significantly increased PD-1+/− CD4+ and PD-1+/− CD8+ T cells infiltrating the intratumoral hemorrhage area. Although these CD8+ T cells in the IM were not exhausted and had a higher capacity of cytotoxicity (higher interferon-γ ratio) than CD8+ T cells in the blood, FasL+ PD-L1+ ocular tissue can strongly inhibit these IM-infiltrating T cells. Conclusions: Immunotherapy can inhibit the disease progression of IM. Enhancing the effects of tumor-infiltrating CD8+ T cells should be one of the highest potentials to improve the visual potential.

Lack of Neuroprotective Effects of High-Density Lipoprotein Therapy in Stroke under Acute Hyperglycemic Conditions.

Introduction: The pleiotropic protective effects of high-density lipoproteins (HDLs) on cerebral ischemia have never been tested under acute hyperglycemic conditions. The aim of this study is to evaluate the potential neuroprotective effect of HDL intracarotid injection in a mouse model of middle cerebral artery occlusion (MCAO) under hyperglycemic conditions. Methods: Forty-two mice were randomized to receive either an intracarotid injection of HDLs or saline. Acute hyperglycemia was induced by an intraperitoneal injection of glucose (2.2 g/kg) 20 min before MCAO. Infarct size (2,3,5-triphenyltetrazolium chloride (TTC)-staining), blood–brain barrier leakage (IgG infiltration), and hemorrhagic changes (hemoglobin assay by ELISA and hemorrhagic transformation score) were analyzed 24 h post-stroke. Brain tissue inflammation (IL-6 by ELISA, neutrophil infiltration and myeloperoxidase by immunohisto-fluorescence) and apoptosis (caspase 3 activation) were also assessed. Results: Intraperitoneal D-glucose injection allowed HDL- and saline-treated groups to reach a blood glucose level of 300 mg/dl in the acute phase of cerebral ischemia. HDL injection did not significantly reduce mortality (19% versus 29% in the saline-injected group) or cerebral infarct size (p = 0.25). Hemorrhagic transformations and inflammation parameters were not different between the two groups. In addition, HDL did not inhibit apoptosis under acute hyperglycemic conditions. Conclusion: We observed a nonsignificant decrease in cerebral infarct size in the HDL group. The deleterious consequences of reperfusion such as hemorrhagic transformation or inflammation were not improved by HDL infusion. In acute hyperglycemia, HDLs are not potent enough to counteract the adverse effects of hyperglycemia. The addition of antioxidants to therapeutic HDLs could improve their neuroprotective capacity.

In vivo two-photon characterization of tumor-associated macrophages and microglia (TAM/M) and CX3CR1 during different steps of brain metastasis formation from lung cancer.

Brain metastases frequently occur in lung cancer and dramatically limit prognosis of affected patients. The influence of tumor-associated macrophages and microglia (TAM/M) and their receptor CX3CR1 on different steps of brain metastasis formation from lung cancer is poorly characterized. We established a syngeneic orthotopic cerebral metastasis model in mice by combining a chronic cranial window with repetitive intravital 2-photon laser scanning microscopy. This allowed in vivo tracking of fluorescence-expressing tumor cells and TAM/M on a single-cell level over weeks. Intracarotid injection of red tdTomato-fluorescent Lewis lung carcinoma cell was performed in transgenic mice either proficient or deficient for CX3CR1. After intracarotid cell injection, intravascular tumor cells extravasated into the brain parenchyma and formed micro- and mature macrometastases. We observed potential phagocytosis of extravasated tumor cells by TAM/M. However, during later steps of metastasis formation, these anti-tumor effects diminished and were paralleled by TAM/M accumulation and activation. Although CX3CR1 deficiency resulted in a lower number of extravasated tumor cells, progression of these extravasated cells into micro metastases was more efficient. Overall, this resulted in a comparable number of mature macrometastases in CX3CR1-deficient and -proficient mice. Our findings indicate that unspecific inhibition of CX3CR1 might not be a suitable therapeutic option to prevent dissemination of lung cancer cells to the brain. Given the close interaction between TAM/M and tumor cells during metastasis formation, other therapeutic approaches targeting TAM/M function may warrant further evaluation. The herein established orthotopic mouse model may be a useful tool to evaluate such concepts in vivo.

BSCI-08. In vivo two-photon characterization of tumor-associated macrophages and microglia (TAM/M) and CX3CR1 during different steps of brain metastasis formation from lung cancer

Abstract Background Brain metastases represent a common complication of lung cancer and dramatically limit prognosis in affected patients. The influence of tumor-associated macrophages and microglia (TAM/M) and their receptor CX3CR1 on different steps of brain metastasis formation from lung cancer is poorly characterized, but might be of therapeutic relevance. Methods We established an orthotopic cerebral metastasis model using CX3CR1-proficient (CX3CR1GFP/wt) and -deficient (CX3CR1GFP/GFP) mice with green-fluorescent TAM/M. A cranial window was prepared, and intracarotid injection of red-fluorescent Lewis Lung Carcinoma-cells (tdtLLC) was performed two weeks later. Formation of brain metastases was followed by repetitive two-photon laser scanning microscopy. Results After intracarotid injection, intravascular tumor cells extravasated into the cerebral parenchyma and eventually formed micrometastases (≤50 cells) and mature macrometastases (>50 cells). We observed phagocytosis of extravasated tumor cells by TAM/M during early steps of metastatic growth. Notably, these anti-tumor effects of TAM/M diminished during later steps of metastasis formation and were accompanied by TAM/M accumulation and activation. CX3CR1-deficiency resulted in a lower number of extravasated tumor cells, and only a small number of TAM/M were visualized during early steps of metastasis formation (extravasation, formation of micrometastases) in such mice. In contrast, progression of extravasated tumor cells into micrometastases was more frequently found in CX3CR1-deficient mice. Overall, these mechanisms resulted in a comparable number of mature macrometastases between CX3CR1-deficient and -proficient mice. Conclusion Our findings indicate that unspecific inhibition of CX3CR1 might not be a suitable therapeutic approach to prevent cerebral dissemination of lung cancer cells. Given the close interaction between TAM/M and tumour cells during metastasis formation, other therapeutic approaches targeting TAM/M function warrant evaluation. Such concepts might be evaluated in vivo using the herein established orthotopic mouse model.

Intracarotid propofol injection for WADA testing in a paediatric patient–A case report

Surgical management of drug-resistant epilepsy is a well-established method and the lateralization of language and/or memory is essential to avoid resection of the eloquent cortex. The Wada test was developed in the 1960s to determine the lateralization of language and memory by injecting a short-acting anaesthetic drug into the carotid artery, narcotising one single hemisphere and testing the performance of the other isolated hemisphere [1]. Originally, sodium amytal was used, but after its production was stopped in the mid-2000s, it was replaced by methohexital as an alternative drug, which also ceased to be available after 2015.

Intracarotid Transplantation of Skin-Derived Precursor Schwann Cells Promotes Functional Recovery After Acute Ischemic Stroke in Rats.

Purpose: Skin-derived Precursor Schwann cells (SKP-SCs) have been reported to provide neuroprotection for the injured and dysmyelinated nervous system. However, little is known about SKP-SCs on acute ischemic stroke (AIS). We aimed to explore the efficacy and the potential mechanism of action of SKP-SCs on AIS in a rat ischemic stroke model.Methods: Adult male Sprague–Dawley rats were subjected to a middle cerebral artery occlusion (MCAO) for 1.5 h on Day 0 and subsequently received an intracarotid injection of 2 × 106 green fluorescent protein (GFP) -labeled SKP-SCs or phosphate buffered saline (PBS) during reperfusion. Neurological function was assessed by behavioral tests on Days 1, 4, 7, 14, and 28. In a satellite cohort, rat brains were harvested and infarct volume was measured with 2,3,5-triphenyltetrazolium chloride (TTC) staining on Days 1 and 7, and migration and survival of SKP-SCs in the brain were traced by monitoring green fluorescence at 6 and12 h on Day 0, and on Days 1, 4, 7, 14, and 28. Histopathology and immunofluorescence staining were used to analyze the morphology, survival and apoptosis of neurons. Additionally, in an in vitro SKP-SC co-culture model using fetal rat primary cortical neurons underwent oxygen glucose deprivation/reoxygenation (OGD/R), Western blot was used to detect the expression of apoptosis indicators including activated caspase-3, Bax, and Bcl-2. TUNEL staining was used to count apoptotic cells.Results: Intracarotid transplantation of SKP-SCs effectively migrated to the periinfarct area and survived for at least 4 weeks. Transplanted SKP-SCs inhibited neuronal apoptosis, reduced infarct volume, and improved neurological recovery in the MCAO rats. Moreover, in vitro data showed that SKP-SCs treatment inhibited OGD/R-induced neuronal apoptosis and promoted survival of the cultured primary cortical neurons.Conclusions: Intracarotid transplantation of SKP-SCs promoted functional recovery in the rat AIS model and possesses the potential to be further developed as a novel therapy to treat ischemic stroke in humans.

Tolerability and adverse effects of propofol in the Wada test

The Wada test consists of the selective and reversible inhibition of a cerebral hemisphere by intracarotid injection of amobarbital in order to evaluate the laterality of language and memory. However, there are other anesthetic drugs such as propofol, as an alternative for the test. The objective of the study was to describe the tolerability and adverse effects (AE) of the use of propofol for the Wada test, during the presurgical study of patients with drug-resistant epilepsy. Consecutive patients with a diagnosis of drug-resistant structural epilepsy were selected who underwent the Wada test during the pre-surgical study in the period from June 2012 to May 2019. The patients were retrospectively evaluated. The AE were described according to the Mikuni classification, modified by Curot. The variables of sex, age, epileptic foci laterality, language laterality, lesional substrate, etiology and dose of administered Propofol were analyzed for any statistical significance. A total of 74 patients, 40 men (54%), were studied. Forty-seven patients (63.5%) had at least one AE. The mean dose of propofol was 9.23 mg. The most frequent AE were tearing, sweating and red eye, corresponding to group I (57%). One patient developed convulsive status epilepticus, an important AE not previously described during the Wada test. Performing the Wada test with propofol causes frequent mild adverse effects, which do not prevent its completion. We describe a case of convulsive status epilepticus as the only serious AE.

Changes in measures of consciousness during anaesthesia of one hemisphere (Wada test)

BackgroundIn the Wada test, one hemisphere is selectively anaesthetised by unilateral intracarotid injection of a fast-acting anaesthetic agent. This gives a unique opportunity to observe the functions and physiological activity of one hemisphere while anaesthetising the other, allowing direct comparisons between brain states and hemispheres that are not possible in any other setting. AimTo test whether potential measures of consciousness would be affected by selective anaesthesia of one hemisphere, and reliably distinguish the states of the anesthetised and non-anesthetised hemispheres. MethodsWe analysed EEG data from 7 patients undergoing Wada-tests in preparation for neurosurgery and computed several measures reported to correlate with the state of consciousness: power spectral density, functional connectivity, and measures of signal diversity. These measures were compared between conditions (normal rest vs. unilateral anaesthesia) and hemispheres (injected vs. non-injected), and used with a support vector machine to classify the state and site of injection objectively from individual patient's recordings. ResultsAlthough brain function, assessed behaviourally, appeared to be substantially altered only on the injected side, we found large bilateral changes in power spectral density for all frequency bands tested, and functional connectivity changed significantly both between and within both hemispheres. Surprisingly, we found no statistically significant differences in the measures of signal diversity between hemispheres or states, for the group of 7 patients, although 4 of the individual patients showed a significant decrease in signal diversity on the injected side. Nevertheless, including signal diversity measures improved the classification results, indicating that these measures carry at least some non-redundant information about the condition and injection site. We propose that several of these results may be explained by conduction of activity, via the corpus callosum, from the injected to the contralateral hemisphere and vice versa, without substantially affecting the function of the receiving hemisphere, thus reflecting what we call “cross-state unreceptiveness”.

Activation of the carotid body increases directly recorded cardiac sympathetic nerve activity and coronary blood flow in conscious sheep.

Activation of the carotid body (CB) using intracarotid potassium cyanide (KCN) injection increases coronary blood flow (CoBF). This increase in CoBF is considered to be mediated by co-activation of both the sympathetic and parasympathetic nerves to the heart. However, whether cardiac sympathetic nerve activity (cardiac SNA) actually increases during CB activation has not been determined previously. We hypothesized that activation of the CB would increase directly recorded cardiac SNA, which would cause coronary vasodilatation. Experiments were conducted in conscious sheep implanted with electrodes to record cardiac SNA and diaphragmatic electromyography (dEMG), flow probes to record CoBF and cardiac output, and a catheter to record arterial pressure. Intracarotid KCN injection was used to activate the CB. To eliminate the contribution of metabolic demand on coronary flow, the heart was paced at a constant rate during CB chemoreflex stimulation. Intracarotid KCN injection resulted in a significant increase in directly recorded cardiac SNA frequency (from 24 ± 2 to 40 ± 4 bursts/min; P < 0.05) as well as a dose-dependent increase in mean arterial pressure (79 ± 15 to 88 ± 14 mmHg; P < 0.01) and CoBF (75 ± 37 vs. 86 ± 42 mL/min; P < 0.05). The increase in CoBF and coronary vascular conductance to intracarotid KCN injection was abolished after propranolol infusion, suggesting that the increased cardiac SNA mediates coronary vasodilatation. The pressor response to activation of the CB was abolished by pretreatment with intravenous atropine, but there was no change in the coronary flow response. Our results indicate that CB activation increases directly recorded cardiac SNA, which mediates vasodilatation of the coronary vasculature.

EXTH-66. NEO100 TRANSIENTLY OPENS UP THE BLOOD BRAIN BARRIER VIA TIGHT JUNCTION INHIBITION

Abstract The blood brain barrier (BBB) prevents effective entry of nearly all therapeutics to the central nervous system (CNS), preventing effective treatment of brain-related malignancies. Intracarotid mannitol injection has been the main technique to transiently open up the BBB, with its attendant variability and complications. A more direct and better tolerated method is needed to open up the BBB. We present our discovery that intraarterial (IA) injection of NEO100, a cGMP-quality form of perillyl alcohol (POH), transiently opens up the BBB in a safe and reversible manner. We used in-vitro models of MDCK1 and patient derived brain endothelial cell (BEC) + astrocyte barriers to determine that NEO100 increased FITC-antibody diffusion across the in-vitro BBB model and decreased trans-epithelial/endothelial electrical resistance (TEER). NEO100 effects on transcellular and paracellular pathways were studied using western blot, flow cytometry, HPLC, fluorescent probes, microarray analysis, and transmission electron microscopy. In-vivo studies were performed using ultrasound-guided intracardiac administration of NEO100 in mice with subsequent intravenous delivery of non-BBB permeable therapeutic agents. We determined that NEO100 transiently disrupts the transcellular pathway by permeabilizing BEC membranes, and the paracellular pathway via delocalization of tight junction proteins. In vivo IA NEO100 administration caused an effective dose- and time-dependent BBB permeabilization, which was reversible and well tolerated by the mice. This was evidenced by the spreading of Evans blue dye, and of therapeutics with different molecular weights, ie methotrexate, anti-PD-1 antibody, and CAR-T cells in the brain. Our results demonstrate that IA NEO100 is able to open the BBB in a controlled and reversible manner, allowing it to facilitate drug delivery to the CNS.

TMOD-01. DEVELOPING CLINICALLY RELEVANT BRAIN METASTATIC TUMOR MODELS FOR TARGETED STEM CELL THERAPEUTICS

Abstract Brain metastasis is associated with increased mortality in patients with cancer, 15-20% of patients will develop brain metastasis during the course of the disease. Characterizing clinically relevant brain metastasis models and exploring different options to deliver drugs across blood brain barrier in such models are fundamental for the development of novel therapies for metastatic brain tumors. We have created imageable breast to brain metastasis tumor models using patient derived brain seeking basal like breast cancer (BLBC) lines. We show a widespread distribution of micro- and macro-metastasis and the close association of tumor cells with blood vessels in the brain thus mimicking the multi-foci metastases observed in the clinics. These models enables us to investigate the pathophysiology of this little-known phenomenon and test the efficacy of new approach targeting the dormant cancer cells in perivascular niche. Next, we assessed the EGFR and DR4/5 levels in BLBC and show that brain seeking BLBC have higher EGFR and DR5 levels than their primary counterparts. We also show that intracarotid artery injection or intrathecal injection of stem cells engineered to express optical imaging markers home to metastatic brain tumors. These findings provide useful models for testing treatments for metastatic brain tumors and also provide insights into the changes in gene expression in metastatic BLBC and means to deliver therapeutics to metastatic tumors in the brain.

Celastrol Dilates and Counteracts Ethanol-Induced Constriction of Cerebral Arteries.

The increasing recognition of the role played by cerebral artery dysfunction in brain disorders has fueled the search for new cerebrovascular dilators. Celastrol, a natural triterpene undergoing clinical trials for treating obesity, exerts neuroprotection, which was linked to its antioxidant/anti-inflammatory activities. We previously showed that celastrol fit pharmacophore criteria for activating calcium- and voltage-gated potassium channels of large conductance (BK channels) made of subunits cloned from cerebrovascular smooth muscle (SM). These recombinant BK channels expressed in a heterologous system were activated by celastrol. Activation of native SM BK channels is well known to evoke cerebral artery dilation. Current data demonstrate that celastrol (1-100 µM) dilates de-endothelialized, ex vivo pressurized middle cerebral arteries (MCAs) from rats, with EC50 = 45 µM and maximal effective concentration (Emax)= 100 µM and with MCA diameter reaching a 10% increase over vehicle-containing, time-matched values (P < 0.05). A similar vasodilatory efficacy is achieved when celastrol is probed on MCA segments with intact endothelium. Selective BK blocking with 1 μM paxilline blunts celastrol vasodilation. Similar blunting is achieved with 0.8 mM 4-aminopirydine, which blocks voltage-gated K+ channels other than BK. Using an in vivo rat cranial window, we further demonstrate that intracarotid injections of 45 μM celastrol into pial arteries branching from MCA mimics celastrol ex vivo action. MCA constriction by ethanol concentrations reached in blood during moderate-heavy alcohol drinking (50 mM), which involves SM BK inhibition, is both prevented and reverted by celastrol. We conclude that celastrol could be an effective cerebrovascular dilator and antagonist of alcohol-induced cerebrovascular constriction, with its efficacy being uncompromised by conditions that disrupt endothelial and/or BK function. SIGNIFICANCE STATEMENT: Our study demonstrates for the first time that celastrol significantly dilates rat cerebral arteries both ex vivo and in vivo and both prevents and reverses ethanol-induced cerebral artery constriction. Celastrol actions are endothelium-independent but mediated through voltage-gated (KV) and calcium- and voltage-gated potassium channel of large conductance (BK) K+ channels. This makes celastrol an appealing new agent to evoke cerebrovascular dilation under conditions in which endothelial and/or BK channel function are impaired.