Intracarotid Injection Research Articles

Intrathecal injection of brilliant blue G, a P2X7 antagonist, attenuates the exercise pressor reflex in rats.

Purinergic 2X (P2X) receptors on the endings of group III and IV afferents play a role in evoking the exercise pressor reflex. Particular attention has been paid to P2X3 receptors because their blockade in the periphery attenuated this reflex. In contrast, nothing is known about the role played by P2X receptors in the spinal cord in evoking the exercise pressor reflex in rats. P2X7 receptors, in particular, may be especially important in this regard because they are found in abundance on spinal glial cells and may communicate with neurons to effect reflexes controlling cardiovascular function. Consequently, we investigated the role played by spinal P2X7 receptors in evoking the exercise pressor reflex in decerebrated rats. We found that intrathecal injection of the P2X7 antagonist brilliant blue G (BBG) attenuated the exercise pressor reflex (blood pressure index: 294 ± 112 mmHg·s before vs. 7 ± 32 mmHg·s after; P < 0.05). Likewise, intrathecal injection of minocycline, which inhibits microglial cell output, attenuated the reflex. In contrast, intrathecal injection of BBG did not attenuate the pressor response evoked by intracarotid injection of sodium cyanide, a maneuver that stimulated carotid chemoreceptors. Moreover, injections of BBG either into the arterial supply of the contracting hindlimb muscles or into the jugular vein did not attenuate the exercise pressor reflex. Our findings support the hypothesis that P2X7 receptors on microglial cells within the spinal cord play a role in evoking the exercise pressor reflex.

The effect of combining Endostar with radiotherapy on blood vessels, tumor-associated macrophages, and T cells in brain metastases of Lewis lung cancer.

BackgroundCombining Endostar (ES) with radiotherapy (RT) has shown a promising therapeutic effect on non-small cell lung carcinoma with brain metastases (BMs) in clinical practice. However, the specific mechanism is not yet fully understood. The present study aimed to investigate the effects of ES on blood vessels, tumor-associated macrophages (TAMs), and T cells in a tumor microenvironment treated with RT.MethodsBM models were established by stereotactic and intracarotid injection of luciferase-Lewis lung cancer (LLC) cells into female C57BL mice. The animals were randomly divided into 4 groups: normal saline (NS), ES, RT, and ES plus radiotherapy (ES + RT) groups. Tumor size was determined with the IVIS imaging system. Tumor specimens were stained with CD34 and α-SMA to investigate tumor vascular changes. The proportions of TAMs, CD4+ T cells, and CD8+ T cells in tumor tissues were determined by flow cytometry and immunofluorescence. The expressions of hypoxia-inducible factor 1α (HIF-1α) and CXCR4 were deduced using western blotting and immunohistochemistry (IHC).ResultsES + RT significantly suppressed tumor growth compared to the other 3 groups. RT decreased M1 and increased M2 in microglial cells and bone marrow-derived macrophages (BMDMs) relative to NS, while ES had the opposite effect. The ratio of CD8+T/CD4+T was increased in the ES + RT group compared to the other 3 groups. Tumor vascular maturity (α-SMA+/CD34+) was increased while HIF-1α was significantly suppressed in the ES + RT group. CXCR4 expression, which is involved in TAM recruitment, increased following RT, whereas, ES attenuated its expression.ConclusionsOur findings suggest that ES can promote the normalization of tumor blood vessels and increase the anti-tumor immune-related immune cells infiltrating the tumor following RT treatment.

The exercise pressor reflex is inhibited by Brilliant Blue G in freely perfused and femoral artery ligated rats following intrathecal, but not hindlimb, treatment.

The exercise pressor reflex (EPR) is mediated in part by purinergic(P)2X receptors which are compromised of seven subtypes. Blockade of the P2X3 subtype on the peripheral endings of group III and IV muscle afferents attenuates the EPR. We investigated whether other P2X receptors were involved in the EPR arc in decerebrate Sprague Dawley rats whose femoral arteries were either patent or were ligated. We antagonized P2X receptors with Brilliant Blue G (BBG), which has highest affinity for P2X7 receptors. We injected BBG in both group of rats either intrathecally (50mg/mL; 25–50uL) or into the superficial epigastric artery, which is a side branch of the femoral artery. Intrathecal injection of BBG attenuated the magnitude of the EPR in both rats with patent femoral arteries and in rats with ligated femoral arteries (P&lt;0.05). Intrathecal injection of BBG had no effect on the pressor response to intra‐carotid injection of sodium cyanide (2ug), which is a potent and selective stimulant to carotid chemoreceptors. This latter finding indicates that intrathecal injection of BBG did not ascend to the medulla and did not impact the spinal sympathetic outflow. BBG injected either into the superficial epigastric artery or intravenously (5mg) had no appreciable effect on the EPR. In addition the effect of intrathecal BBG could not be explained by “neural fatigue” caused by repetitive contractions in rats with ligated femoral arteries. We conclude that intrathecal BBG treatment inhibits the EPR by antagonizing P2X4 or P2X7 receptors within the dorsal horn of the spinal cord.Support or Funding InformationThis study was funded by NIH grants R01 AR 059397 and P01 HL 134609.

Integration of Hypernatremia and Angiotensin II by the Organum Vasculosum of the Lamina Terminalis Regulates Thirst.

The organum vasculosum of the lamina terminalis (OVLT) contains NaCl-sensitive neurons to regulate thirst, neuroendocrine function, and autonomic outflow. The OVLT also expresses the angiotensin II (AngII) type1 receptor, and AngII increases Fos expression in OVLT neurons. The present study tested whether individual OVLT neurons sensed both NaCl and AngII to regulate thirst and body fluid homeostasis. A multifaceted approach, including in vitro whole-cell patch recordings, in vivo single-unit recordings, and optogenetic manipulation of OVLT neurons, was used in adult, male Sprague Dawley rats. First, acute intravenous infusion of hypertonic NaCl or AngII produced anatomically distinct patterns of Fos-positive nuclei in the OVLT largely restricted to the dorsal cap versus vascular core, respectively. However, in vitro patch-clamp recordings indicate 66% (23 of 35) of OVLT neurons were excited by bath application of both hypertonic NaCl and AngII. Similarly, in vivo single-unit recordings revealed that 52% (23 of 44) of OVLT neurons displayed an increased discharge to intracarotid injection of both hypertonic NaCl and AngII. In marked contrast to Fos immunoreactivity, neuroanatomical mapping of Neurobiotin-filled cells from both in vitro and in vivo recordings revealed that NaCl- and AngII-responsive neurons were distributed throughout the OVLT. Next, optogenetic excitation of OVLT neurons stimulated thirst but not salt appetite. Conversely, optogenetic inhibition of OVLT neurons attenuated thirst stimulated by hypernatremia or elevated AngII but not hypovolemia. Collectively, these findings provide the first identification of individual OVLT neurons that respond to both elevated NaCl and AngII concentrations to regulate thirst and body fluid homeostasis.SIGNIFICANCE STATEMENT Body fluid homeostasis requires the integration of neurohumoral signals to coordinate behavior, neuroendocrine function, and autonomic function. Extracellular NaCl concentrations and the peptide hormone angiotensin II (AngII) are two major neurohumoral signals that regulate body fluid homeostasis. Herein, we present the first compelling evidence that individual neurons located in the organum vasculosum of the lamina terminalis detect both NaCl and AngII. Furthermore, optogenetic interrogations demonstrate that these neurons play a pivotal role in the regulation of thirst stimulated by NaCl and AngII. These novel observations lay the foundation for future investigations for how such inputs as well as others converge onto unique organum vasculosum of the lamina terminalis neurons to coordinate body fluid homeostasis and contribute to disorders of fluid balance.

Abstract A011: Preclinical models of breast cancer brain metastasis for drug efficacy studies

Abstract Background: Overall, 10-30% of patients with metastatic breast cancer (BC) will develop brain metastases. Depending on the subtype of the primary breast cancer the incidence of brain metastases varies ranging from 14% (hormone receptor positive BC) to 46% (triple-negative BC) and approximately about 34% for patients with HER2-positive BC. The major impact of the blood-brain-barrier (BBB), BBB efflux pumps and the local microenvironment in the brain represent a challenge for treatment of BC brain metastasis and treatment options remain limited. The aim of this study was to develop a model system of breast cancer brain metastases for evaluation of drugs directed at BC brain metastases. Methods: Models of BC brain metastasis were established by intracranial stereotactic injection of enzymatically digested PDX tumors (ST340, ST941, ST1339, ST1616B, ST1360B, and ST3338) or by intracardiac or intracarotid injection of cell suspensions in nude mice. Contrast-enhanced T1- and T2-weighted magnetic resonance imaging (MRI) or bioluminescence imaging was used to determine tumor take and growth. Drug sensitivity to single agent trastuzumab emtansine (T-DM1) administered intravenously were performed at confirmed tumor take in a subset of the models. Mice were treated with either saline or T-DM1, 10 mg/kg/week x4 or 2 mg/kg/week x4. PET imaging with 18F-FES was applied in estrogen receptor (ER) positive models as a PD biomarker of anti-estrogen receptor endocrine therapy. Results: T-DM1 treatment in the intracranial ST1339 or ST3338 HER2 positive (3+) PDX models inhibited tumor growth and prolonged survival compared to non-treated animals. However, T-DM1 treatment only caused a modest growth delay in the ST3338 model established intracranially compared to a complete response in the model established subcutaneously. Delivery of T-DM1 across the BBB to the intracranial tumors was visualized by 64Cu-trastuzumab PET/CT and high tumor uptake was associated with a response to T-DM1 therapy in the ST1339 model. Models of BC brain metastasis were successfully established by the intracardiac or intracarotid methods for the triple negative BC cell line MDA-MB-231, the HER2 positive cell line BT474, and an ER positive cell line (ST941C). In vivo imaging with 18F-FES PET/CT was able to visualize intracranial ER positive BC tumors, and animals treated with fulvestrant exhibited reduced 18F-FES tumor uptake. Conclusion: We have established model systems of BC brain metastasis and drug efficacy evaluation. Treatment response to T-DM1 was observed in intracranial HER2 positive models and delivery of T-DM1 to the tumors was visualized by 64Cu-trastuzumab PET/CT imaging. Together, the established breast cancer brain metastases models in combination with advanced non-invasive imaging can be used as a relevant translational platform for evaluation of new drugs. Citation Format: Carsten H. Nielsen, Michael J. Wick, Lotte K. Kristensen, Henriette S. Joergensen, Maria Z. Alfsen, Mark U. Juul, Alyssa Moriarty, Kyriakos P. Papadopoulos, Andreas Kjaer. Preclinical models of breast cancer brain metastasis for drug efficacy studies [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A011. doi:10.1158/1535-7163.TARG-19-A011

Microvessel occlusions alter amyloid-beta plaque morphology in a mouse model of Alzheimer’s disease

Vascular dysfunction is correlated to the incidence and severity of Alzheimer’s disease. In a mouse model of Alzheimer’s disease (APP/PS1) using in vivo, time-lapse, multiphoton microscopy, we found that occlusions of the microvasculature alter amyloid-beta (Aβ) plaques. We used several models of vascular injury that varied in severity. Femtosecond laser-induced occlusions in single capillaries generated a transient increase in small, cell-sized, Aβ deposits visualized with methoxy-X04, a label of fibrillar Aβ. After occlusions of penetrating arterioles, some plaques changed morphology, while others disappeared, and some new plaques appeared within a week after the lesion. Antibody labeling of Aβ revealed a transient increase in non-fibrillar Aβ one day after the occlusion that coincided with the disappearance of methoxy-X04-labeled plaques. Four days after the lesion, anti-Aβ labeling decreased and only remained in patches unlabeled by methoxy-X04 near microglia. Histology in two additional models, sparse embolic occlusions from intracarotid injections of beads and infarction from photothrombosis, demonstrated increased labeling intensity in plaques after injury. These results suggest that microvascular lesions can alter the deposition and clearance of Aβ and confirm that Aβ plaques are dynamic structures, complicating the interpretation of plaque burden as a marker of Alzheimer’s disease progression.

Application of Fluorescein Fluorescence in Vascular Neurosurgery.

Background: Fluorescein sodium (FNa) is a fluorescent drug with a long history of use for assessing retinal blood flow in ophthalmology; however, its application in vascular neurosurgery is only now gaining popularity. This review summarizes the current knowledge about using FNa videoangiography in vascular neurosurgery.Methods: We performed a literature review on the usage of FNa for fluorescent videoangiography procedures in neurosurgery. We analyzed methods of injection, dosages of FNa, visualizing platforms, and interpretation of FNa videoangiography. We also reviewed practical applications of FNa videoangiography during various vascular neurosurgeries.Results: FNa videoangiography can be performed with intraarterial (intracarotid) or intravenous dye injections. Both methods provide excellent resolution with enhanced fluorescence that shows intravascular blood flow on top of visible surrounding anatomy, and both allow simultaneous purposeful microsurgical manipulations. Although it is invasive, an intracarotid FNa injection results in faster contrast appearance and higher-intensity fluorescence and requires a lower dose per injection (reported range, 1–50 mg) compared with peripheral intravenous FNa injection (reported range, 75–2,000 mg or 1–1.5 mg/kg body weight). Four optical excitation/detection tools for FNa videoangiography have been successfully used: conventional xenon-light operating microscope with a special filter set, pencil-type light-emitting diode probe with a filter set, laser-illumination operating microscope, and an endoscope with a filter set. FNa videoangiography was reported to be feasible and useful in various clinical scenarios, such as examining the feeders and drainers in arteriovenous malformation surgery, checking the patency of a microvascular anastomosis, and assessing blood flow during aneurysm clipping. FNa videoangiography can be repeated during the same procedure and used along with indocyanine green (ICG) videoangiography.Conclusions: Compared with ICG videoangiography, FNa videoangiography has the advantages of enabling real-time inspection and better visualization at deep locations; however, thick vessel walls limit visualization of FNa in larger vessels. FNa videoangiography is a useful tool in multiple neurovascular scenarios and merits further studies to establish its clinical value.

Safety of oral anticoagulants on experimental brain microbleeding and cognition

This study aims at determining the ability of clinical-based doses of four oral anticoagulants to transform the onset of a cerebral microhemorrhages (CMH) burden into a symptomatic intracerebral hemorrhage (ICH) in the healthy brain, and precipitate cognitive impairment. Wild-type mice were anticoagulated for 10 days using apixaban, rivaroxaban or dabigatran as direct oral anticoagulants (DOACs), or warfarin as vitamin K-antagonist. Meanwhile, a burden of ∼20 CMHs was induced in the Sylvian territory by intra-carotid injection of cyclodextrin nanoparticles. At bleeding onset, only warfarin provoked deadly hematoma, and dramatically increased mortality (+45%). All the DOACs enhanced CMH burden through a greater number of intermediate-sized microhemorrhages (+80% to +180%). Although silent at onset, both baseline- and anticoagulant-enhanced CMH burdens increased mortality (+11% to +58%) along the following year without statistical difference among groups, and despite cessation of anticoagulation and absence of CMH progression or transformation into ICH. All survivor mice exhibited reduction in visual recognition memory from 9 months. In the healthy brain, DOACs preserve the onset of microhemorrhages from transformation into ICH, and do not precipitate cognitive impairment despite enhancement of CMH burden. High CMH burdens should however be considered for early detection and preventive memory care apart from anticoagulation decisions.

Improving orthotopic mouse models of patient-derived breast cancer brain metastases by a\xa0modified intracarotid injection\xa0method\xa0

Breast cancer brain metastasis (BCBM) remains a major clinical problem. Approximately 10–16% of patients with breast cancer develop brain metastases (BCBM). However, no systemic therapy has gained regulatory approval for the specific treatment of BCBM and this remains an area of persistent, unmet medical need. Rapid, predictive and clinically-relevant animal models are critical to study the biology of brain metastases and to identify effective therapeutic approaches for patients with BCBM. Here, we describe a method for efficient establishment of orthotopic mouse models of patient-derived brain metastases via an improved intracarotid injection protocol that permits tumor cell growth in the unique brain microenvironment without compromising the blood-brain barrier (BBB). We demonstrate that our newly improved models of patient-derived brain metastases recapitulate the histologic, molecular, and genetic characteristics of their matched patient tumor specimens and thus represent a potentially powerful tool for pre-clinical and translational research.

Gene Silencing in the Right Place at the Right Time.

Gene Silencing in the Right Place at the Right Time.

Abstract 107: Neurons in the Organum Vasculosum of the Lamina Terminalis Sense NaCl and Angiotensin II to Regulate Sympathetic Nerve Activity and Arterial Blood Pressure

The organum vasculosum of the lamina terminalis (OVLT) contains specialized neurons that sense changes in extracellular NaCl concentrations to regulate sympathetic nerve activity (SNA) and arterial blood pressure (ABP). However, OVLT neurons also abundantly express angiotensin II (AngII)-type I receptors. Therefore, we performed a series of in vitro and in vivo studies in adult male Sprague-Dawley rats to determine whether OVLT neurons sense both NaCl and AngII to impact SNA and ABP. First, in vitro whole-cell recordings revealed the majority of OVLT neurons (66%, 23/35) showed an increased discharge frequency to both +7.5mM NaCl (1.1±0.2 to 2.2±0.3Hz, P&lt;0.001) and 100pM AngII (1.3±0.3 to 2.2±0.3Hz, P&lt;0.001). Second, in vivo single-unit recordings demonstrate the majority of OVLT neurons (61%, 11/18) displayed an increase in cell discharge to intracarotid injection of 0.5M NaCl (3.6±0.6 to 9.0±1.6Hz, P&lt;0.01) and 200ng AngII (3.2±0.7 to 9.2±1.9Hz, P&lt;0.01). Third, optogenetic inhibition of OVLT neurons (rAAV2-CaMKII-eNpHR3.0-mCherry, 5x10 12 particles/mL, 561nm, 10mW) attenuated thirst responses to IV infusion of 2M NaCl (1.25mL/30min; Laser OFF: 6.1±0.5mL vs ON: 2.0±0.7mL, P&lt;0.05) and AngII (Laser OFF 7.2±0.7mL vs ON: 2.2±1.2mL; n=5 per group, P&lt;0.05). Fourth, OVLT injection (20nL) of 0.5M NaCl or 10pmol AngII increased SNA (121±6% or 117±5%, n=4/group; P&lt;0.05 respectively) and mean ABP (7±1 or 9±2mmHg, n=4/group; P&lt;0.05 respectively). To determine whether chronic activation of OVLT neurons produces hypertension, rats received an OVLT injection of rAAV2-hSyn-HM3D(Gq)-mCherry (3x10 12 particles/mL, 50nL) and given access to 0.1% NaCl chow and 0.9% NaCl drinking solution. After 4 weeks, daily administration of clozapine-N-oxide to the drinking solution (3mg/kg/day) significantly increased 24-h fluid intake (baseline: 60±10mL vs Day 7: 178±29mL; n=6, P&lt;0.01) and 24-h mean ABP (baseline: 97±2mmHg vs Day 7: 108±1 mmHg; n=6, P&lt;0.05). Ganglionic blockade with hexamethonium (30mg/kg, ip) produced a greater fall in mean ABP at Day 7 (baseline: -38±6 mmHg vs Day 7: -53±5 mmHg, P&lt;0.05). These findings suggest OVLT neurons sense both NaCl and AngII to regulate body fluid homeostasis and SNA to produce a sympathetically-mediated hypertension.

Invited Article: Visualizing protein synthesis in mice with in vivo labeling of deuterated amino acids using vibrational imaging

Proteins are one of the major components of biological systems, and understanding their metabolism is critical to study various biochemical processes in living systems. Despite extensive efforts to study protein metabolism such as autoradiography, mass spectrometry, and fluorescence microscopy, visualizing the spatial distribution of overall protein metabolism in mammals at subcellular resolution is still challenging. A recent study from our group reported imaging newly synthesized proteins in cultured mammalian cells, tissues, or even in mice using stimulated Raman scattering (SRS) microscopy coupled with metabolic labeling of deuterated amino acids (dAA). However, our previous method of dAA administration via drinking water, albeit convenient, is insufficient for in vivo studies. This is due to poor labeling efficiency and limited access to many important organs such as the brain, pancreas, or tumor. In this study, we have significantly improved and optimized the in vivo administration method by intra-carotid arterial injection of dAA in mice and obtained imaging contrast of protein metabolic activity in many more organs and tissues, such as cerebral and cerebellar cortex and hippocampal regions in the mouse brain. We also imaged newly formed proteins in the choroid plexus and pancreas at different time points, illustrating the metabolic dynamics of proteins in these important secretory organs. In addition, we visualized the metabolic heterogeneity of protein synthesis in colon tumor xenografts, which can be used to distinguish tumor and normal tissues. In summary, this combination of a new dAA administration technique and SRS imaging platform demonstrates an effective tool for the in vivo study of complex protein metabolism in mammals, in both physiological and pathological states.

Red blood cell-hitchhiking boosts delivery of nanocarriers to chosen organs by orders of magnitude

Drug delivery by nanocarriers (NCs) has long been stymied by dominant liver uptake and limited target organ deposition, even when NCs are targeted using affinity moieties. Here we report a universal solution: red blood cell (RBC)-hitchhiking (RH), in which NCs adsorbed onto the RBCs transfer from RBCs to the first organ downstream of the intravascular injection. RH improves delivery for a wide range of NCs and even viral vectors. For example, RH injected intravenously increases liposome uptake in the first downstream organ, lungs, by ~40-fold compared with free NCs. Intra-carotid artery injection of RH NCs delivers >10% of the injected NC dose to the brain, ~10× higher than that achieved with affinity moieties. Further, RH works in mice, pigs, and ex vivo human lungs without causing RBC or end-organ toxicities. Thus, RH is a clinically translatable platform technology poised to augment drug delivery in acute lung disease, stroke, and several other diseases.

Neurons in the Organum Vasculosum of the Lamina Terminalis Sense Both Angiotensin II and NaCl to Regulate Thirst

Angiotensin II (AngII) and NaCl are sensed by hypothalamic circumventricular organs to subsequently regulate body fluid homeostasis and blood pressure. AngII predominantly acts within the subfornical organ (SFO), whereas NaCl is sensed by neurons in the organum vasculosum of the lamina terminalis (OVLT). However, OVLT neurons also abundantly express AngII‐type I receptors. Therefore, we performed a series of in vitro and in vivo experiments in adult male Sprague‐Dawley rats to determine whether OVLT neurons sense both AngII and NaCl to impact body fluid homeostasis. First, in vitro whole‐cell recordings were performed on OVLT neurons in the presence of kynurenic acid (1mM) and bicuculline (20μM) to assess discharge responses to +7.5 mM NaCl (3 min) versus 100pM AngII (90s) in a randomized order. The majority of OVLT neurons (66%, 23/35) demonstrated an increase in discharge frequency to both AngII (1.31±0.25Hz to 2.19±0.31Hz; p&lt;0.001) and hypertonic NaCl (1.11±0.18Hz to 2.16±0.27 Hz; p&lt;0.001). Smaller proportions of OVLT neurons were either excited by NaCl alone (3/35), AngII alone (4/35), neither stimulus (3/35), or were inhibited by NaCl (2/35). In a second set of experiments, in vivo single‐unit recordings of OVLT neurons were performed to assess discharge responses to intracarotid injection (50uL over 15 s) of hypertonic NaCl (0.5M NaCl) or AngII (200ng). Half of OVLT neurons (50%, 6/12) displayed an increase in discharge to intracarotid injection of AngII (3.2±0.7 to 9.2±1.9 Hz, P&lt;0.01) and 0.5M NaCl (3.6±0.6 to 9.0±1.6 Hz, P&lt;0.01). The remainder of OVLT neurons showed an increase in discharge to hypertonic NaCl only (n=1) or AngII only (5/12). In a final set of experiments, we employed an optogenetic approach to determine the extent by which inhibition of OVLT neurons would attenuate thirst stimulated by NaCl versus AngII. rAAV2‐CamKII‐eNPHR3.0‐mCherry (5×1012 particles/mL, 50nL) was injected into the OVLT, and an optical ferrule (200um OD) was implanted 300um dorsal to the injection site. Approximately 4 weeks later, optogenetic inhibition of OVLT neurons (561nm, 10mW) significantly reduced water intake in response to intravenous infusion (1.25 mL per 30 min) of 2M NaCl (control: 5.1±0.9 mL vs laser: 1.6±0.9 mL; n=4, P&lt;0.01). In addition, optogenetic inhibition of OVLT neurons also attenuated hypotension‐induced thirst produced by intravenous injection of 25 mg/kg diazoxide (control: 4.5±0.8 mL vs laser: 1.3±0.7 mL; n=4, P&lt;0.01). These findings suggest that individual OVLT neurons can sense both NaCl and AngII to functionally regulate thirst to both stimuli.Support or Funding InformationNIH R01 HL113270 and AHA Established Investigator AwardThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Tolerability of propofol and adverse effects during wada test

Background: The Wada test consists of a selective intracarotid injection of a fast acting barbiturate drug, generally sodium amobarbital, which reversibly inhibits the ipsilateral cerebral hemisphere, to isolate the contralateral hemisphere and evaluate its activity. Intracarotid administration of propofol has been used as an alternative drug to amobarbital for evaluation of memory and language, avoiding postsurgical amnesic and aphasic syndromes.

NaCl and osmolarity produce different responses in organum vasculosum of the lamina terminalis neurons, sympathetic nerve activity and blood pressure.

Changes in extracellular osmolarity stimulate thirst and vasopressin secretion through a central osmoreceptor; however, central infusion of hypertonic NaCl produces a greater sympathoexcitatory and pressor response than infusion of hypertonic mannitol/sorbitol. Neurons in the organum vasculosum of the lamina terminalis (OVLT) sense changes in extracellular osmolarity and NaCl. In this study, we discovered that intracerebroventricular infusion or local OVLT injection of hypertonic NaCl increases lumbar sympathetic nerve activity, adrenal sympathetic nerve activity and arterial blood pressure whereas equi-osmotic mannitol/sorbitol did not alter any variable. In vitro whole-cell recordings demonstrate the majority of OVLT neurons are responsive to hypertonic NaCl or mannitol. However, hypertonic NaCl stimulates a greater increase in discharge frequency than equi-osmotic mannitol. Intracarotid or intracerebroventricular infusion of hypertonic NaCl evokes a greater increase in OVLT neuronal discharge frequency than equi-osmotic sorbitol. Collectively, these novel data suggest that subsets of OVLT neurons respond differently to hypertonic NaCl versus osmolarity and subsequently regulate body fluid homeostasis. These responses probably reflect distinct cellular mechanisms underlying NaCl- versus osmo-sensing. Systemic or central infusion of hypertonic NaCl and other osmolytes readily stimulate thirst and vasopressin secretion. In contrast, central infusion of hypertonic NaCl produces a greater increase in arterial blood pressure (ABP) than equi-osmotic mannitol/sorbitol. Although these responses depend on neurons in the organum vasculosum of the lamina terminalis (OVLT), these observations suggest OVLT neurons may sense or respond differently to hypertonic NaCl versus osmolarity. The purpose of this study was to test this hypothesis in Sprague-Dawley rats. First, intracerebroventricular (icv) infusion (5μl/10min) of 1.0m NaCl produced a significantly greater increase in lumbar sympathetic nerve activity (SNA), adrenal SNA and ABP than equi-osmotic sorbitol (2.0osmoll-1 ). Second, OVLT microinjection (20nl) of 1.0m NaCl significantly raised lumbar SNA, adrenal SNA and ABP. Equi-osmotic sorbitol did not alter any variable. Third, in vitro whole-cell recordings demonstrate that 50% (18/36) of OVLT neurons display an increased discharge to both hypertonic NaCl (+7.5mm) and mannitol (+15mm). Of these neurons, 56% (10/18) displayed a greater discharge response to hypertonic NaCl vs mannitol. Fourth, in vivo single-unit recordings revealed that intracarotid injection of hypertonic NaCl produced a concentration-dependent increase in OVLT cell discharge, lumbar SNA and ABP. The responses to equi-osmotic infusions of hypertonic sorbitol were significantly smaller. Lastly, icv infusion of 0.5m NaCl produced significantly greater increases in OVLT discharge and ABP than icv infusion of equi-osmotic sorbitol. Collectively, these findings indicate NaCl and osmotic stimuli produce different responses across OVLT neurons and may represent distinct cellular processes to regulate thirst, vasopressin secretion and autonomic function.

Abstract 1090: Preclinical evaluation of combination therapy with dasatinib and sorafenib for the treatment of brain metastases

Abstract Brain metastases, the most common type of intracranial tumors in adults, carry a dismal prognosis with a median survival of less than 18 months regardless of primary status or treatment given. As part of an ongoing project, this study is concerned with the preclinical evaluation of targeted combination therapy for the treatment of brain metastases. The cell line used in this study was the H1915 (ATCC® CRL-5904TM) human lung cancer cell line, which was originally derived from a brain metastasis from primary lung adenocarcinoma. The cell line was stably transfected with a plasmid that allows expression of firefly luciferase (i.e., H1915-luc) so that the tumor growth in a mouse orthotopic model of brain metastases can be assessed in real time using the Xenogen in vivo bioluminescence imaging (BLI). Our previous study has demonstrated that the synergistic effect of combined dasatinib and sorafenib treatment was cell-line dependent. In this study, the cytotoxicity of dasatinib and sorafenib, when used alone or in combination at the fixed molar ratios of 3:1, 1:1 and 1:3 (dasatinib:sorafenib), was further examined in H1915-luc cells using the MTT assay. The results demonstrated that simultaneous and continuous exposure of H1915-luc cells to dasatinib and sorafenib at the fixed concentration ratio of 1:1 and 1:3 for 72 h showed synergism (the combination index value between 0.7 and 0.8) at effect levels of 40 and 80% inhibition of cell viability. For the in vivo study, an orthotopic mouse model of brain metastases was established by injecting H1915-luc cells into the right general carotid artery of individual athymic nude mice followed by permanent ligation. The preliminary study showed that following the intracarotid injection of H1915-luc cells, brain metastases were formed and readily to be detected by BLI. The obtained whole brain samples were fixed in OCT and stored at -80oC. The brain sections are to be subjected to the immunofluorescence double staining of CD31 and firefly luciferase to assess the potential effect of the dasatinib-sorafenib combination on microscopic brain metastasis formation and progression and vessel co-option. Given the fact that brain metastases usually have a poor response to chemotherapeutic agents in part due to the presence of blood-brain barrier, a pharmacokinetic study is underway to characterize the distribution of sorafenib and dasatinib in normal brain tissues and brain metastases using the serial blood sampling and brain microdialysis technique. Overall, results from the in vitro cytotoxicity study demonstrated the synergistic effect of dasatinib and sorafenib on H1915-luc human lung cancer cells, providing a rationale for the in vivo evaluation the potential of this combination for the treatment of brain metastases from lung carcinomas. Citation Format: Qingyu Stephanie Zhou, Xiaofang Guo. Preclinical evaluation of combination therapy with dasatinib and sorafenib for the treatment of brain metastases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1090. doi:10.1158/1538-7445.AM2017-1090

Intracarotid Cancer Cell Injection to Produce Mouse Models of Brain Metastasis.

Metastasis, the spread and growth of malignant cells at secondary sites within a patient's body, accounts for > 90% of cancer-related mortality. Recently, impressive advances in novel therapies have dramatically prolonged survival and improved quality of life for many cancer patients. Sadly, incidence of brain metastatic recurrences is fast rising, and all current therapies are merely palliative. Hence, good experimental animal models are urgently needed to facilitate in-depth studies of the disease biology and to assess novel therapeutic regimens for preclinical evaluation. However, the standard in vivo metastasis assay via tail vein injection of cancer cells produces predominantly lung metastatic lesions; animals usually succumb to the lung tumor burden before any meaningful outgrowth of brain metastasis. Intracardiac injection of tumor cells produces metastatic lesions to multiple organ sites including the brain; however, the variability of tumor growth produced with this model is large, dampening its utility in evaluating therapeutic efficacy. To generate reliable and consistent animal models for brain metastasis study, here we describe a procedure for producing experimental brain metastasis in the house mouse (Mus musculus) via intracarotid injection of tumor cells. This approach allows one to produce large number of brain metastasis-bearing mice with similar growth and mortality characteristics, thus facilitating research efforts to study basic biological mechanisms and to assess novel therapeutic agents.

Transient Receptor Potential Canonical 3 (TRPC3) Channels Are Required for Hypothalamic Glucose Detection and Energy Homeostasis.

The mediobasal hypothalamus (MBH) contains neurons capable of directly detecting metabolic signals such as glucose to control energy homeostasis. Among them, glucose-excited (GE) neurons increase their electrical activity when glucose rises. In view of previous work, we hypothesized that transient receptor potential canonical type 3 (TRPC3) channels are involved in hypothalamic glucose detection and the control of energy homeostasis. To investigate the role of TRPC3, we used constitutive and conditional TRPC3-deficient mouse models. Hypothalamic glucose detection was studied in vivo by measuring food intake and insulin secretion in response to increased brain glucose level. The role of TRPC3 in GE neuron response to glucose was studied by using in vitro calcium imaging on freshly dissociated MBH neurons. We found that whole-body and MBH TRPC3-deficient mice have increased body weight and food intake. The anorectic effect of intracerebroventricular glucose and the insulin secretory response to intracarotid glucose injection are blunted in TRPC3-deficient mice. TRPC3 loss of function or pharmacological inhibition blunts calcium responses to glucose in MBH neurons in vitro. Together, the results demonstrate that TRPC3 channels are required for the response to glucose of MBH GE neurons and the central effect of glucose on insulin secretion and food intake.

Intracarotid Etomidate Decreases the Interhemispheric Synchronization in Electroencephalogram (EEG) During the Wada Test.

The Wada test is a diagnostic method to anesthetize 1 cerebral hemisphere and assess the language and memory functions of awake contralateral hemisphere before temporal lobectomy in patients with epilepsy. The effects of an ipsilateral injection of etomidate on the functional properties of contralateral hemisphere are not known. Our aim was to characterize the electroencephalographic (EEG) effects after intracarotid injection of etomidate and to determine the changes in synchronization between the ipsilateral and contralateral hemispheres. We retrospectively analyzed EEG data from 15 patients who underwent Wada test with etomidate. Continuous EEG data from 4 electrodes (2 anterior [F3, F4] and 2 posterior [P3, P4]) were analyzed. After artifact rejection, we measured the amplitudes of delta (1 to 4 Hz) and alpha (7 to 13 Hz) waves and the changes in the interhemispheric synchronization before, during, and after etomidate injection. Ipsilateral injection of etomidate causes EEG changes in both hemispheres with increased amplitude of delta waves anteriorly and the alpha waves posteriorly. However this effect of etomidate is not uniform. Although there was a decrease in the interhemispheric synchronization of delta waves, there was no change in the synchronization of the alpha waves. Our study showed that the EEG effects of intracarotid ipsilateral injection of etomidate is not uniform in both hemispheres. Decrease in the interhemispheric synchronization of delta waves might serve as an indicator of ipsilateral effect of etomidate on the brain. Additional studies are needed to confirm our findings.