Early molecular events of autosomal-dominant Alzheimer's disease in marmosets with PSEN1 mutations.

INTRODUCTION: Fundamental questions remain about the key mechanisms that initiate Alzheimer's disease (AD) and the factors that promote its progression. Here we report the successful generation of the first genetically engineered marmosets that carry knock-in (KI) point mutations in the presenilin 1 (PSEN1) gene that can be studied from birth throughout lifespan. METHODS: CRISPR/Cas9 was used to generate marmosets with C410Y or A426P point mutations in PSEN1. Founders and their germline offspring are comprehensively studied longitudinally using non-invasive measures including behavior, biomarkers, neuroimaging, and multiomics signatures. RESULTS: Prior to adulthood, increases in plasma amyloid beta were observed in PSEN1 mutation carriers relative to non-carriers. Analysis of brain revealed alterations in several enzyme-substrate interactions within the gamma secretase complex prior to adulthood. DISCUSSION: Marmosets carrying KI point mutations in PSEN1 provide the opportunity to study the earliest primate-specific mechanisms that contribute to the molecular and cellular root causes of AD onset and progression. HIGHLIGHTS: We report the successful generation of genetically engineered marmosets harboring knock-in point mutations in the PSEN1 gene. PSEN1 marmosets and their germline offspring recapitulate the early emergence of AD-related biomarkers. Studies as early in life as possible in PSEN1 marmosets will enable the identification of primate-specific mechanisms that drive disease progression.

An open-source MRI compatible frame for multimodal presurgical mapping in macaque and capuchin monkeys.

BACKGROUND: High-precision neurosurgical targeting in nonhuman primates (NHPs) often requires presurgical anatomical mapping with noninvasive neuroimaging techniques (MRI, CT, PET), allowing for translation of individual anatomical coordinates to surgical stereotaxic apparatus. Given the varied tissue contrasts that these imaging techniques produce, precise alignment of imaging-based coordinates to surgical apparatus can be cumbersome. MRI-compatible stereotaxis with radiopaque fiducial markers offer a straight-forward and reliable solution, but existing commercial options do not fit in conformal head coils that maximize imaging quality. NEW METHOD: We developed a compact MRI-compatible stereotaxis suitable for a variety of NHP species (Macaca mulatta, Macaca fascicularis, and Cebus apella) that allows multimodal alignment through technique-specific fiducial markers. COMPARISON WITH EXISTING METHODS: With the express purpose of compatibility with clinically available MRI, CT, and PET systems, the frame is no larger than a human head, while allowing for imaging NHPs in the supinated position. This design requires no marker implantation, special software, or additional knowledge other than the operation of a common large animal stereotaxis. RESULTS: We demonstrated the applicability of this 3D-printable apparatus across a diverse set of experiments requiring presurgical planning: 1) We demonstrate the accuracy of the fiducial system through a within-MRI cannula insertion and subcortical injection of a viral vector. 2) We also demonstrated accuracy of multimodal (MRI and CT) alignment and coordinate transfer to guide a surgical robot electrode implantation for deep-brain electrophysiology. CONCLUSIONS: The computer-aided design files and engineering drawings are publicly available, with the modular design allowing for low cost and manageable manufacturing.

Noninvasive focal transgene delivery with viral neuronal tracers in the marmoset monkey.

We establish a reliable method for selectively delivering adeno-associated viral vectors (AAVs) across the blood-brain barrier (BBB) in the marmoset without the need for neurosurgical injection. We focally perturbed the BBB (∼1 × 2 mm) in area 8aD of the frontal cortex in four adult marmoset monkeys using low-intensity transcranial focused ultrasound aided by microbubbles. Within an hour of opening the BBB, either AAV2 or AAV9 was delivered systemically via tail-vein injection. In all four marmosets, fluorescence-encoded neurons were observed at the site of BBB perturbation, with AAV2 showing a sparse distribution of transduced neurons when compared to AAV9. The results are compared to direct intracortical injections of anterograde tracers into area 8aD and similar (albeit sparser) long-range connectivity was observed. With evidence of transduced neurons specific to the region of BBB opening as well as long-distance tracing, we establish a framework for focal noninvasive transgene delivery to the marmoset brain.

Direct interhemispheric cortical communication via thalamic commissures: a new white matter pathway in the primate brain.

Cortical neurons of eutherian mammals project to the contralateral hemisphere, crossing the midline primarily via the corpus callosum and the anterior, posterior, and hippocampal commissures. We recently reported and named the thalamic commissures (TCs) as an additional interhemispheric axonal fiber pathway connecting the cortex to the contralateral thalamus in the rodent brain. Here, we demonstrate that TCs also exist in primates and characterize the connectivity of these pathways with high-resolution diffusion-weighted MRI, viral axonal tracing, and fMRI. We present evidence of TCs in both New World (Callithrix jacchus and Cebus apella) and Old World primates (Macaca mulatta). Further, like rodents, we show that the TCs in primates develop during the embryonic period, forming anatomical and functionally active connections of the cortex with the contralateral thalamus. We also searched for TCs in the human brain, showing their presence in humans with brain malformations, although we could not identify TCs in healthy subjects. These results pose the TCs as a vital fiber pathway in the primate brain, allowing for more robust interhemispheric connectivity and synchrony and serving as an alternative commissural route in developmental brain malformations.

Ex vivo comparison of pin placement with patient-specific drill guides or freehand technique in canine cadaveric spines.

OBJECTIVE: To compare vertebral implant placement in the canine thoracolumbar spine between 3D-printed patient-specific drill guides (3DPG) and the conventional freehand technique (FH). STUDY DESIGN: Ex vivo study. ANIMALS: Cadaveric canine spines (n = 24). METHODS: Implant trajectories were established for the left and right sides of the T10 through L6 vertebrae based on computed tomography (CT) imaging. Customized drill guides were created for each vertebra of interest. Each cadaver was randomly assigned to one of six veterinarians with varying levels of experience placing vertebral implants. Vertebrae were randomly assigned a surgical order and technique (3DPG or FH) for both sides. Postoperative CT images were acquired. A single, blinded observer assessed pin placement using a modified Zdichavsky classification. RESULTS: A total of 480 implants were placed in 240 vertebrae. Three sites were excluded from the analysis; therefore, a total of 238 implants were evaluated using the FH technique and 239 implants using 3DPG. When evaluating implant placement, 152/239 (63.6%) of 3DPG implants were considered to have an acceptable placement in comparison with 115/248 (48.32%) with FH. Overall, pin placement using 3DPG was more likely to provide acceptable pin placement (p < .001) in comparison with the FH technique for surgeons at all levels of experience. CONCLUSION: The use of 3DPG was shown to be better than the conventional freehand technique regarding acceptable placement of implants in the thoracolumbar spine of canine cadavers. CLINICAL SIGNIFICANCE: Utilizing 3DPG can be considered better than the traditional FH technique when placing implants in the canine thoracolumbar spine.

Bridging the rodent to human translational gap: Marmosets as model systems for the study of Alzheimer's disease.

Introduction: Our limited understanding of the mechanisms that trigger the emergence of Alzheimer's disease (AD) has contributed to the lack of interventions that stop, prevent, or fully treat this disease. We believe that the development of a non-human primate model of AD will be an essential step toward overcoming limitations of other model systems and is crucial for investigating primate-specific mechanisms underlying the cellular and molecular root causes of the pathogenesis and progression of AD. Methods: A new consortium has been established with funding support from the National Institute on Aging aimed at the generation, characterization, and validation of Marmosets As Research Models of AD (MARMO-AD). This consortium will study gene-edited marmoset models carrying genetic risk for AD and wild-type genetically diverse aging marmosets from birth throughout their lifespan, using non-invasive longitudinal assessments. These include characterizing the genetic, molecular, functional, behavioral, cognitive, and pathological features of aging and AD. Results: The consortium successfully generated viable founders carrying PSEN1 mutations in C410Y and A426P using CRISPR/Cas9 approaches, with germline transmission demonstrated in the C410Y line. Longitudinal characterization of these models, their germline offspring, and normal aging outbred marmosets is ongoing. All data and resources from this consortium will be shared with the greater AD research community. Discussion: By establishing marmoset models of AD, we will be able to investigate primate-specific cellular and molecular root causes that underlie the pathogenesis and progression of AD, overcome limitations of other model organisms, and support future translational studies to accelerate the pace of bringing therapies to patients.

Noninvasive disruption of the blood-brain barrier in the marmoset monkey.

The common marmoset monkey (Callithrix jacchus) is a species of rising prominence in the neurosciences due to its small size, ease of handling, fast breeding, and its shared functional and structural brain characteristics with Old World primates. With increasing attention on modeling human brain diseases in marmosets, understanding how to deliver therapeutic or neurotropic agents to the marmoset brain noninvasively is of great preclinical importance. In other species, including humans, transcranial focused ultrasound (tFUS) aided by intravenously injected microbubbles has proven to be a transient, reliable, and safe method for disrupting the blood-brain barrier (BBB), allowing the focal passage of therapeutic agents that do not otherwise readily traverse the tight endothelial junctions of the BBB. The critical gap that we address here is to document parameters to disrupt the BBB reliably and safely in marmosets using tFUS. By integrating our marmoset brain atlases and the use of a marmoset-specific stereotactic targeting system, we conduct a series of systematic transcranial sonication experiments in nine marmosets. We demonstrate the effects of center frequency, acoustic pressure, burst period, and duration, establish a minimum microbubble dose, estimate microbubble clearance time, and estimate the duration that the BBB remains open to passage. Successful BBB disruption is reported in vivo with MRI-based contrast agents, as well as Evans blue staining assessed ex vivo. Histology (Hematoxylin and Eosin staining) and immunohistochemistry indicate that the BBB can be safely and reliably opened with the parameters derived from these experiments. The series of experiments presented here establish methods for safely, reproducibly, and focally perturbing the BBB using tFUS in the common marmoset monkey that can serve as a basis for noninvasive delivery of therapeutic or neurotropic agents.

Direct interhemispheric cortical communication via thalamic commissures: a new white-matter pathway in the primate brain.

Cortical neurons of eutherian mammals project to the contralateral hemisphere, crossing the midline primarily via the corpus callosum and the anterior, posterior, and hippocampal commissures. We recently reported an additional commissural pathway in rodents, termed the thalamic commissures (TCs), as another interhemispheric axonal fiber pathway that connects cortex to the contralateral thalamus. Here, we demonstrate that TCs also exist in primates and characterize the connectivity of these pathways with high-resolution diffusion-weighted magnetic resonance imaging, viral axonal tracing, and functional MRI. We present evidence of TCs in both New World (Callithrix jacchus and Cebus apella) and Old World primates (Macaca mulatta). Further, like rodents, we show that the TCs in primates develop during the embryonic period, forming anatomical and functionally active connections of the cortex with the contralateral thalamus. We also searched for TCs in the human brain, showing their presence in humans with brain malformations, although we could not identify TCs in healthy subjects. These results pose the TCs as an important fiber pathway in the primate brain, allowing for more robust interhemispheric connectivity and synchrony and serving as an alternative commissural route in developmental brain malformations.

Evaluation of Nasal Oxygen Administration at Various Flow Rates and Concentrations in Conscious, Standing Adult Horses.

This study evaluated the effects of various flow rates and fractions of oxygen on arterial blood gas parameters and on the fraction of inspired oxygen (FIO2) delivered to the distal trachea. Oxygen was administered to 6 healthy, conscious, standing, adult horses via single nasal cannula positioned within the nasopharynx. Three flow rates (5, 15, 30 L/min) and fractions of oxygen (21, 50, 100%) were delivered for 15 minutes, each in a randomized order. FIO2 was measured at the level of the nares and distal trachea. Adverse reactions were not observed with any flow rate. FIO2 (nares and trachea) and PaO2 increased with increasing flow rate and fraction of oxygen (P < .0001). FIO2 (trachea) was significantly less than FIO2 (nares) at 50% and 100% oxygen at all flow rates (P < .0001). Differences in PaO2 were not observed between 100% oxygen-5L/min and 50% oxygen-15L/min and or between 100% oxygen-15L/min and 50% oxygen-30L/min. Tracheal FIO2 for 100% oxygen-15L/min was increased compared to 50% oxygen-30L/min (P < .0001). Respiratory rate, ETCO2, PaCO2, and pH did not differ between treatments. Administration of 50% oxygen via nasal cannula at 15 and 30 L/min effectively increased in PaO2 and was well tolerated in conscious, standing, healthy horses. While these results can be used guide therapy in hypoxemic horses, evaluation of the administration of 50% oxygen to horses with respiratory disease is warranted.

Frontoparietal connectivity as a product of convergent evolution in rodents and primates: functional connectivity topologies in grey squirrels, rats, and marmosets.

Robust frontoparietal connectivity is a defining feature of primate cortical organization. Whether mammals outside the primate order, such as rodents, possess similar frontoparietal functional connectivity organization is a controversial topic. Previous work has primarily focused on comparing mice and rats to primates. However, as these rodents are nocturnal and terrestrial, they rely much less on visual input than primates. Here, we investigated the functional cortical organization of grey squirrels which are diurnal and arboreal, thereby better resembling primate ecology. We used ultra-high field resting-state fMRI data to compute and compare the functional connectivity patterns of frontal regions in grey squirrels (Sciurus carolinensis), rats (Rattus norvegicus), and marmosets (Callithrix jacchus). We utilized a fingerprinting analysis to compare interareal patterns of functional connectivity from seeds across frontal cortex in all three species. The results show that grey squirrels, but not rats, possess a frontoparietal connectivity organization that resembles the connectivity pattern of marmoset lateral prefrontal cortical areas. Since grey squirrels and marmosets have acquired an arboreal way of life but show no common arboreal ancestor, the expansion of the visual system and the formation of a frontoparietal connectivity architecture might reflect convergent evolution driven by similar ecological niches in primates and tree squirrels.

Robot Assisted Neurosurgery for High-Accuracy, Minimally-Invasive Deep Brain Electrophysiology in Monkeys.

Traditional methods to access subcortical structures involve the use of anatomical atlases and high precision stereotaxic frames but suffer from significant variations in implantation accuracy. Here, we leveraged the use of the ROSA One(R) Robot Assistance Platform in non-human primates to study electrophysiological interactions of the corticospinal tract with spinal cord circuits. We were able to target and stimulate the corticospinal tract within the internal capsule with high accuracy and efficiency while recording spinal local field potentials and multi-unit spikes. Our method can be extended to any subcortical structure and allows implantation of multiple deep brain stimulation probes at the same time. Clinical Relevance- Our method will allow us to elucidate further roles of the corticospinal tract and its interactions with other processing centers in intact animals and in motor syndromes in the future.

The effect of repeated freezing and thawing on the suture pull-out strength in equine arytenoid and cricoid cartilages.

OBJECTIVE: To assess the effect of repeated freezing and thawing on the suture pull-out strength in arytenoid and cricoid cartilages subjected to the laryngoplasty (LP) procedure. STUDY DESIGN: Ex vivo experimental study. SAMPLE POPULATION: Ten grossly normal equine cadaveric larynges. METHODS: Bilateral LP constructs were created using a standard LP technique. One hemilarynx was randomly allocated to the single freeze and thaw group and the other allocated to the repeated freeze and thaw (3 complete cycles) group. The suture ends of each LP construct were attached to a load frame and subjected to monotonic loading until construct failure. Mean load (N) and displacement (mm) at LP construct failure were compared between groups. RESULTS: All LP constructs failed by suture pull through the arytenoid cartilage. The mean load at failure was similar between groups (118.9 ± 25.5 N in the single freeze and thaw group and 113.4 ± 20.5 N in the repeated freeze and thaw group, P = .62). The mean displacement at failure was similar between groups (54.4 ± 15.1 mm in the single freeze and thaw group and 54.4 ± 15.4 mm in the repeated freeze and thaw group, P = .99). CONCLUSION: Repeated freezing and thawing did not affect the suture pullout strength of the arytenoid and cricoid cartilages. CLINICAL SIGNIFICANCE: Laryngeal specimens that have been subjected to repeated freezing and thawing can be utilized in the experimental evaluation of LP procedures because there is no alteration of the suture pull-out strength of the relevant cartilages.

Kinetic Analysis in Horses With Deep Digital Flexor Tendinopathy Within the Digit Diagnosed by Magnetic Resonance Imaging.

Objectives: To determine the stance duration and ground reaction forces (GRF) of horses with deep digital flexor (DDF) tendinopathy at the level of the foot and compare the stance duration and GRF to those of clinically sound horses. Design: Prospective clinical study. Animals: Sixteen horses (seven horses with bilateral forelimb lameness, four horses with unilateral forelimb lameness, and five horses with no lameness). Procedures: Analyses of kinetic variables were performed on both forelimbs from sound horses and horses diagnosed with chronic DDF tendinopathy. Stance duration and longitudinal and vertical components of the GRF were determined for the limbs of clinically sound horses and limbs of horses with DDF tendinopathy. Separate Spearman correlation analyses were used to assess potential association within groups (combined left and right forelimbs of clinically sound horses, lamest limbs of horses with DDF tendinopathy, and contralateral limbs of horses with DDF tendinopathy) and with the set of kinetic variables. Analysis of variance on mean ranks of tied values was used to determine differences in kinetic variables between groups (PROC GLIMMIX) using the kinetic values of the clinically sound horses as the reference group. Results: There were a total of 11 lame horses. Seven horses had bilateral forelimb lameness and four had unilateral lameness. Of the 11 horses, there were 15 DDF tendinopathies. There were eight dorsal border DDF tendinopathies, five core DDF tendinopathies, and two sagittal/parasagittal splits DDF tendinopathies. The most lame limbs of horses with DDF tendinopathy had significantly smaller values for peak vertical force and time of peak braking force than did forelimbs of clinically sound horses. Also, the most lame limbs of horses with DDF tendinopathy had significantly larger values for the time of peak vertical force than did forelimbs of clinically sound horses. Conclusions and Clinical Relevance: Horses with chronic DDF tendinopathies develop certain alterations of GRF parameters. This information can be used in future studies to determine if particular kinetic variable changes in horses with DDF tendinopathies differ from those of horses with other pathologies within the foot and therefore could be diagnostic.

Chlorpyrifos should be banned in agriculture and livestock production in Colombia

Chlorpyrifos (CPF) is a pesticide widely used in Colombia´s agriculture, including crops, farm animals and pets, despite it has been banned for use in the European Union and the United States. Studies demonstrate that even low blood levels of CPF -which do not inhibit blood acetylcholinesterase- can lead to child developmental and neurological disorders such as smaller head circumference and brain alterations, and psychomotor and cognitive deficits related to learning ability, attention and memory. In adults, CPF is an endocrine disruptor and breast carcinogen. High direct and indirect economic costs have been associated with CPF exposure. Not only farmers and their families -who have the highest exposures- but the general population consuming crops sprayed with CPF are also at risk. For these reasons CPF was recently banned by the European Union (2020) and the USA (2021). Pesticide regulation policies vary greatly depending on which and how scientific studies are used to assess health risks. Pesticide evaluations funded by the chemical industry should be rectified to avoid conflicts of interest. Furthermore, political alignment with the interests of the industry should not take precedence over independent scientific evidence. It is discouraging, to say the least, that until stricter health laws are passed in Colombia, CPFs and related pesticides will continue to be imported from those countries that have already banned them. Colombian scientists should raise their voice to challenge blind acceptance of profits over unintended consequences, and efforts to prevent pesticide´s abuse should be encouraged.

El clorpirifos (CPF) es un pesticida ampliamente utilizado en la agricultura de Colombia, incluidos cultivos, animales de granja y mascotas, a pesar de haber sido prohibido en la Unión Europea y Estados Unidos. Los estudios han demostrado que incluso niveles bajos de CPF en sangre -que no inhiben la acetilcolinesterasa sanguínea- pueden provocar trastornos neurológicos y del desarrollo infantil, como menor circunferencia de la cabeza y alteraciones cerebrales, y déficits psicomotores y cognitivos relacionados con la capacidad de aprendizaje, la atención y la memoria. En adultos, el CPF es un disruptor endocrino y causante de cáncer de mama. Altos costos económicos directos e indirectos se han asociado con la exposición al CPF. No solo los trabajadores agrícolas y sus familias, que están más expuestos, sino también la población en general que consume cultivos rociados con CPF también están en riesgo. Por estas razones el CPF fue prohibido recientemente por la Unión Europea (2020) y los EE. UU. (2021). Las políticas de regulación de plaguicidas varían mucho según los estudios científicos escogidos para evaluar los riesgos para la salud. Las evaluaciones de plaguicidas financiadas por la industria química deben rectificarse para evitar conflictos de interés. Además, ante la evidencia científica independiente no debería prevalecer la alineación política con los intereses de dicha industria. Es desalentador, por decir lo menos, que hasta que se aprueben leyes de salud más estrictas en Colombia se seguirán importando CPF y pesticidas relacionados desde aquellos países que ya los han prohibido. Los científicos colombianos deben alzar la voz para desafiar la aceptación ciega de ganancias por encima de las consecuencias no deseadas en salud pública, y se deben alentar los esfuerzos para prevenir el abuso de pesticidas.

Clorpirifós (CPF) é um pesticida registrado amplamente utilizado na agricultura colombiana, incluindo lavouras, animais de fazenda e animais de estimação, apesar de ter sido proibido na União Europeia e nos Estados Unidos. Estudos têm demonstrado que mesmo níveis baixos de CPF no sangue -que não inibem a acetilcolinesterase sanguínea-podem levar a distúrbios neurológicos e de desenvolvimento em crianças, como menor perímetro cefálico e alterações cerebrais, além de déficits psicomotores e cognitivos relacionados à capacidade de aprendizagem, atenção e memoria. Em adultos, o CPF é um desregulador endócrino e cancerígeno da mama. Altos custos econômicos diretos (devido ao tratamento) e indiretos (devido à perda de produtividade) têm sido associados à exposição ao CPF. Não apenas os trabalhadores agrícolas e suas famílias, que têm as maiores exposições, mas a população em geral que consome culturas pulverizadas com CPF também estão em risco. Por essas razões, o CPF foi recentemente proibido pela União Europeia (2020) e pelos EUA (2021). As políticas de regulamentação de pesticidas variam muito, dependendo de quais (e como) os estudos científicos são usados para avaliar os riscos à saúde. As avaliações de pesticidas financiadas pela indústria química devem ser retificadas para evitar conflitos de interesse. Além disso, o alinhamento político com os interesses da indústria não deve ter precedência sobre as evidências científicas independentes. É desanimador - para dizer o mínimo - que até que leis de saúde mais rígidas sejam aprovadas na Colômbia, o CPF e tóxicos relacionados continuarão a ser importados dos países que já os proibiram.

Three-dimensional hydrogel culture systems support growth and determination of chemosensitivity of feline sarcoma and carcinoma cell lines.

OBJECTIVE: To evaluate feline injection site-associated sarcoma (FISAS) and oral squamous cell carcinoma (FOSCC) cells in 3-D hydrogel-based cell cultures to determine chemosensitivity to carboplatin at concentrations comparable to those eluted from carboplatin-impregnated calcium sulfate hemihydrate (C-ICSH) beads. SAMPLE: 2 immortalized cell lines, each from a histologically confirmed primary FISAS and FOSCC. PROCEDURES: Hydrogels (10% wt/vol) were formed via UV exposure from methacrylamide-functionalized gelatin dissolved in PBSS. For each cell line, approximately 100,000 cells were encapsulated per hydrogel. Three cell-seeded 3-D hydrogels were evaluated for each carboplatin concentration (0, 150, 300, 450, and 600 µM) across 3 experiments. Drug efficacy was assessed by luminescence assay 72 hours after treatment. Growth of tumor cells treated with 300 µM or 600 µM carboplatin was evaluated using live-cell morphology imaging and confocal microscopy at 3, 7, and 14 days after treatment. RESULTS: Mean half-maximal inhibitory concentration (IC50) values for FISAS and FOSCC cells ranged from 123 to 171 µM and 155 to 190 µM, respectively, based on luminescence assay. Viability at 3, 7, and 14 days for both cell lines at 300 µM carboplatin was 50%, 25%, and 5% and at 600 µM carboplatin was 25%, 10%, and < 5%. CLINICAL RELEVANCE: 3-D hydrogel cell culture systems supported growth of feline tumor cells for determination of in vitro chemosensitivity. IC50s of each cell line were within the range of carboplatin concentrations eluted from C-ICSH beads. Cells from FISAS and FOSCC cell lines treated with carboplatin showed dose-dependent and time-dependent decreases in viability.

Three-dimensional hydrogel culture systems support growth and determination of chemosensitivity of feline sarcoma and carcinoma cell lines.

OBJECTIVE: To evaluate feline injection site-associated sarcoma (FISAS) and oral squamous cell carcinoma (FOSCC) cells in 3-D hydrogel-based cell cultures to determine chemosensitivity to carboplatin at concentrations comparable to those eluted from carboplatin-impregnated calcium sulfate hemihydrate (C-ICSH) beads. SAMPLE: 2 immortalized cell lines, each from a histologically confirmed primary FISAS and FOSCC. PROCEDURES: Hydrogels (10% wt/vol) were formed via UV exposure from methacrylamide-functionalized gelatin dissolved in PBSS. For each cell line, approximately 100,000 cells were encapsulated per hydrogel. Three cell-seeded 3-D hydrogels were evaluated for each carboplatin concentration (0, 150, 300, 450, and 600 µM) across 3 experiments. Drug efficacy was assessed by luminescence assay 72 hours after treatment. Growth of tumor cells treated with 300 µM or 600 µM carboplatin was evaluated using live-cell morphology imaging and confocal microscopy at 3, 7, and 14 days after treatment. RESULTS: Mean half-maximal inhibitory concentration (IC50) values for FISAS and FOSCC cells ranged from 123 to 171 µM and 155 to 190 µM, respectively, based on luminescence assay. Viability at 3, 7, and 14 days for both cell lines at 300 µM carboplatin was 50%, 25%, and 5% and at 600 µM carboplatin was 25%, 10%, and < 5%. CLINICAL RELEVANCE: 3-D hydrogel cell culture systems supported growth of feline tumor cells for determination of in vitro chemosensitivity. IC50s of each cell line were within the range of carboplatin concentrations eluted from C-ICSH beads. Cells from FISAS and FOSCC cell lines treated with carboplatin showed dose-dependent and time-dependent decreases in viability.

An open access resource for functional brain connectivity from fully awake marmosets.

The common marmoset (Callithrix jacchus) is quickly gaining traction as a premier neuroscientific model. However, considerable progress is still needed in understanding the functional and structural organization of the marmoset brain to rival that documented in longstanding preclinical model species, like mice, rats, and Old World primates. To accelerate such progress, we present the Marmoset Functional Brain Connectivity Resource (marmosetbrainconnectome.org), currently consisting of over 70 h of resting-state fMRI (RS-fMRI) data acquired at 500 µm isotropic resolution from 31 fully awake marmosets in a common stereotactic space. Three-dimensional functional connectivity (FC) maps for every cortical and subcortical gray matter voxel are stored online. Users can instantaneously view, manipulate, and download any whole-brain functional connectivity (FC) topology (at the subject- or group-level) along with the raw datasets and preprocessing code. Importantly, researchers can use this resource to test hypotheses about FC directly - with no additional analyses required - yielding whole-brain correlations for any gray matter voxel on demand. We demonstrate the resource's utility for presurgical planning and comparison with tracer-based neuronal connectivity as proof of concept. Complementing existing structural connectivity resources for the marmoset brain, the Marmoset Functional Brain Connectivity Resource affords users the distinct advantage of exploring the connectivity of any voxel in the marmoset brain, not limited to injection sites nor constrained by regional atlases. With the entire raw database (RS-fMRI and structural images) and preprocessing code openly available for download and use, we expect this resource to be broadly valuable to test novel hypotheses about the functional organization of the marmoset brain.

Effect of TrisEDTA and Chlorhexidine 0.12% on an In Vitro-Defined Biofilm Representing the Subgingival Plaque Biofilm of the Dog.

This study was designed to investigate the effects of chlorhexidine 0.12%, TrisEDTA (tromethamine ethylenediamintetraacetic acid), and a combination of chlorhexidine 0.12% and TrisEDTA on an in vitro plaque biofilm model comprised of three bacterial species commonly found in canine subgingival plaque. Porphyromonas gulae, Actinomyces canis, and Neisseria canis were grown in a biofilm on polished hydroxyapatite coated titanium alloy pucks for 72 h prior to exposure to one of four test solutions: TrisEDTA, chlorhexidine 0.12%, a combination of TrisEDTA and chlorhexidine 0.12%, or sterile deionized water as a control. Following exposure to the test solution, a sample was collected of the biofilm either immediately or following 24 h of additional incubation in a broth medium. Lower numbers of CFU/mL of Porphyromonas gulae resulted when the biofilm was treated with a solution of chlorhexidine 0.12% and TrisEDTA compared to with chlorhexidine 0.12% alone, TrisEDTA alone, or the control and so this solution can be said to be synergistic against Porphyromonas gulae in this controlled in vitro model. Greater reductions in the numbers of CFU/mL of Actinomyces canis and Neisseria canis resulted from treatment with chlorhexidine 0.12% alone than if treated with the combination of TrisEDTA and chlorhexidine 0.12%. When treated biofilm samples were allowed 24 h of additional growth in fresh media, greater variance resulted and this variance highlights the complex dynamics involved in bacterial growth within a biofilm.

Functional Organization of Frontoparietal Cortex in the Marmoset Investigated with Awake Resting-State fMRI.

Frontoparietal networks contribute to complex cognitive functions in humans and macaques, such as working memory, attention, task-switching, response suppression, grasping, reaching, and eye movement control. However, there has been no comprehensive examination of the functional organization of frontoparietal networks using functional magnetic resonance imaging in the New World common marmoset monkey (Callithrix jacchus), which is now widely recognized as a powerful nonhuman primate experimental animal. In this study, we employed hierarchical clustering of interareal blood oxygen level-dependent signals to investigate the hypothesis that the organization of the frontoparietal cortex in the marmoset follows the organizational principles of the macaque frontoparietal system. We found that the posterior part of the lateral frontal cortex (premotor regions) was functionally connected to the anterior parietal areas, while more anterior frontal regions (frontal eye field [FEF]) were connected to more posterior parietal areas (the region around the lateral intraparietal area [LIP]). These overarching patterns of interareal organization are consistent with a recent macaque study. These findings demonstrate parallel frontoparietal processing streams in marmosets and support the functional similarities of FEF-LIP and premotor-anterior parietal pathways between marmoset and macaque.

Simultaneous functional MRI of two awake marmosets.

Social cognition is a dynamic process that requires the perception and integration of a complex set of idiosyncratic features between interacting conspecifics. Here we present a method for simultaneously measuring the whole-brain activation of two socially interacting marmoset monkeys using functional magnetic resonance imaging. MRI hardware (a radiofrequency coil and peripheral devices) and image-processing pipelines were developed to assess brain responses to socialization, both on an intra-brain and inter-brain level. Notably, the brain activation of a marmoset when viewing a second marmoset in-person versus when viewing a pre-recorded video of the same marmoset-i.e., when either capable or incapable of socially interacting with a visible conspecific-demonstrates increased activation in the face-patch network. This method enables a wide range of possibilities for potentially studying social function and dysfunction in a non-human primate model.