Full- versus Sub-Regional Quantification of Amyloid-Beta Load on Mouse Brain Sections

Extracellular accumulation of amyloid-beta (Aβ) plaques is one of the major pathological hallmarks of Alzheimer\u27s disease (AD), and is the target of the only FDA-approved disease-modifying treatment for AD. Accordingly, the use of transgenic mouse models that overexpress the amyloid precursor protein and thereby accumulate cerebral Aβ plaques are widely used to model human AD in mice. Therefore, immunoassays, including enzyme-linked immunosorbent assay (ELISA) and immunostaining, commonly measure the Aβ load in brain tissues derived from AD transgenic mice. Though the methods for Aβ detection and quantification have been well established and documented, the impact of the size of the region of interest selected in the brain tissue on Aβ load measurements following immunostaining has not been reported. Therefore, the current protocol aimed to compare the Aβ load measurements across the full- and sub-regions of interest using an image analysis software. The steps involved in brain tissue preparation, free-floating brain section immunostaining, imaging, and quantification of Aβ load in full- versus sub-regions of interest are described using brain sections derived from 13-month-old APP/PS1 double transgenic male mice. The current protocol and the results provide valuable information about the impact of the size of the region of interest on Aβ-positive area quantification, and show a strong correlation between the Aβ-positive area obtained using the full- and sub-regions of interest analyses for brain sections derived from 13-month-old male APP/PS1 mice that show widespread Aβ deposition

Comparative Studies Between the Murine Immortalized Brain Endothelial Cell Line (bEnd.3) and Induced Pluripotent Stem Cell-Derived Human Brain Endothelial Cells for Paracellular Transport

Brain microvascular endothelial cells, forming the anatomical site of the blood-brain barrier (BBB), are widely used as in vitro complements to in vivo BBB studies. Among the immortalized cells used as in vitro BBB models, the murine-derived bEnd.3 cells offer culturing consistency and low cost and are well characterized for functional and transport assays, but result in low transendothelial electrical resistance (TEER). Human-induced pluripotent stem cells differentiated into brain microvascular endothelial cells (ihBMECs) have superior barrier properties, but the process of differentiation is time-consuming and can result in mixed endothelial-epithelial gene expression. Here we performed a side-by-side comparison of the ihBMECs and bEnd.3 cells for key paracellular diffusional transport characteristics. The TEER across the ihBMECs was 45- to 68-fold higher than the bEnd.3 monolayer. The ihBMECs had significantly lower tracer permeability than the bEnd.3 cells. Both, however, could discriminate between the paracellular permeabilities of two tracers: sodium fluorescein (MW: 376 Da) and fluorescein isothiocyanate (FITC)–dextran (MW: 70 kDa). FITC-dextran permeability was a strong inverse-correlate of TEER in the bEnd.3 cells, whereas sodium fluorescein permeability was a strong inverse-correlate of TEER in the ihBMECs. Both bEnd.3 cells and ihBMECs showed the typical cobblestone morphology with robust uptake of acetylated LDL and strong immuno-positivity for vWF. Both models showed strong claudin-5 expression, albeit with differences in expression location. We further confirmed the vascular endothelial- (CD31 and tube-like formation) and erythrophagocytic-phenotypes and the response to inflammatory stimuli of ihBMECs. Overall, both bEnd.3 cells and ihBMECs express key brain endothelial phenotypic markers, and despite differential TEER measurements, these in vitro models can discriminate between the passage of different molecular weight tracers. Our results highlight the need to corroborate TEER measurements with different molecular weight tracers and that the bEnd.3 cells may be suitable for large molecule transport studies despite their low TEER

Acute and Chronic Dosing of a High-Affinity Rat/Mouse Chimeric Transferrin Receptor Antibody in Mice

Non-invasive brain delivery of neurotherapeutics is challenging due to the blood-brain barrier. The revived interest in transferrin receptor antibodies (TfRMAbs) as brain drug-delivery vectors has revealed the effect of dosing regimen, valency, and affinity on brain uptake, TfR expression, and Fc-effector function side effects. These studies have primarily used monovalent TfRMAbs with a human constant region following acute intravenous dosing in mice. The effects of a high-affinity bivalent TfRMAb with a murine constant region, without a fusion partner, following extravascular dosing in mice are, however, not well characterized. Here we elucidate the plasma pharmacokinetics and safety of a high-affinity bivalent TfRMAb with a murine constant region following acute and chronic subcutaneous dosing in adult C57BL/6J male mice. Mice received a single (acute dosing) 3 mg/kg dose, or were treated for four weeks (chronic dosing). TfRMAb and control IgG1 significantly altered reticulocyte counts following acute and chronic dosing, while other hematologic parameters showed minimal change. Chronic TfRMAb dosing did not alter plasma- and brain-iron measurements, nor brain TfR levels, however, it significantly increased splenic-TfR and -iron. Plasma concentrations of TfRMAb were significantly lower in mice chronically treated with IgG1 or TfRMAb. Overall, no injection related reactions were observed in mic

The consistency of invasive and non-invasive arterial blood pressure for the assessment of dynamic cerebral autoregulation in NICU patients

BackgroundStudies of the clinical application of dynamic cerebral autoregulation show considerable variations, and differences in blood pressure devices may be one of the reasons for this variation. Few studies have examined the consistency of invasive and non-invasive arterial blood pressure for evaluating cerebral autoregulation. We attempted to investigate the agreement between invasive and non-invasive blood pressure methods in the assessment of dynamic cerebral autoregulation with transfer function analysis.MethodsContinuous cerebral blood flow velocity and continuous invasive and non-invasive arterial blood pressure were simultaneously recorded for 15 min. Transfer function analysis was applied to derive the phase shift, gain and coherence function at all frequency bands from the first 5, 10, and 15 min of the 15-min recordings. The consistency was assessed with Bland–Altman analysis and intraclass correlation coefficient.ResultsThe consistency of invasive and noninvasive blood pressure methods for the assessment of dynamic cerebral autoregulation was poor at 5 min, slightly improved at 10 min, and good at 15 min. The values of the phase shift at the low-frequency band measured by the non-invasive device were higher than those measured with invasive equipment. The coherence function values measured by the invasive technique were higher than the values derived from the non-invasive method.ConclusionBoth invasive and non-invasive arterial blood pressure methods have good agreement in evaluating dynamic cerebral autoregulation when the recording duration reaches 15 min. The phase shift values measured with non-invasive techniques are higher than those measured with invasive devices. We recommend selecting the most appropriate blood pressure device to measure cerebral autoregulation based on the disease, purpose, and design

Biologic TNF-α Inhibitors Reduce Microgliosis, Neuronal Loss, and Tau Phosphorylation in a Transgenic Mouse Model of Tauopathy

Background Tumor necrosis factor-α (TNF-α) plays a central role in Alzheimer’s disease (AD) pathology, making biologic TNF-α inhibitors (TNFIs), including etanercept, viable therapeutics for AD. The protective effects of biologic TNFIs on AD hallmark pathology (Aβ deposition and tau pathology) have been demonstrated. However, the effects of biologic TNFIs on Aβ-independent tau pathology have not been reported. Existing biologic TNFIs do not cross the blood–brain barrier (BBB), therefore we engineered a BBB-penetrating biologic TNFI by fusing the extracellular domain of the type-II human TNF-α receptor (TNFR) to a transferrin receptor antibody (TfRMAb) that ferries the TNFR into the brain via receptor-mediated transcytosis. The present study aimed to investigate the effects of TfRMAb-TNFR (BBB-penetrating TNFI) and etanercept (non-BBB-penetrating TNFI) in the PS19 transgenic mouse model of tauopathy. Methods Six-month-old male and female PS19 mice were injected intraperitoneally with saline (n = 12), TfRMAb-TNFR (1.75 mg/kg, n = 10) or etanercept (0.875 mg/kg, equimolar dose of TNFR, n = 10) 3 days/week for 8 weeks. Age-matched littermate wild-type mice served as additional controls. Blood was collected at baseline and 8 weeks for a complete blood count. Locomotion hyperactivity was assessed by the open-field paradigm. Brains were examined for phosphorylated tau lesions (Ser202, Thr205), microgliosis, and neuronal health. The plasma pharmacokinetics were evaluated following a single intraperitoneal injection of 0.875 mg/kg etanercept or 1.75 mg/kg TfRMAb-TNFR or 1.75 mg/kg chronic TfRMAb-TNFR dosing for 4 weeks. Results Etanercept significantly reduced phosphorylated tau and microgliosis in the PS19 mouse brains of both sexes, while TfRMAb-TNFR significantly reduced these parameters in the female PS19 mice. Both TfRMAb-TNFR and etanercept treatment improved neuronal health by significantly increasing PSD95 expression and attenuating hippocampal neuron loss in the PS19 mice. The locomotion hyperactivity in the male PS19 mice was suppressed by chronic etanercept treatment. Equimolar dosing resulted in eightfold lower plasma exposure of the TfRMAb-TNFR compared with etanercept. The hematological profiles remained largely stable following chronic biologic TNFI dosing except for a significant increase in platelets with etanercept. Conclusion Both TfRMAb-TNFR (BBB-penetrating) and non-BBB-penetrating (etanercept) biologic TNFIs showed therapeutic effects in the PS19 mouse model of tauopathy

Efficacy and Safety of a Brain-Penetrant Biologic TNF-α Inhibitor in Aged APP/PS1 Mice

Tumor necrosis factor alpha (TNF-α) plays a vital role in Alzheimer’s disease (AD) pathology, and TNF-α inhibitors (TNFIs) modulate AD pathology. We fused the TNF-α receptor (TNFR), a biologic TNFI that sequesters TNF-α, to a transferrin receptor antibody (TfRMAb) to deliver the TNFI into the brain across the blood–brain barrier (BBB). TfRMAb-TNFR was protective in 6-month-old transgenic APP/PS1 mice in our previous work. However, the effects and safety following delayed chronic TfRMAb-TNFR treatment are unknown. Herein, we initiated the treatment when the male APP/PS1 mice were 10.7 months old (delayed treatment). Mice were injected intraperitoneally with saline, TfRMAb-TNFR, etanercept (non-BBB-penetrating TNFI), or TfRMAb for ten weeks. Biologic TNFIs did not alter hematology indices or tissue iron homeostasis; however, TfRMAb altered hematology indices, increased splenic iron transporter expression, and increased spleen and liver iron. TfRMAb-TNFR and etanercept reduced brain insoluble-amyloid beta (Aβ) 1-42, soluble-oligomeric Aβ, and microgliosis; however, only TfRMAb-TNFR reduced Aβ peptides, Thioflavin-S-positive Aβ plaques, and insoluble-oligomeric Aβ and increased plaque-associated phagocytic microglia. Accordingly, TfRMAb-TNFR improved spatial reference memory and increased BBB-tight junction protein expression, whereas etanercept did not. Overall, despite delayed treatment, TfRMAb-TNFR resulted in a better therapeutic response than etanercept without any TfRMAb-related hematology- or iron-dysregulation in aged APP/PS1 mice

Research on Dynamics and Stability in the Stairs-Climbing of a Tracked Mobile Robot

Aiming at the functional requirement of climbing up the stairs, the dynamics and stability during a tracked mobile robot's climbing of stairs is studied. First, from the analysis of its cross-country performance, the mechanical structure of the tracked mobile robot is designed and the hardware composition of its control system is given. Second, based on the analysis to its stairs-climbing process, the dynamical model of stairs-climbing is established by using the classical mechanics method. Next, the stability conditions for its stairs-climbing are determined and an evaluation method of its stairs-climbing stability is proposed, based on a mechanics analysis on the robot's backwards tumbling during the stairs-climbing process. Through simulation and experiments, the effectiveness of the dynamical model and the stability evaluation method of the tracked mobile robot in stairs-climbing is verified, which can provide design and analysis foundations for the tracked mobile robots' stairs-climbing

Clinical outcomes and risk factors of secondary extraintestinal manifestation in ulcerative colitis: results of a multicenter and long-term follow-up retrospective study

Background Extraintestinal manifestations (EIM) are common in ulcerative colitis (UC). In Shanghai, China, data on the incidence rate and risk factors of EIM in UC patients remain scarce. Methods The study population consisted of UC patients who were identified from a prospectively maintained, institutional review board-approved database at our institutes from June 1986 to December 2018. The demographic and clinical characteristics of the study participants were analyzed. The study included secondary EIM in UC patients and follow-up, while primary EIM was excluded. The diagnosis of EIM was based on clinical, radiological, endoscopic, and immunologic examination and histological findings. Results In total, 271 eligible patients were included in the current study, with a median follow-up time of 13.0 years (interquartile range, 9.0–17.0), and including 31 cases (11.4%) that developed EIM. EIM was associated with clinical outcomes in UC patients and the following factors were identified as contributing factors for the development of EIM: a disease duration of >5 years (odds ratio (OR), 3.721; 95% confidence interval (CI) [1.209–11.456]), age at diagnosis >40 years (OR, 2.924, 95% CI [1.165–7.340]), refractory clinical symptoms (OR, 4.119; 95% CI [1.758–9.650]), and moderate or severe anemia (OR, 2.592; 95% CI [1.047–6.413]). Conclusion In this study, approximately 11.4% UC patients go on to develop at least one EIM. Clinicians should prioritize early control of the disease and treatment of anemia in UC in order to prevent the development of EIM and improve disease prognosis