Cerebral hemodynamics as biomarkers for neuropathic pain in rats: a longitudinal study using a spinal nerve ligation model.

ABSTRACT: Neuropathic pain is one of the most challenging types of pain to diagnose and treat, a problem exacerbated by the lack of a quantitative biomarker. Recently, several clinical and preclinical studies have shown that neuropathic pain induces cerebral hemodynamic changes as a result of neuroplasticity in the brain. Our hypothesis in this study is that neuropathic pain leads to cerebral hemodynamic changes over postoperative time in a spinal nerve ligation (SNL) rat model, which has not been longitudinally explored previously. Furthermore, by identifying multiple regional hemodynamic features that are the most distinct between SNL and sham groups, where the sham group underwent only an incision without SNL, it may be possible to classify the SNL group regardless of when the onset of pain occurs. We investigate cerebral hemodynamic changes using dynamic susceptibility contrast magnetic resonance imaging in a rat model up to 28 days after ligating L5/L6 spinal nerves. We trained a linear support vector machine with relative cerebral blood volume data from different brain regions and found that the prediction model trained on the nucleus accumbens, motor cortex, pretectal area, and thalamus classified the SNL group and sham group at a 79.27% balanced accuracy, regardless of when the onset of pain occurred (SNL/sham: 60/45 data points). From the use of the SNL model without prior knowledge of the onset time of pain, the current findings highlight the potential of relative cerebral blood volume in the 4 highlighted brain regions as a biomarker for neuropathic pain.

User-designed device with programmable release profile for localized treatment.

Three-dimensional printing enables precise and on-demand manufacture of customizable drug delivery systems to advance healthcare toward the goal of personalized medicine. However, major challenges remain in realizing personalized drug delivery that fits a patient-specific drug dosing schedule using local drug delivery systems. In this study, a user-designed device is developed as implantable therapeutics that can realize personalized drug release kinetics by programming the inner structural design on the microscale. The drug release kinetics required for various treatments, including dose-dense therapy and combination therapy, can be implemented by controlling the dosage and combination of drugs along with the rate, duration, initiation time, and time interval of drug release according to the device layer design. After implantation of the capsular device in mice, the in vitro-in vivo and pharmacokinetic evaluation of the device is performed, and the therapeutic effect of the developed device is achieved through the local release of doxorubicin. The developed user-designed device provides a novel platform for developing next-generation drug delivery systems for personalized and localized therapy.

MRI investigation of vascular remodeling for heterogeneous edema lesions in subacute ischemic stroke rat models: Correspondence between cerebral vessel structure and function.

The spatial heterogeneity in the temporal occurrence of pseudo-normalization of MR apparent diffusion coefficient values for ischemic lesions may be related to morphological and functional vascular remodeling. As the area of accelerated pseudo-normalization tends to expand faster and more extensively into the chronic stage, detailed vascular characterization of such areas is necessary. During the subacute stage of transient middle cerebral artery occlusion rat models, the morphological size of the macrovasculature, microvascular vessel size index (VSI), and microvessel density (MVD) were quantified along with functional perfusion measurements of the relative cerebral blood flow (rCBF) and mean transit time (rMTT) of the corresponding areas (33 cases for each parameter). When compared with typical pseudo-normalization lesions, early pseudo-normalization lesions exhibited larger VSI and rCBF (p < 0.001) at reperfusion days 4 and 7, along with reduced MVD and elongated rMTT (p < 0.001) at reperfusion days 1, 4, and 7. The group median VSI and rCBF exhibited a strong positive correlation (r = 0.92), and the corresponding MVD and rMTT showed a negative correlation (r = -0.48). Light sheet fluorescence microscopy images were used to quantitatively validate the corresponding MRI-derived microvascular size, density, and cerebral blood volume.

Model-free leakage index estimation of the blood-brain barrier using dual dynamic susceptibility contrast MRI acquisition.

Pharmacokinetic K2 mapping from dynamic susceptibility contrast (DSC)-MRI can be a sensitive technique for evaluating the vascular permeability of the subtly damaged blood-brain barrier (BBB) in ischemic regions. However, the K2 values of ischemic lesions depend upon the selection of the intact BBB reference region. As previous observations suggest that the ΔR2* curve of pre-loaded DSC-MRI is not significantly affected by the extravasation of contrast agent, dual DSC-MRI acquisitions can be performed to derive the BBB leakage index from the voxel-wise reference input function for ischemic regions. This study aims to demonstrate the robustness of such model-free leakage index estimation in ischemic brains. By configuring the relationship between dual ΔR2* curves of the intact contralateral brain, the deviation of the measured ΔR2* curve from the unloaded DSC-MRI with respect to the non-deviated ΔR2* curve in the pre-loaded DSC-MRI can be quantified as the BBB leakage index. Such model-free leakage index values from rats with transient middle carotid artery occlusion (tMCAO) (n = 17) and normal controls (n = 3) were evaluated and compared with conventional K2 values with multiple reference regions. Inter-subject leakage index values were also compared with the corresponding ΔT1 map. Evans-blue-stained images were used to validate the leakage index. For the tMCAO group, leakage index values correlated well with ΔT1 (Pearson's r = 0.828). The hyperintense area on the leakage index map matched well with the corresponding Evans-blue-stained area (Dice correlation = 0.626). The slopes of the scatter-plot from the leakage index (0.97-1.00) were observed to be more robust against changes in the reference region than those from conventional K2 values (0.94-1.07). In a subtly damaged BBB tMCAO model, model-free evaluation of vascular permeability using dual DSC-MRIs would provide a consistent measure of inter-subject vascular permeability.

Target-switchable Gd(III)-DOTA/protein cage nanoparticle conjugates with multiple targeting affibody molecules as target selective T1 contrast agents for high-field MRI.

Magnetic resonance imaging (MRI) is a non-invasive in vivo imaging tool, providing high enough spatial resolution to obtain both the anatomical and the physiological information of patients. However, MRI generally suffers from relatively low sensitivity often requiring the aid of contrast agents (CA) to enhance the contrast of vessels and/or the tissues of interest from the background. The targeted delivery of diagnostic probes to the specific lesion is a powerful approach for early diagnosis and signal enhancement leading to the effective treatment of various diseases. Here, we established targeting ligand switchable nanoplatforms using lumazine synthase protein cage nanoparticles derived from Aquifex aeolicus (AaLS) by genetically introducing the SpyTag peptide (ST) to the C-terminus of the AaLS subunits to form an ST-displaying AaLS (AaLS-ST). Conversely, multiple targeting ligands were constructed by genetically fusing SpyCatcher protein (SC) to either HER2 or EGFR targeting affibody molecules (SC-HER2Afb or SC-EGFRAfb). Gd(III)-DOTA complexes were chemically attached to the AaLS-ST and the external surface of the Gd(III)-DOTA conjugated AaLS-ST (Gd(III)-DOTA-AaLS-ST) were successfully decorated with either the HER2Afb or the EGFRAfb. The resulting Gd(III)-DOTA-AaLS/HER2Afb and Gd(III)-DOTA-AaLS/EGFR2Afb exhibited high r1 relaxivity values of 57 and 25 mM-1 s-1 at 1.4 and 7 T, respectively, which were 10-fold or higher than those of the clinically used Dotarem. Their target-selective contrast enhancements were confirmed with in vitro cell-based MRI scans and the in vivo MR imaging of tumor-bearing mouse models at 7 T. A target-switchable AaLS-based nanoplatform that was developed in this study might serve as a promising T1 CA developing platform at a high magnetic field to detect various tumor sites in a target-specific manner in future clinical applications.

Mapping of microvascular architecture in the brain of an Alzheimer's disease mouse model using MRI.

Increasing evidence suggests that alterations in cerebral microvasculature play a critical role in the pathogenesis of Alzheimer's disease (AD). The objective of this study was to characterize and evaluate the cerebral microvascular architecture of AD transgenic (Tg) mice and compare it with that of non-Tg mice using brain microvascular indices obtained by MRI. Seven non-Tg mice and 10 5xFAD Tg mice were scanned using a 7-T animal MRI system to measure the transverse relaxation rates of R2 and R2* before and after the injection of the monocrystalline iron oxide nanoparticle contrast agent. After calculating ΔR2* and ΔR2, the vessel size index (VSI), mean vessel diameter (mVD), mean vessel density, mean vessel-weighted image (MvWI) and blood volume fraction (BVf) were mapped. Voxel-based analyses and region of interest (ROI)-based analyses were performed to compare the indices of the non-Tg and Tg groups. Voxel comparisons showed that BVf, mVD, VSI and MvWI were greater in the Tg group than in the non-Tg group. Additionally, the ROI-based analysis showed that ΔR2*, BVf, mVD, MvWI and VSI increased in several brain regions of the Tg group compared with those in the non-Tg group. VSI and mVD increased in Tg mice; these findings indicated microvascular disruption in the brain that could be related to damage to the neurovascular unit in AD caused by cerebral amyloid angiopathy.

MRI Visualization of Whole Brain Macro- and Microvascular Remodeling in a Rat Model of Ischemic Stroke: A Pilot Study.

Using superparamagnetic iron oxide nanoparticles (SPION) as a single contrast agent, we investigated dual contrast cerebrovascular magnetic resonance imaging (MRI) for simultaneously monitoring macro- and microvasculature and their association with ischemic edema status (via apparent diffusion coefficient [ADC]) in transient middle cerebral artery occlusion (tMCAO) rat models. High-resolution T1-contrast based ultra-short echo time MR angiography (UTE-MRA) visualized size remodeling of pial arteries and veins whose mutual association with cortical ischemic edema status is rarely reported. ΔR2-ΔR2*-MRI-derived vessel size index (VSI) and density indices (Q and MVD) mapped morphological changes of microvessels occurring in subcortical ischemic edema lesions. In cortical ischemic edema lesions, significantly dilated pial veins (p = 0.0051) and thinned pial arteries (p = 0.0096) of ipsilateral brains compared to those of contralateral brains were observed from UTE-MRAs. In subcortical regions, ischemic edema lesions had a significantly decreased Q and MVD values (p < 0.001), as well as increased VSI values (p < 0.001) than normal subcortical tissues in contralateral brains. This pilot study suggests that MR-based morphological vessel changes, including but not limited to venous blood vessels, are directly related to corresponding tissue edema status in ischemic stroke rat models.

Pattern recognition analysis of directional intravoxel incoherent motion MRI in ischemic rodent brains.

This study aimed to demonstrate a reliable automatic segmentation method for independently separating reduced diffusion and decreased perfusion areas in ischemic stroke brains using constrained nonnegative matrix factorization (cNMF) pattern recognition in directional intravoxel incoherent motion MRI (IVIM-MRI). First, the feasibility of cNMF-based segmentation of IVIM signals was investigated in both simulations and in vivo experiments. The cNMF analysis was independently performed for S0 -normalized and scaled (by the difference between the maximum and minimum) IVIM signals, respectively. Segmentations of reduced diffusion (from S0 -normalized IVIM signals) and decreased perfusion (from scaled IVIM signals) areas were performed using the corresponding cNMF pattern weight maps. Second, Monte Carlo simulations were performed for directional IVIM signals to investigate the relationship between the degree of vessel alignment and the direction of the diffusion gradient. Third, directional IVIM-MRI experiments (x, y and z diffusion-gradient directions, 20 b values at 7 T) were performed for normal (n = 4), sacrificed (n = 1, no flow) and ischemic stroke models (n = 4, locally reduced flow). The results showed that automatic segmentation of the hypoperfused lesion using cNMF analysis was more accurate than segmentation using the conventional double-exponential fitting. Consistent with the simulation, the double-exponential pattern of the IVIM signals was particularly strong in white matter and ventricle regions when the directional flows were aligned with the applied diffusion-gradient directions. cNMF analysis of directional IVIM signals allowed robust automated segmentation of white matter, ventricle, vascular and hypoperfused regions in the ischemic brain. In conclusion, directional IVIM signals were simultaneously sensitive to diffusion and aligned flow and were particularly useful for automatically segmenting ischemic lesions via cNMF-based pattern recognition.

Pattern recognition analysis of dynamic susceptibility contrast (DSC)-MRI curves automatically segments tissue areas with intact blood-brain barrier in a rat stroke model: A feasibility and comparison study.

BACKGROUND: The manual segmentation of intact blood-brain barrier (BBB) regions in the stroke brain is cumbersome, due to the coexistence of infarction, large blood vessels, ventricles, and intact BBB regions, specifically in areas with weak signal enhancement following contrast agent injection. HYPOTHESIS: That from dynamic susceptibility contrast (DSC)-MRI alone, without user intervention, regions of weak BBB damage can be segmented based on the leakage-related parameter K 2 and the extent of intact BBB regions, needed to estimate K 2 values, determined. STUDY TYPE: Feasibility. ANIMAL MODEL: Ten female Sprague-Dawley rats (SD, 200-250g) underwent 1-hour middle carotid artery occlusion (MCAO) and 1-day reperfusion. Two SD rats underwent 1-hour MCAO with 3-day and 5-day reperfusion. FIELD STRENGTH/SEQUENCE: 7T; ADC and T1 maps using diffusion-weighted echo planar imaging (EPI) and relaxation enhancement (RARE) with variable repetition time (TR), respectively. dynamic contrast-enhanced (DCE)-MRI using FLASH. DSC-MRI using gradient-echo EPI. ASSESSMENT: Constrained nonnegative matrix factorization (cNMF) was applied to the dynamic ΔR2* -curves of DSC-MRI (<4 min) in a BBB-disrupted rat model. Areas of voxels with intact BBB, classified by automated cNMF analyses, were then used in estimating K 1 and K 2 values, and compared with corresponding values from manually-derived areas. STATISTICAL TESTS: Mean ± standard deviation of ΔT1 -differences between ischemic and healthy areas were displayed with unpaired Student's t-tests. Scatterplots were displayed with slopes and intercepts and Pearson's r values were evaluated between K 2 maps obtained with automatic (cNMF)- and manually-derived regions of interest (ROIs) of the intact BBB region. RESULTS: Mildly BBB-damaged areas (indistinguishable from DCE-MRI (10 min) parameters) were automatically segmented. Areas of voxels with intact BBB, classified by automated cNMF, matched closely the corresponding, manually-derived areas when respective areas were used in estimating K 2 maps (Pearson's r = 0.97, 12 slices). DATA CONCLUSION: Automatic segmentation of short DSC-MRI data alone successfully identified areas with intact and compromised BBB in the stroke brain and compared favorably with manual segmentation. LEVEL OF EVIDENCE: 3 Technical Efficacy: Stage 1 J. Magn. Reson. Imaging 2020;51:1369-1381.

Cerebral blood perfusion deficits using dynamic susceptibility contrast MRI with gadolinium chelates in rats with post-ischemic reperfusion without significant dynamic contrast-enhanced MRI-derived vessel permeabilities: A cautionary note.

In this study, we quantified perfusion deficits using dynamic susceptibility contrast magnetic resonance imaging (DSC-MRI) with an extravasating contrast agent (CA). We also investigated the efficacy of leakage compensation from CA pre-load in brains from post-ischemic rat models without significant dynamic contrast-enhanced MRI (DCE-MRI)-derived vessel wall permeability. DSC measurements were obtained using fast (0.3 s) echo-planar imaging in both normal rats and rats with transient middle carotid artery occlusion (MCAO) (1-h MCAO, 24-h reperfusion) after successive administrations of gadoterate meglumine (Dotarem) and intravascular superparamagnetic iron oxide nanoparticles (SPION). The relative cerebral blood volume (CBV) and cerebral blood flow (CBF) values acquired using Dotarem were significantly underestimated (~20%) when compared to those acquired using SPION in ipsilesional post-ischemic brain regions. A slight overestimation of relative mean transit time was observed. Areas with underestimated CBV and CBF values from the corresponding error maps encompassed the area of infarcted tissue (apparent diffusion coefficient < 500 µm2/s) and mostly coincided with the area wherein conspicuous longitudinal relaxation time differences were observed pre- vs. post-injection of Dotarem. The DSC measurements with significant pre-load (0.3 mmol·kg-1) of Dotarem displayed minimal perfusion deficits when compared to those determined using the reference intravascular SPION.