Skeletal muscle regeneration with 3D bioprinted hyaluronate/gelatin hydrogels incorporating MXene nanoparticles.

There has been significant progress in the field of three-dimensional (3D) bioprinting technology, leading to active research on creating bioinks capable of producing structurally and functionally tissue-mimetic constructs. Ti3C2Tx MXene nanoparticles (NPs), promising two-dimensional nanomaterials, are being investigated for their potential in muscle regeneration due to their unique physicochemical properties. In this study, we integrated MXene NPs into composite hydrogels made of gelatin methacryloyl (GelMA) and hyaluronic acid methacryloyl (HAMA) to develop bioinks (namely, GHM bioink) that promote myogenesis. The prepared GHM bioinks were found to offer excellent printability with structural integrity, cytocompatibility, and microporosity. Additionally, MXene NPs within the 3D bioprinted constructs encouraged the differentiation of C2C12 cells into skeletal muscle cells without additional support of myogenic agents. Genetic analysis indicated that representative myogenic markers both for early and late myogenesis were significantly up-regulated. Moreover, animal studies demonstrated that GHM bioinks contributed to enhanced regeneration of skeletal muscle while reducing immune responses in mice models with volumetric muscle loss (VML). Our results suggest that the GHM hydrogel can be exploited to craft a range of strategies for the development of a novel bioink to facilitate skeletal muscle regeneration because these MXene-incorporated composite materials have the potential to promote myogenesis.

Recent Advances in Marine Biomaterials Tailored and Primed for the Treatment of Damaged Soft Tissues.

The inherent self-repair abilities of the body often fall short when it comes to addressing injuries in soft tissues like skin, nerves, and cartilage. Tissue engineering and regenerative medicine have concentrated their research efforts on creating natural biomaterials to overcome this intrinsic healing limitation. This comprehensive review delves into the advancement of such biomaterials using substances and components sourced from marine origins. These marine-derived materials offer a sustainable alternative to traditional mammal-derived sources, harnessing their advantageous biological traits including sustainability, scalability, reduced zoonotic disease risks, and fewer religious restrictions. The use of diverse engineering methodologies, ranging from nanoparticle engineering and decellularization to 3D bioprinting and electrospinning, has been employed to fabricate scaffolds based on marine biomaterials. Additionally, this review assesses the most promising aspects in this field while acknowledging existing constraints and outlining necessary future steps for advancement.

MXene and Xene: promising frontier beyond graphene in tissue engineering and regenerative medicine.

The emergence of 2D nanomaterials (2D NMs), which was initiated by the isolation of graphene (G) in 2004, revolutionized various biomedical applications, including bioimaging and -sensing, drug delivery, and tissue engineering, owing to their unique physicochemical and biological properties. Building on the success of G, a novel class of monoelemental 2D NMs, known as Xenes, has recently emerged, offering distinct advantages in the fields of tissue engineering and regenerative medicine. In this review, we focus on the comparison of G and Xene materials for use in fabricating tissue engineering scaffolds. After a brief introduction to the basic physicochemical properties of these materials, recent representative studies are classified in terms of the engineered tissue, i.e., bone, cartilage, neural, muscle, and skin tissues. We analyze several methods of improving the clinical potential of Xene-laden scaffolds using state-of-the-art fabrication technologies and innovative biomaterials. Despite the considerable advantages of Xene materials, critical concerns, such as biocompatibility, biodistribution and regulatory challenges, should be considered. This review and collaborative efforts should advance the field of Xene-based tissue engineering and enable innovative, effective solutions for use in future tissue regeneration.

3D printed membranes of polylactic acid and graphene oxide for guided bone regeneration.

We fabricated graphene oxide (GO)-incorporated polylactic acid (PLA) (GO-PLA) films by using three-dimensional (3D) printing to explore their potential benefits as barrier membranes for guided bone regeneration (GBR). Our results showed that the 3D printed GO-PLA films provided highly favorable matrices for preosteoblasts and accelerated new bone formation in rat calvarial bone defect models.

Towards a biomarker for acute arterial thrombosis using complete blood count and white blood cell differential parameters in mice.

There is no blood biomarker diagnostic of arterial thrombosis. We investigated if arterial thrombosis per se was associated with alterations in complete blood count (CBC) and white blood cell (WBC) differential count in mice. Twelve-week-old C57Bl/6 mice were used for FeCl3-mediated carotid thrombosis (n = 72), sham-operation (n = 79), or non-operation (n = 26). Monocyte count (/µL) at 30-min after thrombosis (median 160 [interquartile range 140-280]) was ~ 1.3-fold higher than at 30-min after sham-operation (120 [77.5-170]), and twofold higher than in non-operated mice (80 [47.5-92.5]). At day-1 and -4 post-thrombosis, compared with 30-min, monocyte count decreased by about 6% and 28% to 150 [100-200] and 115 [100-127.5], which however were about 2.1-fold and 1.9-fold higher than in sham-operated mice (70 [50-100] and 60 [30-75], respectively). Lymphocyte counts (/µL) at 1- and 4-days after thrombosis (mean ± SD; 3513 ± 912 and 2590 ± 860) were ~ 38% and ~ 54% lower than those in the sham-operated mice (5630 ± 1602 and 5596 ± 1437, respectively), and ~ 39% and ~ 55% lower than those in non-operated mice (5791 ± 1344). Post-thrombosis monocyte-lymphocyte-ratio (MLR) was substantially higher at all three time-points (0.050 ± 0.02, 0.046 ± 0.025, and 0.050 ± 0.02) vs. sham (0.003 ± 0.021, 0.013 ± 0.004, and 0.010 ± 0.004). MLR was 0.013 ± 0.005 in non-operated mice. This is the first report on acute arterial thrombosis-related alterations in CBC and WBC differential parameters.

Role of Graphene Family Nanomaterials in Skin Wound Healing and Regeneration.

Owing to astonishing properties such as the large surface area to volume ratio, mechanical stability, antimicrobial property, and collagen crosslinking, graphene family nanomaterials (GFNs) have been widely used in various biomedical applications including tissue regeneration. Many review literatures are available to compile the role of GFNs in cardiac, bone, and neuronal tissue regeneration. However, the contribution of GFNs in skin wound healing and tissue regeneration was not yet discussed. In the present review, we have highlighted the properties of GFNs and their application in skin wound healing. In addition, we have included challenges and future directions of GFNs in skin tissue regeneration in the portion of conclusion and perspectives.

Functional Graphene Nanomaterials-Based Hybrid Scaffolds for Osteogenesis and Chondrogenesis.

With the emerging trends and recent advances in nanotechnology, it has become increasingly possible to overcome current hurdles for bone and cartilage regeneration. Among the wide type of nanomaterials, graphene (G) and its derivatives (graphene-based materials, GBMs) have been highlighted due to the specific physicochemical and biological properties. In this review, we present the recent development of GBM-based scaffolds for bone and cartilage engineering, focusing on the formulation/shape/size-dependent characteristics, types of scaffold and modification, biocompatibility, bioactivity and underlying mechanism, drawback and prospect of each study. From the findings described herein, mechanical property, biocompatibility, osteogenic and chondrogenic property of GBM-based scaffolds could be significantly enhanced through various scaffold fabrication methods and conjugation with polymers/nanomaterials/drugs. In conclusion, the results presented in this review support the promising prospect of using GBM-based scaffolds for improved bone and cartilage tissue engineering. Although GBM-based scaffolds have some limitations to be overcome by future research, we expect further developments to provide innovative results and improve their clinical potential for bone and cartilage regeneration.

Potential of Carbon-Based Nanocomposites for Dental Tissue Engineering and Regeneration.

While conventional dental implants focus on mechanical properties, recent advances in functional carbon nanomaterials (CNMs) accelerated the facilitation of functionalities including osteoinduction, osteoconduction, and osseointegration. The surface functionalization with CNMs in dental implants has emerged as a novel strategy for reinforcement and as a bioactive cue due to their potential for mechanical reinforcing, osseointegration, and antimicrobial properties. Numerous developments in the fabrication and biological studies of CNMs have provided various opportunities to expand their application to dental regeneration and restoration. In this review, we discuss the advances in novel dental implants with CNMs in terms of tissue engineering, including material combination, coating strategies, and biofunctionalities. We present a brief overview of recent findings and progression in the research to show the promising aspect of CNMs for dental implant application. In conclusion, it is shown that further development of surface functionalization with CNMs may provide innovative results with clinical potential for improved osseointegration after implantation.

Spontaneously promoted osteogenic differentiation of MC3T3-E1 preosteoblasts on ultrathin layers of black phosphorus.

Recently, black phosphorus (BP) has garnered great attention as one of newly emerging two-dimensional nanomaterials. Especially, the degraded platelets of BP in the physiological environment were shown to be nontoxic phosphate anions, which are a component of bone tissue and can be used for mineralization. Here, our study presents the potential of BP as biofunctional and biocompatible nanomaterials for the application to bone tissue engineering and regeneration. An ultrathin layer of BP nanodots (BPNDs) was created on a glass substrate by using a flow-enabled self-assembly process, which yielded a highly uniform deposition of BPNDs in a unique confined geometry. The BPND-coated substrates represented unprecedented favorable topographical microenvironments and supportive matrices suitable for the growth and survival of MC3T3-E1 preosteoblasts. The prepared substrates promoted the spontaneous osteodifferentiation of preosteoblasts, which had been confirmed by determining alkaline phosphatase activity and extracellular calcium deposition as early- and late-stage markers of osteogenic differentiation, respectively. Furthermore, the BPND-coated substrates upregulated the expression of some specific genes (i.e., RUNX2, OCN, OPN, and Vinculin) and proteins, which are closely related to osteogenesis. Conclusively, our BPND-coating strategy suggests that a biologically inert surface can be readily activated as a cell-favorable nanoplatform enabled with excellent biocompatibility and osteogenic ability.

Short-Term Cessation of Dabigatran Causes a Paradoxical Prothrombotic State.

OBJECTIVE: It is unclear if stopping treatment with dabigatran, a new oral anticoagulant (NOAC), induces a paradoxical rebound prothrombotic state. We investigated if short-term (1-3 days) dabigatran cessation is associated with a higher thrombus volume than expected from a simple reversal of the anticoagulant effect. METHODS: Ten-week-old C57Bl/6 mice (n = 338) received one of the following oral treatments: phosphate-buffered saline (PBS), dabigatran for 7 days with or without 1 to 4 day cessation, and aspirin in either a single dose or daily for 7 days. Some of the animals that ceased dabigatran for 1 to 3 days received single-dose aspirin. Thereafter, we induced FeCl3 -mediated carotid thrombosis in 130 mice, after which we performed micro computed tomography thrombus imaging. The other 208 mice underwent coagulation assays or platelet function tests. As an explorative pilot study, we reviewed the medical records of 18 consecutive patients with NOAC cessation-related cerebral infarction in a large acute stroke cohort. RESULTS: We observed a ~ 40% higher volume of carotid thrombus after dabigatran cessation at 1 to 3 days than after vehicle treatment and showed that this effect could be prevented by single-dose aspirin pretreatment. Dabigatran cessation unduly increased platelet aggregability for 2 days after drug cessation, an effect mediated through thrombin or arachidonic acid, which effect was significantly attenuated by single-dose aspirin pretreatment. In patients, short-term (≤ 3 days) cessation of NOAC therapy, compared with longer-term (≥ 5 days) cessation, tended to be associated with relatively high stroke severity. INTERPRETATION: We provide the first preclinical evidence that a rebound prothrombotic state follows short-term cessation of dabigatran therapy. ANN NEUROL 2021;89:444-458.

Effects of exercise training and detraining on atheromatous matrix metalloproteinase activity in mice.

BACKGROUND AND AIMS: Exercise training (ET) helps treat atherosclerosis. However, many patients stop regular ET for various reasons. The effect of detraining on atherosclerosis is not well studied. We examined the effects of ET vs. short-term detraining on atheromatous matrix-metalloproteinase (MMP) activity in preexisting plaque and circulating cytokines/lipids. METHODS AND RESULTS: Eighteen-week-old apolipoprotein-E-/- mice (n = 56) on a Western diet underwent: 1) ET for 6-weeks (ET5+1), 2) ET for 5-weeks and detraining for 1-week (ET5+0), 3) ET for the last 1-week (ET0+1), or 4) no treadmill ET at all for 6-weeks (ET0+0). Atheromatous MMP-activity was visualized using molecular imaging with an MMP-2/9-activatable near-infrared-fluorescent probe. Compared with no ET (ET0+0), regular ET (ET5+1) decreased carotid atheromatous MMP activity, but this protective effect was significantly blunted by short-term detraining (ET5+0). Short-term detraining after longer-term ET showed a reduction in MMP-activity similar to short-term ET (ET0+1). Blood levels of lipids and cytokines paralleled the molecular imaging results: exercise caused higher levels of high-density lipoprotein, adiponectin, and interleukin-10 and lower levels of vascular cell adhesion molecule, monocyte chemoattractant protein-1, interleukin-1ß, and low-density lipoprotein. However, this beneficial effect was short-lived, with the ET5+0 group being similar to the ET0+0 group, and the ET0+1 group being similar to the ET5+1 group. The effect of exercise can be modeled with an exponential-decay of the protective factor of about 15%/day. CONCLUSIONS: Even short-term detraining reduces atheroprotective effects, and tips the balance towards atherosclerosis. This suggests that ET, to be effective, needs to be prolonged and regular, and that detraining should be avoided.

Synthesis of Monolayer Gold Nanorings Sandwich Film and Its Higher Surface-Enhanced Raman Scattering Intensity.

Most previous studies relating to surface-enhanced Raman spectroscopy (SERS) signal enhancement were focused on the interaction between the light and the substrate in the x-y axis. 3D SERS substrates reported in the most of previous papers could contribute partial SERS enhancement via z axis, but the increases of the surface area were the main target for those reports. However, the z axis is also useful in achieving improved SERS intensity. In this work, hot spots along the z axis were specifically created in a sandwich nanofilm. Sandwich nanofilms were prepared with self-assembly and Langmuir-Blodgett techniques, and comprised of monolayer Au nanorings sandwiched between bottom Ag mirror and top Ag cover films. Monolayer Au nanorings were formed by self-assembly at the interface of water and hexane, followed by Langmuir-Blodgett transfer to a substrate with sputtered Ag mirror film. Their hollow property allows the light transmitted through a cover film. The use of a Ag cover layer of tens nanometers in thickness was critical, which allowed light access to the middle Au nanorings and the bottom Ag mirror, resulting in more plasmonic resonance and coupling along perpendicular interfaces (z-axis). The as-designed sandwich nanofilms could achieve an overall ~8 times SERS signals amplification compared to only the Au nanorings layer, which was principally attributed to enhanced electromagnetic fields along the created z-axis. Theoretical simulations based on finite-difference time-domain (FDTD) method showed consistent results with the experimental ones. This study points out a new direction to enhance the SERS intensity by involving more hot spots in z-axis in a designer nanostructure for high-performance molecular recognition and detection.

Dual-Modal Imaging-Guided Precise Tracking of Bioorthogonally Labeled Mesenchymal Stem Cells in Mouse Brain Stroke.

Noninvasive and precise stem cell tracking after transplantation in living subject is very important to monitor both stem cell destinations and their in vivo fate, which is closely related to their therapeutic efficacy. Herein, we developed bicyclo[6.1.0]nonyne (BCN)-conjugated glycol chitosan nanoparticles (BCN-NPs) as a delivery system of dual-modal stem cell imaging probes. Near-infrared fluorescent (NIRF) dye Cy5.5 was chemically conjugated to the BCN-NPs, and then oleic acid-coated superparamagnetic iron oxide nanoparticles (OA-Fe3O4 NPs) were encapsulated into BCN-NPs, resulting in Cy5.5-labeled and OA-Fe3O4 NP-encapsulated BCN-NPs (BCN-dual-NPs). For bioorthogonal labeling of human adipose-derived mesenchymal stem cells (hMSCs), first, hMSCs were treated with tetra-acetylated N-azidoacetyl-d-mannosamine (Ac4ManNAz) for generating azide (-N3) groups onto their surface via metabolic glycoengineering. Second, azide groups on the cell surface were successfully chemically labeled with BCN-dual-NPs via bioorthogonal click chemistry in vitro. This bioorthogonal labeling of hMSCs could greatly increase the cell labeling efficiency, safety, and imaging sensitivity, compared to only nanoparticle-derived labeling technology. The dual-modal imaging-guided precise tracking of bioorthogonally labeled hMSCs was tested in the photothrombotic stroke mouse model via intraparenchymal injection. Finally, BCN-dual-NPs-labeled hMSCs could be effectively tracked by their migration from the implanted site to the brain stroke lesion using NIRF/T2-weighted magnetic resonance (MR) dual-modal imaging for 14 days. Our observation would provide a potential application of bioorthogonally labeled stem cell imaging in regenerative medicine by providing safety and high labeling efficiency in vitro and in vivo.

Synthesis of octahedral gold tip-blobbed nanoparticles and their dielectric sensing properties.

Site-selective synthesis of nanostructures is an important topic in the nanoscience community. Normally, the difference between seeds and deposition atoms in terms of crystallinity triggers the deposition atoms to grow initially at the specific site of nucleation. It is more challenging to control the deposition site of atoms that have the same composition as the seeds because the atoms tend to grow epitaxially, covering the whole surface of the seed nanoparticles. Gold (Au) nano-octahedrons used as seeds in this study possess obvious hierarchical surface energies depending on whether they are at vertices, edges, or terraces. Although vertices of Au nano-octahedrons have the highest surface energy, it remains a challenge to selectively deposit Au atoms at the vertices but not at the edges and faces; this selectivity is required to meet the ever-increasing demands of engineered nanomaterial properties. This work demonstrates an easy and robust method to precisely deposit Au nanoparticles at the vertices of Au nano-octahedrons via wet-chemical seed-mediated growth. The successful synthesis of octahedral Au tip-blobbed nanoparticles (Oh Au TBPs) benefited from the cooperative use of thin silver (Ag) layers at the surface of Au nano-octahedron seeds and iodide ions in the Au growth solution. As-synthesized Au nanostructures (i.e., Au TBPs) gave rise to hybrid optical properties, as evidenced from the UV-vis-NIR extinction spectra, in which a new extinction peak appeared after Au nanoparticles were formed at the vertices of Au nano-octahedrons. A sensitivity evaluation toward dielectric media of a mixture of dimethyl sulfoxide and water suggested that Au TBPs were more optically sensitive compared to the original Au nano-octahedrons. The method demonstrated in this work is promising in the synthesis of advanced Au nanostructures with hybrid optical properties for versatile applications, by engineering the surface energy of vertex-bearing Au nanostructures to trigger site-selective overgrowth of congener Au atoms.

Bimetallic junction mediated synthesis of multilayer graphene edges towards ultrahigh capacity for lithium ion batteries.

In this work, we report on a novel strategy to synthesize high-density graphene edges on a vertically-aligned nanorod array substrate based on multiple segmented Ni-Au units. The growth of graphene layers on Ni and Au was performed by chemical vapor deposition (CVD) leading to the effective generation of edge-rich multilayer graphene due to the distinct carbon solubility. The composite material was applied as an anode in a lithium ion battery (LIB) whose discharging capacity was found to closely depend on the total number of Ni-Au junctions within the vertical nanorods. Graphene deposited on the 19-junction composite Ni-(Au-Ni)9 exhibited an ultrahigh capacity of 86.3 µAh cm-2 at 50 µA cm-2 which was much higher than graphene deposited on 1-junction, 2-junction and pure Ni nanorods. This ultrahigh capacity was mainly ascribed to the generation of high-density graphene edges engineered by the bimetallic junction. The proposed strategy opens new appealing routes to synthesize high-density graphene edges using bimetallic junctions, which is promising for increasing the performance of LIBs and other electrochemical energy systems (supercapacitors, fuel cells, etc.).

Fourier Transform Surface Plasmon Resonance (FTSPR) with Gyromagnetic Plasmonic Nanorods.

An unprecedented active and dynamic sensing platform based on a LSPR configuration that is modulated by using an external magnetic field is reported. Electrochemically synthesized Au/Fe/Au nanorods exhibited plasmonically active behavior through plasmonic coupling, and the middle ferromagnetic Fe block responded to a magnetic impetus, allowing the nanorods to be modulated. The shear force variation induced by the specific binding events between antigens and antibodies on the nanorod surface is used to enhance the sensitivity of detection of antigens in the plasmonics-based sensor application. As a proof-of-concept, influenza A virus (HA1) was used as a target protein. The limit of detection was enhanced by two orders of magnitude compared to that of traditional LSPR sensing.

Cytokine Response to Diet and Exercise Affects Atheromatous Matrix Metalloproteinase-2/9 Activity in Mice.

BACKGROUND: The aim of this study is to identify the principal circulating factors that modulate atheromatous matrix metalloproteinase (MMP) activity in response to diet and exercise.Methods and Results:Apolipoprotein-E knock-out (ApoE-/-) mice (n=56) with pre-existing plaque, fed either a Western diet (WD) or normal diet (ND), underwent either 10 weeks of treadmill exercise or had no treatment. Atheromatous MMP activity was visualized using molecular imaging with a MMP-2/9 activatable near-infrared fluorescent (NIRF) probe. Exercise did not significantly reduce body weight, visceral fat, and plaque size in either WD-fed animals or ND-fed animals. However, atheromatous MMP-activity was different; ND animals that did or did not exercise had similarly low MMP activities, WD animals that did not exercise had high MMP activity, and WD animals that did exercise had reduced levels of MMP activity, close to the levels of ND animals. Factor analysis and path analysis showed that soluble vascular cell adhesion molecule (sVCAM)-1 was directly positively correlated to atheromatous MMP activity. Adiponectin was indirectly negatively related to atheromatous MMP activity by way of sVCAM-1. Resistin was indirectly positively related to atheromatous MMP activity by way of sVCAM-1. Visceral fat amount was indirectly positively associated with atheromatous MMP activity, by way of adiponectin reduction and resistin elevation. MMP-2/9 imaging of additional mice (n=18) supported the diet/exercise-related anti-atherosclerotic roles for sVCAM-1. CONCLUSIONS: Diet and exercise affect atheromatous MMP activity by modulating the systemic inflammatory milieu, with sVCAM-1, resistin, and adiponectin closely interacting with each other and with visceral fat.

A close-packed 3D plasmonic superlattice of truncated octahedral gold nanoframes.

We report the surface-enhanced Raman scattering (SERS) enhancement of three-dimensional (3D) close-packed plasmonic superlattices of truncated octahedral gold nanostructures. Experimentally, we resolved two different types of hot spots, one originates from the face-to-face contacts and the other is from the edge-to-edge contacts among the 3D close-packed plasmonic superlattice of gold nanostructures. The high degree of homogeneity of truncated octahedral Au@Pt nanoparticles (TOh Au@Pt NPs) and truncated octahedral Pt@Au nanoframes (TOh Pt@Au NFs) allowed them to self-assemble into remarkable 3D close-packed plasmonic superlattices. The morphological evolution and the corresponding optical behavior of TOh Pt@Au NFs were systematically monitored during each synthetic process. The measured SERS enhancement on the hot spots from the edge-to-edge contacts of TOh Pt@Au NFs was five times greater than the SERS response from the analogous face-to-face contacts of TOh Au@Pt NPs under the same given experimental conditions. The hollow interior and well-defined structural morphology of TOh Pt@Au NFs will allow researchers to design highly programmable 3D plasmonic superlattices for efficient SERS enhancement.

Quantitative Imaging of Cerebral Thromboemboli In Vivo: The Effects of Tissue-Type Plasminogen Activator.

BACKGROUND AND PURPOSE: Quantitative imaging for the noninvasive assessment of thrombolysis is needed to advance basic and clinical thrombosis-related research and tailor tissue-type plasminogen activator (tPA) treatment for stroke patients. We quantified the evolution of cerebral thromboemboli using fibrin-targeted glycol chitosan-coated gold nanoparticles and microcomputed tomography, with/without tPA therapy. METHODS: We injected thrombi into the distal internal carotid artery in mice (n=50). Fifty-five minutes later, we injected fibrin-targeted glycol chitosan-coated gold nanoparticles, and 5 minutes after that, we treated animals with tPA or not (25 mg/kg). We acquired serial microcomputed tomography images for 24 hours posttreatment. RESULTS: Thrombus burden at baseline was 784×103±59×103 µm2 for the tPA group (n=42) and 655×103±103×103 µm2 for the saline group (n=8; P=0.37). Thrombus shrinkage began at 0.5 to 1 hour after tPA therapy, with a maximum initial rate of change at 4603±957 µm2/min. The rate of change lowered to ≈61% level of the initial in hours 1 to 2, followed by ≈29% and ≈1% in hours 2 to 3 and 3 to 24, respectively. Thus, 85% of total thrombolysis over 24 hours (≈500 µm2, equivalent to 64% of the baseline thrombus burden) occurred within the first 3 hours of treatment. Thrombus burden at 24 hours could be predicted at around 1.5 to 2 hours. Saline treatment was not associated with significant changes in the thrombus burden. Infarct size was smaller in the tPA group versus saline group (18.1±2.3 versus 45.8±3.3 mm2; P<0.01). Infarct size correlated to final thrombus burden (r=0.71; P<0.01). Time to thrombolysis, completeness of thrombolysis, and tPA therapy were independent predictors of infarct size. CONCLUSIONS: Thromboembolic burden and the efficacy of tPA therapy can be assessed serially, noninvasively, and quantitatively using high-resolution microcomputed tomography and a fibrin-binding nanoparticle imaging agent.

Green-channel autofluorescence imaging: A novel and sensitive technique to delineate infarcts.

BACKGROUND: We have found that infarcted brain regions exhibit green channel autofluorescence (GCAF). Here, we compare ex vivo GCAF-imaging with 2,3,5-triphenylteterazolium chloride (TTC)-staining. NEW METHOD: C57BL/6 mice (n=120) underwent GCAF-imaging after transient or permanent middle cerebral artery occlusion (tMCAO or pMCAO). COMPARISON WITH EXISTING METHODS: TTC-staining may not reflect subtle ischemic injury. TTC-stained tissues, when reused, are prone to processing artifacts related to prior TTC-staining. GCAF imaging requires little experimental manipulation of animals and brain tissues, and allows for more consistent measurements of infarct volume and reliable reuse of the fresh unstained tissues. RESULTS: Lesion volumes measured at 24-h after 1-h tMCAO by using GCAF-images were similar to those using TTC-staining: 87.6±13.6mm3 vs. 83.8±12.8mm3 in 1mm-thick sections (n=9 mice, 10 slices/mouse, p=0.88; Pearson's r=0.91, p<0.001) and 75.1±7.6mm3 vs. 73.6±6.7mm3 in 2 mm-thick sections (n=9 mice, 5 slices/mouse, p=0.99; Pearson's r=0.87, p<0.001), respectively. In serial ex vivo imaging performed at 1, 2, 3, 6, 12, and 24-h after tMCAO, GCAF-imaging correlated well with TTC-staining at all time-points. In the pMCAO model however, the correlation was strong at later time-points (6-24-h); but at time points up to 3-h, GCAF-imaging was more sensitive than TTC-staining to detect ischemic areas, as verified by histology, where ischemic damage was observed in the GCAF-positive areas of the cerebral cortex and striatum, even in the face of normal TTC-staining. CONCLUSION: GCAF-imaging is a reliable alternative to TTC-staining in the qualitative and quantitative assessments of focal brain ischemia, and more sensitive for detecting early ischemic damage in pMCAO.