Long-distance growth and connectivity of neural stem cells after severe spinal cord injury.

Neural stem cells (NSCs) expressing GFP were embedded into fibrin matrices containing growth factor cocktails and grafted to sites of severe spinal cord injury. Grafted cells differentiated into multiple cellular phenotypes, including neurons, which extended large numbers of axons over remarkable distances. Extending axons formed abundant synapses with host cells. Axonal growth was partially dependent on mammalian target of rapamycin (mTOR), but not Nogo signaling. Grafted neurons supported formation of electrophysiological relays across sites of complete spinal transection, resulting in functional recovery. Two human stem cell lines (566RSC and HUES7) embedded in growth-factor-containing fibrin exhibited similar growth, and 566RSC cells supported functional recovery. Thus, properties intrinsic to early-stage neurons can overcome the inhibitory milieu of the injured adult spinal cord to mount remarkable axonal growth, resulting in formation of new relay circuits that significantly improve function. These therapeutic properties extend across stem cell sources and species.

Computed tomography-based structural rigidity analysis can assess tumor- and treatment-induced changes in rat bones with metastatic lesions.

BACKGROUND: Breast cancer (BrCa) is a predominant malignancy, with metastasis occurring in one in eight patients, nearly half of which target the bone, leading to serious complications such as pain, fractures, and compromised mobility. Structural rigidity, crucial for bone strength, becomes compromised with osteolytic lesions, highlighting the vulnerability and increased fracture risk in affected areas. Historically, two-dimensional radiographs have been employed to predict these fracture risks; however, their limitations in capturing the three-dimensional structural and material changes in bone have raised concerns. Recent advances in CT-based Structural Rigidity Analysis (CTRA), offer a promising, more accurate non-invasive 3D approach. This study aims to assess the efficacy of CTRA in monitoring osteolytic lesions' progression and response to therapy, suggesting its potential superiority over existing methodologies in guiding treatment strategies. METHODS: Twenty-seven female nude rats underwent femoral intra-medullary inoculation with MDA-MB-231 human breast cancer cells or saline control. They were divided into Control, Cancer Control, Ibandronate, and Paclitaxel groups. Osteolytic progression was monitored weekly using biplanar radiography, quantitative computed tomography (QCT), and dual-energy X-ray absorptiometry (DEXA). CTRA was employed to predict fracture risk, normalized using the contralateral femur. Statistical analyses, including Kruskal-Wallis and ANOVA, assessed differences in outcomes among groups and over time. RESULTS: Biplanar radiographs showed treatment benefits over time; however, only certain time-specific differences between the Control and other treatment groups were discernible. Notably, observer subjectivity in X-ray scoring became evident, with significant inter-operator variations. DEXA measurements for metaphyseal Bone Mineral Content (BMC) did not exhibit notable differences between groups. Although diaphyseal BMC highlighted some variance, it did not reveal significant differences between treatments at specific time points, suggesting a limited ability for DEXA to differentiate between treatment effects. In contrast, the CTRA consistently demonstrated variations across different treatments, effectively capturing bone rigidity changes over time, and the axial- (EA), bending- (EI), and torsional rigidity (GJ) outcomes from the CTRA method successfully distinguished differences among treatments at specific time points. CONCLUSION: Traditional approaches, such as biplanar radiographs and DEXA, have exhibited inherent limitations, notably observer bias and time-specific inefficacies. Our study accentuates the capability of CTRA in capturing real-time, progressive changes in bone structure, with the potential to predict fractures more accurately and provide a more objective analysis. Ultimately, this innovative approach may bridge the existing gap in clinical guidelines, ushering in enhanced Clinical Decision Support Tool (CDST) for both surgical and non-surgical treatments.

Behavioral and axon histomorphometric analyses after intraneural transplantation of human skin- and nerve-derived Schwann cells in nude rats.

INTRODUCTION/AIMS: We have recently isolated and expanded skin-derived Schwann cells (Sk-SCs) from human skin and showed that they are largely similar to nerve-derived Schwann cells (N-SCs). Here, we extend our investigation into functional assessments of the nude rats that received human Sk-SCs and N-SCs after intraneural delivery into crushed and decellularized tibial nerve in adult nude rats. METHODS: Sk-SCs, N-SCs, dermal fibroblasts, or control culture medium was injected into the crushed and decellularized tibial nerve using in situ repeated freeze-thaw cycles. Animals were then subjected to a ladder rung walking test, nociceptive von Frey testing, and walking gait analysis weekly. Animals were euthanized 6 weeks after surgery, gastrocnemius and soleus muscles were weighed, distal nerves were harvested, and whole semithin cross-sections were analyzed using segmentation software. RESULTS: N-SC-injected and dermal fibroblast-injected animals improved significantly at 4 to 6 weeks postinjury in nociceptive assessment compared with medium-injected controls. Sk-SCs recovered more rapidly in tibial functional index at 2 weeks postinjury compared with medium-injected controls. No significant difference was observed for the ladder rung walking test or muscle weight ratio. Histologically, the number of myelinated axons was significantly higher in all cell injection groups compared with medium-injected controls. No significant difference was observed in g ratio, axon diameter, or myelin thickness. DISCUSSION: Cell injection significantly improved axon regeneration across an in situ decellularized nerve segment. However, a more human cell-permissive animal model is required to delineate functional differences between cell types for preclinical transplantation studies.

Comparison of demineralized bone matrix with different cycling crushing times in posterolateral fusion model of athymic rats.

Decalcified bone matrix (DBM) is a widely used alternative material for bone transplantation. In the DBM production process, an effective particle size and the highest utilization rate of raw materials can be achieved only through multiple high-speed circulating comminution. The rat posterolateral lumbar fusion model (PLF) is the most mature small animal model for the initial evaluation of the efficacy of graft materials for bone regeneration and spinal fusion. To evaluate the differences in the in vivo osteogenic effects of DBM pulverization through 1, 5, 9, and 14 high-speed cycles, sixty athymic rats were divided into six groups: single cycling crushing (CC1), 5 cycles of crushing (CC5), 9 cycles of crushing (CC9), 13 cycles of crushing (CC13), autogenous bone graft (ABG) and negative control (NC). Posterolateral lumbar fusion was performed. Six weeks after surgery, the bilateral lumbar fusion of athymic rats was evaluated through manual palpation, X-ray, micro-CT and histological sections. Rank data were tested by the rank-sum test, and nonparametric data were tested by the Kruskal‒Wallis H test. The manual palpation and X-ray results showed that the fusion rate did not significantly differ between the CC1, CC5, CC9, CC13 and ABG groups. However, cavities appeared in CC9 and CC13 on the micro-CT image. The bone mass (BV/TV) of CC1, CC5, CC9 and CC13 was better than that of the ABG group, while almost no osteogenesis was observed in the NC group. Histologically, there was no obvious difference between the four groups except that the CC9 group and CC13 group had more fibrous tissues in the new bone. In conclusion, DMB with different cycling crushing times has no obvious difference in fusion rate of PLF, but it is slightly better than the ABG group.

Transmural myocardial repair with engineered heart muscle in a rat model of heterotopic heart transplantation - A proof-of-concept study.

Engineered heart muscle (EHM) can be implanted epicardially to remuscularize the failing heart. In case of a severely scarred ventricle, excision of scar followed by transmural heart wall replacement may be a more desirable application. Accordingly, we tested the hypothesis that allograft (rat) and xenograft (human) EHM can also be administered as transmural heart wall replacement in a heterotopic, volume-loaded heart transplantation model. We first established a novel rat model model to test surgical transmural left heart wall repair. Subsequently and in continuation of our previous allograft studies, we tested outcome after implantation of contractile engineered heart muscle (EHM) and non-contractile engineered connective tissue (ECT) as well as engineered mesenchymal tissue (EMT) allografts as transmural heart wall replacement. Finally, proof-of-concept for the application of human EHM was obtained in an athymic nude rat model. Only in case of EHM implantation, remuscularization of the surgically created transmural defect was observed with palpable graft vascularization. Taken together, feasibility of transmural heart repair using bioengineered myocardial grafts could be demonstrated in a novel rat model of heterotopic heart transplantation.

Exosome-educated macrophages and exosomes differentially improve ligament healing.

Recently, our group used exosomes from mesenchymal stromal/stem cells (MSCs) to simulate an M2 macrophage phenotype, that is, exosome-educated macrophages (EEMs). These EEMs, when delivered in vivo, accelerated healing in a mouse Achilles tendon injury model. For the current study, we first tested the ability of EEMs to reproduce the beneficial healing effects in a different rodent model, that is, a rat medial collateral ligament (MCL) injury model. We hypothesized that treatment with EEMs would reduce inflammation and accelerate ligament healing, similar to our previous tendon results. Second, because of the translational advantages of a cell-free therapy, exosomes alone were also examined to promote MCL healing. We hypothesized that MSC-derived exosomes could also alter ligament healing to reduce scar formation. Similar to our previous Achilles tendon results, EEMs improved mechanical properties in the healing ligament and reduced inflammation, as indicated via a decreased endogenous M1/M2 macrophage ratio. We also showed that exosomes improved ligament remodeling as indicated by changes in collagen production and organization, and reduced scar formation but without improved mechanical behavior in healing tissue. Overall, our findings suggest EEMs and MSC-derived exosomes improve healing but via different mechanisms. EEMs and exosomes each have attractive characteristics as therapeutics. EEMs as a cell therapy are terminally differentiated and will not proliferate or differentiate. Alternatively, exosome therapy can be used as a cell free, shelf-stable therapeutic to deliver biologically active components. Results herein further support using EEMs and/or exosomes to improve ligament healing by modulating inflammation and promoting more advantageous tissue remodeling.

Do Liquid Nitrogen-treated Tumor-bearing Nerve Grafts Have the Capacity to Regenerate, and Do They Pose a Risk of Local Recurrence? A Study in Rats.

BACKGROUND: Under most circumstances, the resection of soft tissue sarcomas of the extremities can be limb-sparing, function-preserving oncologic resections with adequate margins. However, en bloc resection may require resection of the major peripheral nerves, causing poor function in the extremities. Although liquid nitrogen treatment has been used to sterilize malignant bone tumors, its use in the preparation of nerve grafts has, to our knowledge, not been reported. Hence, this study aimed to investigate the tumor recurrence and function after peripheral nerve reconstruction using liquid nitrogen-treated tumor-bearing nerves in a rat model. QUESTIONS/PURPOSES: (1) Do liquid nitrogen-treated frozen autografts have regeneration capabilities? (2) Do liquid nitrogen-treated tumor-bearing nerves cause any local recurrences in vivo in a rat model? METHODS: Experiment 1: Twelve-week-old female Wistar rats, each weighing 250 g to 300 g, were used. A 10-mm-long section of the right sciatic nerve was excised; the prepared nerve grafts were bridge-grafted through end-to-end suturing. The rats were grouped as follows: an autograft group, which underwent placement of a resected sciatic nerve after it was sutured in the reverse orientation, and a frozen autograft group, which underwent bridging of the nerve gap using a frozen autograft. The autograft was frozen in liquid nitrogen, thawed at room temperature, and then thawed in distilled water before application. The third group was a resection group in which the nerve gap was not reconstructed. Twenty-four rats were included in each group, and six rats per group were evaluated at 4, 12, 24, and 48 weeks postoperatively. To assess nerve regeneration after reconstruction using the frozen nerve graft in the nontumor rat model, we evaluated the sciatic functional index, tibialis anterior muscle wet weight ratio, electrophysiologic parameters (amplitude and latency), muscle fiber size (determined with Masson trichrome staining), lower limb muscle volume, and immunohistochemical findings (though neurofilament staining and S100 protein produced solely and uniformly by Schwann cells associated with axons). Lower limb muscle volume was calculated via CT before surgery (0 weeks) and at 4, 8, 12, 16, 20, 24, 32, 40, and 48 weeks after surgery. Experiment 2: Ten-week-old female nude rats (F344/NJcl-rnu/rnu rats), each weighing 100 g to 150 g, were injected with HT1080 (human fibrosarcoma) cells near the bilateral sciatic nerves. Two weeks after injection, the tumor grew to a 10-mm-diameter mass involving the sciatic nerves. Subsequently, the tumor was resected with the sciatic nerves, and tumor-bearing sciatic nerves were obtained. After liquid nitrogen treatment, the frozen tumor-bearing nerve graft was trimmed to a 5-mm-long tissue and implanted into another F344/NJcl-rnu/rnu rat, in which a 5-mm-long section of the sciatic nerve was resected to create a nerve gap. Experiment 2 was performed with 12 rats; six rats were evaluated at 24 and 48 weeks postoperatively. To assess nerve regeneration and tumor recurrence after nerve reconstruction using frozen tumor-bearing nerve grafts obtained from the nude rat with human fibrosarcoma involving the sciatic nerve, the sciatic nerve's function and histologic findings were evaluated in the same way as in Experiment 1. RESULTS: Experiment 1: The lower limb muscle volume decreased once at 4 weeks in the autograft and frozen autograft groups and gradually increased thereafter. The tibialis anterior muscle wet weight ratio, sciatic functional index, muscle fiber size, and electrophysiologic evaluation showed higher nerve regeneration potential in the autograft and frozen autograft groups than in the resection group. The median S100-positive areas (interquartile range [IQR]) in the autograft group were larger than those in the frozen autograft group at 12 weeks (0.83 [IQR 0.78 to 0.88] versus 0.57 [IQR 0.53 to 0.61], difference of medians 0.26; p = 0.04) and at 48 weeks (0.86 [IQR 0.83 to 0.99] versus 0.74 [IQR 0.69 to 0.81], difference of median 0.12; p = 0.03). Experiment 2: Lower limb muscle volume decreased at 4 weeks and gradually increased thereafter. The median muscle fiber size increased from 0.89 (IQR 0.75 to 0.90) at 24 weeks to 1.20 (IQR 1.08 to 1.34) at 48 weeks (difference of median 0.31; p< 0.01). The median amplitude increased from 0.60 (IQR 0.56 to 0.67) at 24 weeks to 0.81 (IQR 0.76 to 0.90) at 48 weeks (difference of median 0.21; p < 0.01). Despite tumor involvement and freezing treatment, tumor-bearing frozen grafts demonstrated nerve regeneration activity, with no local recurrence observed at 48 weeks postoperatively in nude rats. CONCLUSION: Tumor-bearing frozen nerve grafts demonstrated nerve regeneration activity, and there was no tumor recurrence in rats in vivo. CLINICAL RELEVANCE: A frozen nerve autograft has a similar regenerative potential to that of a nerve autograft. Although the findings in a rat model do not guarantee efficacy in humans, if they are substantiated by large-animal models, clinical trials will be needed to evaluate the efficacy of tumor-bearing frozen nerve grafts in humans.

In vivo evidence for the cellular basis of central hypoventilation of Rett syndrome and pharmacological correction in the rat model.

Rett syndrome (RTT) is a neurodevelopmental disorder caused mostly by mutations in the MECP2 gene. RTT patients show periodical hypoventilation attacks. The breathing disorder contributing to the high incidence of sudden death is thought to be due to depressed central inspiratory (I) activity via unknown cellular processes. Demonstration of such processes may lead to targets for pharmacological control of the RTT-type hypoventilation. We performed in vivo recordings from medullary respiratory neurons on the RTT rat model. To our surprise, both I and expiratory (E) neurons in the ventral respiratory column (VRC) increased their firing activity in Mecp2-null rats with severe hypoventilation. These I neurons including E-I phase-spanning and other I neurons remained active during apneas. Consistent with enhanced central I drive, ectopic phrenic discharges during expiration as well as apnea were observed in the Mecp2-null rats. Considering the increased I neuronal firing and ectopic phrenic activity, the RTT-type hypoventilation does not seem to be caused by depression in central I activity, neither reduced medullary I premotor output. This as well as excessive E neuronal firing as shown in our previous studies suggests inadequate synaptic inhibition for phase transition. We found that the abnormal respiratory neuronal firing, ectopic phrenic discharge as well as RTT-type hypoventilation all can be corrected by enhancing GABAergic inhibition. More strikingly, Mecp2-null rats reaching humane endpoints with severe hypoventilation can be rescued by GABAergic augmentation. Thus, defective GABAergic inhibition among respiratory neurons is likely to play a role in the RTT-type hypoventilation, which can be effectively controlled with pharmacological agents.

The inflammatory role of dysregulated IRS2 in pulmonary vascular remodeling under hypoxic conditions.

Pulmonary hypertension (PH) is a devastating disease characterized by progressive elevation of pulmonary vascular resistance, right ventricular failure, and ultimately death. We have shown previously that insulin receptor substrate 2 (IRS2), a molecule highly critical to insulin resistance and metabolism, has an anti-inflammatory role in Th2-skewed lung inflammation and pulmonary vascular remodeling. Here, we investigated the hypothesis that IRS2 has an immunomodulatory role in human and experimental PH. Expression analysis showed that IRS2 was significantly decreased in the pulmonary vasculature of patients with pulmonary arterial hypertension and in rat models of PH. In mice, genetic ablation of IRS2 enhanced the hypoxia-induced signaling pathway of Akt and Forkhead box O1 (FOXO1) in the lung tissue and increased pulmonary vascular muscularization, proliferation, and perivascular macrophage recruitment. Furthermore, mice with homozygous IRS2 gene deletion showed a significant gene dosage-dependent increase in pulmonary vascular remodeling and right ventricular hypertrophy in response to hypoxia. Functional studies with bone marrow-derived macrophages isolated from homozygous IRS2 gene-deleted mice showed that hypoxia exposure led to enhancement of the Akt and ERK signaling pathway followed by increases in the pro-PH macrophage activation markers, vascular endothelial growth factor-A and arginase 1. Our data suggest that IRS2 contributes to anti-inflammatory effects by regulating macrophage activation and recruitment, which may limit the vascular inflammation, remodeling, and right ventricular hypertrophy that are seen in PH pathology. Restoring the IRS2 pathway may be an effective therapeutic approach for the treatment of PH and right heart failure.

Exosomal miR-335 derived from mature dendritic cells enhanced mesenchymal stem cell-mediated bone regeneration of bone defects in athymic rats.

BACKGROUND: Transplantation of bone marrow-derived mesenchymal stem cells (BM-MSCs) embedded in a bio-compatible matrix has been demonstrated as a promising strategy for the treatment of bone defects. This study was designed to explore the effect and mechanism of exosomes derived from mature dendritic cells (mDC-Exo) on the BM-MSCs-mediated bone regeneration using the matrix support in an athymic rat model of femoral bone defect. METHODS: The BM-MSCs were isolated from rats and incubated with osteoblast induction medium, exosomes derived from immature DCs (imDC-Exo), mDC-Exo, and miR-335-deficient mDC-Exo. BM-MSCs treated without or with mDC-Exo were embedded in a bio-compatible matrix (Orthoss®) and then implanted into the femoral bone defect of athymic rats. RESULTS: mDC-Exo promoted the proliferation and osteogenic differentiation of BM-MSCs by transferring miR-335. Mechanistically, exosomal miR-335 inhibited Hippo signaling by targeting large tongue suppressor kinase 1 (LATS1) and thus promoted the proliferation and osteogenic differentiation of BM-MSCs. Animal experiments showed that mDC-Exo enhanced BM-MSCs-mediated bone regeneration after bone defect, and this effect was abrogated when miR-335 expression was inhibited in mDC-Exo. CONCLUSION: mDC-Exo promoted osteogenic differentiation of BM-MSCs and enhanced BM-MSCs-mediated bone regeneration after femoral bone defect in athymic rats by transferring miR-335.

SIRT1-mediated deacetylation of NF-κB inhibits the MLCK/MLC2 pathway and the expression of ET-1, thus alleviating the development of coronary artery spasm.

Coronary artery spasm (CAS) is an intense vasoconstriction of coronary arteries that causes total or subtotal vessel occlusion. The cardioprotective effect of sirtuin-1 (SIRT1) has been extensively highlighted in coronary artery diseases. The aims within this study include the investigation of the molecular mechanism by which SIRT1 alleviates CAS. SIRT1 expression was first determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot analysis in an endothelin-1 (ET-1)-induced rat CAS model. Interaction among SIRT1, nuclear factor-kappaB (NF-κB), myosin light chain kinase/myosin light chain-2 (MLCK/MLC2), and ET-1 was analyzed using luciferase reporter assay, RT-qPCR, and Western blot analysis. After ectopic expression and depletion experiments in vascular smooth muscle cells (VSMCs), contraction and proliferation of VSMCs and expression of contraction-related proteins (α-SMA, calponin, and SM22α) were measured by collagen gel contraction, 5-ethynyl-2'-deoxyuridine (EdU) assay, RT-qPCR, and Western blot analysis. The obtained results showed that SIRT1 expression was reduced in rat CAS models. However, overexpression of SIRT1 inhibited the contraction and proliferation of VSMCs in vitro. Mechanistic investigation indicated that SIRT1 inhibited NF-κB expression through deacetylation. Moreover, NF-κB could activate the MLCK/MLC2 pathway and upregulate ET-1 expression by binding to their promoter regions, thus inducing VSMC contraction and proliferation in vitro. In vivo experimental results also revealed that SIRT1 alleviated CAS through regulation of the NF-κB/MLCK/MLC2/ET-1 signaling axis. Collectively, our data suggested that SIRT1 could mediate the deacetylation of NF-κB, disrupt the MLCK/MLC2 pathway, and inhibit the expression of ET-1 to relieve CAS, providing a theoretical basis for the prospect of CAS treatment and prevention.NEW & NOTEWORTHY Rat coronary artery spasm models exhibit reduced expression of SIRT1. Overexpression of SIRT1 inhibits contraction and proliferation of VSMCs. SIRT1 inhibits NF-κB through deacetylation to modulate VSMC contraction and proliferation. NF-κB activates the MLCK/MLC2 pathway. NF-κB upregulates ET-1 to modulate VSMC contraction and proliferation.

In vivo characterization of physiological and metabolic changes related to isocitrate dehydrogenase 1 mutation expcression by multiparametric MRI and MRS in a rat model with orthotopically grafted human-derived glioblastoma cell lines.

The physiological mechanism induced by the isocitrate dehydrogenase 1 (IDH1) mutation, associated with better treatment response in gliomas, remains unknown. The aim of this preclinical study was to characterize the IDH1 mutation through in vivo multiparametric MRI and MRS. Multiparametric MRI, including the measurement of blood flow, vascularity, oxygenation, permeability, and in vivo MRS, was performed on a 4.7 T animal MRI system in rat brains grafted with human-derived glioblastoma U87 cell lines expressing or not the IDH1 mutation by the CRISPR/Cas9 method, and secondarily characterized with additional ex vivo HR-MAS and histological analyses. In univariate analyses, compared with IDH1-, IDH1+ tumors exhibited higher vascular density (p < 0.01) and better perfusion (p = 0.02 for cerebral blood flow), but lower vessel permeability (p < 0.01 for time to peak (TTP), p = 0.04 for contrast enhancement) and decreased T1 map values (p = 0.02). Using linear discriminant analysis, vascular density and TTP values were found to be independent MRI parameters for characterizing the IDH1 mutation (p < 0.01). In vivo MRS and ex vivo HR-MAS analysis showed lower metabolites of tumor aggressiveness for IDH1+ tumors (p < 0.01). Overall, the IDH1 mutation exhibited a higher vascularity on MRI, a lower permeability, and a less aggressive metabolic profile. These MRI features may prove helpful to better pinpoint the physiological mechanisms induced by this mutation.

Toward a doxorubicin-loaded bioinspired bone cement for the localized treatment of osteosarcoma.

Aims: Osteosarcoma represents the second most common cause of death in children and young adults. No biomaterial allowing local drug delivery has been specifically developed. However, a biocompatible bioactive implantable material could prevent some amputations, and the local release of an antitumor agent could limit risks of relapse and metastasis. Methods: We propose a proof of concept of a self-setting paste combining amorphous calcium phosphate and doxorubicin-loaded particles of bone-like carbonated nanocrystalline apatite, as a means of local release. Results: The cement formulation and doping, first with folic acid and then with doxorubicin, was successful. Its physicochemistry was scrutinized. Preliminary in vivo data on an invasive osteosarcoma rat model suggest a limiting effect on metastatic events in the lungs without signs of toxicity.

Scaffold-free tissue-engineered arterial grafts derived from human skeletal myoblasts.

Tissue-engineered vascular grafts (TEVGs) are in urgent demand for both adult and pediatric patients. Although several approaches have utilized vascular smooth muscle cells (SMCs) and endothelial cells as cell sources for TEVGs, these cell sources have a limited proliferative capacity that results in an inability to reconstitute neotissues. Skeletal myoblasts are attractive cell sources as they possess high proliferative capacity, and they are already being tested in clinical trials for patients with ischemic cardiomyopathy. Our previous study demonstrated that periodic hydrostatic pressurization (PHP) promoted fibronectin fibrillogenesis in vascular SMCs, and that PHP-induced extracellular matrix (ECM) arrangements enabled the fabrication of implantable arterial grafts derived from SMCs without using a scaffold material. We assessed the molecular response of human skeletal myoblasts to PHP exposure, and aimed to fabricate arterial grafts from the myoblasts by exposure to PHP. To examine the PHP-response genes, human skeletal myoblasts were subjected to bulk RNA-sequencing after PHP exposure. Gene-set enrichment analysis revealed significant positive correlations between PHP exposure and vascular development-related genes. Real-time polymerase chain reaction (RT-PCR) demonstrated that PHP significantly upregulated collagen and elastic fiber formation-related gene expression, such as fibronectin, lysyl oxidase, collagen type I α1, collagen type IV α1, and tropoelastin. Based on these findings showing the potential role of PHP in vessel formation, we fabricated arterial grafts by repeated cell seeding and exposure to PHP every 24 hours. The resultant 15-layered myoblast grafts had high collagen content, which provided a tensile rupture strength of 899 ± 104 mm Hg. Human skeletal myoblast grafts were implanted as patch grafts in the aorta of immunosuppressed rats and found to be endothelialized and completely patent until the endpoint of 60 postoperative days. Implanted human myoblasts were gradually replaced by host-derived cells, which successfully formed vascular neotissues with layered elastic fibers. These findings suggest that human skeletal myoblasts have the potential to be a feasible cell source for scaffold-free implantable arterial grafts under PHP culture conditions.

Changes in Phospholipid/Ceramide Profiles and Eicosanoid Levels in the Plasma of Rats Irradiated with UV Rays and Treated Topically with Cannabidiol.

Chronic UV radiation causes oxidative stress and inflammation of skin and blood cells. Therefore, in this study, we assessed the effects of cannabidiol (CBD), a natural phytocannabinoid with antioxidant and anti-inflammatory properties, on the phospholipid (PL) and ceramide (CER) profiles in the plasma of nude rats irradiated with UVA/UVB and treated topically with CBD. The results obtained showed that UVA/UVB radiation increased the levels of phosphatidylcholines, lysophospholipids, and eicosanoids (PGE2, TxB2), while downregulation of sphingomyelins led to an increase in CER[NS] and CER[NDS]. Topical application of CBD to the skin of control rats significantly upregulated plasma ether-linked phosphatidylethanolamines (PEo) and ceramides. However, CBD administered to rats irradiated with UVA/UVB promoted further upregulation of CER and PEo and led to significant downregulation of lysophospholipids. This was accompanied by the anti-inflammatory effect of CBD, manifested by a reduction in the levels of proinflammatory PGE2 and TxB2 and a dramatic increase in the level of anti-inflammatory LPXA4. It can therefore be suggested that topical application of CBD to the skin of rats exposed to UVA/UVB radiation prevents changes in plasma phospholipid profile resulting in a reduction of inflammation by reducing the level of LPE and LPC species and increasing antioxidant capacity due to upregulation of PEo species.

Macrophage Activation in the Dorsal Root Ganglion in Rats Developing Autotomy after Peripheral Nerve Injury.

Autotomy, self-mutilation of a denervated limb, is common in animals after peripheral nerve injury (PNI) and is a reliable proxy for neuropathic pain in humans. Understanding the occurrence and treatment of autotomy remains challenging. The objective of this study was to investigate the occurrence of autotomy in nude and Wistar rats and evaluate the differences in macrophage activation and fiber sensitization contributing to the understanding of autotomy behavior. Autotomy in nude and Wistar rats was observed and evaluated 6 and 12 weeks after sciatic nerve repair surgery. The numbers of macrophages and the types of neurons in the dorsal root ganglion (DRG) between the two groups were compared by immunofluorescence studies. Immunostaining of T cells in the DRG was also assessed. Nude rats engaged in autotomy with less frequency than Wistar rats. Autotomy symptoms were also relatively less severe in nude rats. Immunofluorescence studies revealed increased macrophage accumulation and activation in the DRG of Wistar rats. The percentage of NF200+ neurons was higher at 6 and 12 weeks in Wistar rats compared to nude rats, but the percentage of CGRP+ neurons did not differ between two groups. Additionally, macrophages were concentrated around NF200-labeled A fibers. At 6 and 12 weeks following PNI, CD4+ T cells were not found in the DRG of the two groups. The accumulation and activation of macrophages in the DRG may account for the increased frequency and severity of autotomy in Wistar rats. Our results also suggest that A fiber neurons in the DRG play an important role in autotomy.

Preliminary Study of New Gallium-68 Radiolabeled Peptide Targeting NRP-1 to Detect Brain Metastases by Positron Emission Tomography.

Due to their very poor prognosis and a fatal outcome, secondary brain tumors are one of the biggest challenges in oncology today. From the point of view of the early diagnosis of these brain micro- and macro-tumors, the sensitivity and specificity of the diagnostic tools constitute an obstacle. Molecular imaging, such as Positron Emission Tomography (PET), is a promising technique but remains limited in the search for cerebral localizations, given the commercially available radiotracers. Indeed, the [18F]FDG PET remains constrained by the physiological fixation of the cerebral cortex, which hinders the visualization of cerebral metastases. Tumor angiogenesis is recognized as a crucial phenomenon in the progression of malignant tumors and is correlated with overexpression of the neuropilin-1 (NRP-1) receptor. Here, we describe the synthesis and the photophysical properties of the new gallium-68 radiolabeled peptide to target NRP-1. The KDKPPR peptide was coupled with gallium-68 anchored into a bifunctional NODAGA chelating agent, as well as Cy5 for fluorescence detection. The Cy5 absorbance spectra did not change, whereas the molar extinction coefficient (ε) decreased drastically. An enhancement of the fluorescence quantum yield (φF) could be observed due to the better water solubility of Cy5. [68Ga]Ga-NODAGA-K(Cy5)DKPPR was radiosynthesized efficiently, presented hydrophilic properties (log D = -1.86), and had high in vitro stability (>120 min). The molecular affinity and the cytotoxicity of this new chelated radiotracer were evaluated in vitro on endothelial cells (HUVEC) and MDA-MB-231 cancer cells (hormone-independent and triple-negative line) and in vivo on a brain model of metastasis in a nude rat using the MDA-MB-231 cell line. No in vitro toxicity has been observed. The in vivo preliminary experiments showed promising results, with a high contrast between the healthy brain and metastatic foci for [68Ga]Ga-NODAGA-K(Cy5)DKPPR.

Isolation of LMX1a Ventral Midbrain Progenitors Improves the Safety and Predictability of Human Pluripotent Stem Cell-Derived Neural Transplants in Parkinsonian Disease.

Human pluripotent stem cells (hPSCs) are a promising resource for the replacement of degenerated ventral midbrain dopaminergic (vmDA) neurons in Parkinson's disease. Despite recent advances in protocols for the in vitro generation of vmDA neurons, the asynchronous and heterogeneous nature of the differentiations results in transplants of surprisingly low vmDA neuron purity. As the field advances toward the clinic, it will be optimal, if not essential, to remove poorly specified and potentially proliferative cells from donor preparations to ensure safety and predictable efficacy. Here, we use two novel hPSC knock-in reporter lines expressing GFP under the LMX1A and PITX3 promoters, to selectively isolate vm progenitors and DA precursors, respectively. For each cell line, unsorted, GFP+, and GFP- cells were transplanted into male or female Parkinsonian rodents. Only rats receiving unsorted cells, LMX1A-eGFP+, or PITX3-eGFP- cell grafts showed improved motor function over 6 months. Postmortem analysis revealed small grafts from PITX3-eGFP+ cells, suggesting that these DA precursors were not compatible with cell survival and integration. In contrast, LMX1A-eGFP+ grafts were highly enriched for vmDA neurons, and importantly excluded expansive proliferative populations and serotonergic neurons. These LMX1A-eGFP+ progenitor grafts accelerated behavioral recovery and innervated developmentally appropriate forebrain targets, whereas LMX1A-eGFP- cell grafts failed to restore motor deficits, supported by increased fiber growth into nondopaminergic target nuclei. This is the first study to use an hPSC-derived reporter line to purify vm progenitors, resulting in improved safety, predictability of the graft composition, and enhanced motor function.SIGNIFICANCE STATEMENT Clinical trials have shown functional integration of transplanted fetal-derived dopamine progenitors in Parkinson's disease. Human pluripotent stem cell (hPSC)-derived midbrain progenitors are now being tested as an alternative cell source; however, despite current differentiation protocols generating >80% correctly specified cells for implantation, resultant grafts contain a small fraction of dopamine neurons. Cell-sorting approaches, to select for correctly patterned cells before implantation, are being explored yet have been suboptimal to date. This study provides the first evidence of using 2 hPSC reporter lines (LMX1A-GFP and PITX3-GFP) to isolate correctly specified cells for transplantation. We show LMX1A-GFP+, but not PITX3-GFP+, cell grafts are more predictable, with smaller grafts, enriched in dopamine neurons, showing appropriate integration and accelerated functional recovery in Parkinsonian rats.

T cell infiltration and upregulation of MHCII in microglia leads to accelerated neuronal loss in an α-synuclein rat model of Parkinson's disease.

BACKGROUND: Parkinson's disease (PD) is the second most prevalent movement disorder characterized by up to 80% loss of dopamine (DA) neurons and accumulation of Lewy body deposits composed of α-synuclein (α-syn). Accumulation of α-syn is associated with microglial activation, leading to a pro-inflammatory environment linked with the pathogenesis of PD. Along with microglia, CD4 and CD8 T cells are observed in SNpc. The contribution of T-cells to PD development remains unclear with studies demonstrating that they may mediate neurodegeneration or act in a neuroprotective manner. METHODS: Here, we assessed the contribution of T cells to PD neurodegeneration using an adeno-associated virus (AAV) coding human wild-type α-syn or GFP injected into the substantia nigra pars compacta (SNpc) in T cell deficient (athymic nude) and T cell competent (heterozygous) rats. The rats were behaviorally assessed with cylinder test to test paw bias. Following behavior testing, brains were collected and analyzed for markers of dopamine neuron, microglial activation, T cells, and α-syn expression. RESULTS: Injection of AAV9-α-syn unilaterally into the SN of T cell competent rats resulted in a significant paw bias in comparison to the controls at 60 days post-injection. Conversely, T cell-deficient rats injected with AAV9-α-syn showed no deficit in paw bias. As expected, injected T cell competent rats demonstrated a significant increase in microglial activation (MHCII staining) as well as significant dopaminergic neuron loss. In contrast, the T cell-deficient counterparts did not show a significant increase in microglial activation or significant neuron loss compared to the control animals. We also observed CD4 and CD8 T cells in SNpc following microglial MHCII expression and dopaminergic neuron loss. The time course of T cell entry correlates with upregulation of MHCII and the peak loss of TH+ cells in the SNpc. CONCLUSION: These data demonstrate that T cell infiltration and microglial upregulation of MHCII are involved in α-synuclein-mediated DA neuron loss in this rat model of PD.

Targeted imaging of orthotopic prostate cancer by using clinical transformable photoacoustic molecular probe.

BACKGROUND: To obtain high-yield histological samples by targeted prostate cancer (PCa) biopsy is the current trend compared with transrectal ultrasound (TRUS)-guided systematic histological biopsy, which is regarded as the gold standard for prostate cancer (PCa) diagnosis. In this paper, we present a targeted PCa imaging strategy using a real-time molecular photoacoustic imaging system integrated with a handheld US probe (PAI/US) and synthesized an integrin αvß3 targeted probe based on ICG (cRGD-ICG). METHODS: To prepare cRGD-ICG, ICG-NHS was linked to cRGD through carboxyl-co-reaction. In vitro PA imaging ability of cRGD-ICG was tested. Orthotopic PCa-bearing rats were used as animal models. After injected with either cRGD-ICG or non-targeted probe, rats were implemented with PA imaging to confirm the specific accumulation of cRGD-ICG at tumor region. Moreover, pathological frozen slices were made to observe distribution of the probe in prostate tissue ex vivo. RESULTS: A small molecular PAI probe was synthesized and exhibited excellent targeted imaging ability in vitro. In vivo photoacoustic imaging was carried out after intravenous injection of cRGD-ICG in orthotopic PCa-bearing rats under the facilitation of the PAI/US system. Maximum molecular photoacoustic signals were observed in the tumor area in vivo after the probe injection, which showed 3.8-fold higher signal enhancement than that in the control group (P < 0.05). Significantly higher cRGD-ICG accumulation was observed under confocal microscopy in the tumor region than in normal prostate tissue. CONCLUSIONS: All our results showed that the comprehensive strategy provided a high-yield and reliable method for PCa diagnosis and targeted prostate biopsy, with great clinical translation potential.