Vascular anatomy-based localization of intervertebral discs assisting needle puncture for constructing a mouse model of mechanical injury-induced lumbar intervertebral disc degeneration.

Intervertebral disc degeneration (IDD) may be the primary cause of low back pain. Potential therapeutics for IDD must be validated in animal models, and their effectiveness quantified using functional metrics. Needle puncture of intervertebral discs (IVDs) has been used to induce IDD in mice and rats. Due to operational challenges, most animal IDD models are constructed using needle puncture of the caudal IVDs in mice, or by using larger animals, such as rats and rabbits. However, mouse IDD models involving lumbar IVD puncture are preferable because mice are genetically similar to humans and are the most commonly used transgenic animals, and because human IDD commonly affects the lumbar spine. We constructed a needle puncture-based mouse IDD model that relies on vascular anatomy to pinpoint lumbar IVDs. We evaluated the morphological and molecular changes in this model by using radiological, pathological, and immunostaining examinations. In our mechanical injury-induced IDD model, lumbar IVDs were accurately localized by injecting colored perfusates into the common iliac artery and vein, and right iliolumbar vein, which helped to visualize puncture positions, avoid neuromuscular injury, shorten the operation time, and decrease bleeding. Nucleus pulposus cells, defined by Krt19, and the disc height index gradually decreased after the surgery, and the degenerative effects peaked at 1 week. In conclusion, we established a mouse IDD model by performing precise puncture of lumbar IVDs via the ventral anterior approach assisted by vessel position. Our model effectively simulated the effects of IDD, and may serve as an efficient research tool.

Novel immunosuppressive effect of FK506 by upregulation of PD-L1 via FKBP51 in heart transplantation.

The calcineurin inhibitor-FK506-is a first-line immunosuppressant that regulates T cell secretion of IL-2 and other cytokines. However, the mechanism of its protective effect on target cells and its role on tumour recurrence and interaction with anti-tumour immune checkpoint inhibitors, such as PD-L1 blocking, are still unclear. Here, in a murine heart transplantation model, we observed the upregulation of programmed death-ligand 1 (PD-L1) expression by FK506 in both dendritic cells (DCs) and allografts. Blocking PD-L1 during FK506 treatment increased IFN-γ and TNF-α expression, enhanced CD4+ and CD8+ T cell proliferation, and suppressed Treg differentiation. Moreover, PD-L1 decreased T cell infiltration and induced T cell apoptosis in both the spleen and graft. PD-L1 was not only required in FK506-mediated immunosuppression but also upregulated by FK506. Treatment with SAFit2, a FKBP51 selective inhibitor, reduced the expression of PD-L1 on DCs and the grafts and interfered with the immunosuppressive effect of FK506, suggesting that the mechanism depends on FK506-binding protein (FKBP) 51 expression. Overall, our results add new insights into the role of FK506, not only on T cell cytokine secretion but also on co-inhibitory molecular regulation and target cell immune privilege.

Propane-2-sulfonic acid octadec-9-enyl-amide, a novel PPARα/γ dual agonist, attenuates molecular pathological alterations in learning memory in AD mice.

OBJECTIVE: Previous studies have revealed that Propane-2-sulfonic acid octadec-9-enyl-amide(N15) exerts a protective role in the inflammatory response after ischemic stroke and in neuronal damage. However, little is known about N15 in Alzheimer's disease (AD). The aim of this study was to investigate the effects of N15 on AD and explore the underlying molecular mechanism. METHODS: AD mice model was established by lateral ventricular injection with Aß25-35. N15 was daily intraperitoneal administered for 28 days. Morris Water Maze was used to evaluate the neurocognitive function of the mice. The expression of PPARα/γ, brain-derived neurotrophic factor (BDNF), Neurotrophin-3 (NT3), ADAM10, PS1 and BACE1 were measured by qPCR. Aß amyloid in the hippocampus was measured by Congo red assay. Toluidine blue staining was used to detect the neuronal apoptosis. Protein levels of ADAM10, PS1 and BACE1 were determined using immunoblotting. RESULTS: N15 treatment significantly reduced neurocognitive dysfunction, which also significantly activated the expression of PPARα/γ at an optimal dose of 200 mg/kg. Administration of N15 alleviated the formation of Aß amyloid in the hippocampus of AD mice, enhanced the BDNF mRNA expression, decreased the mRNA and protein levels of PS1 and BACE1, upregulated ADAM10 mRNA and protein levels. CONCLUSION: N15 exerts its neuroprotective effects through the activation of PPARα/γ and may be a potential drug for the treatment of AD.

Oleoylethanolamide Protects against Acute Ischemic Stroke by Promoting PPARα-Mediated Microglia/Macrophage M2 Polarization.

Oleoylethanolamide (OEA) has been demonstrated to be a feasible protectant in ischemic stroke. However, the mechanism for OEA-afforded neuroprotection remains elusive. The present study aimed to investigate the neuroprotective effects of OEA on peroxisome proliferator-activated receptor α (PPARα)-mediated microglia M2 polarization after cerebral ischemia. Transient middle cerebral artery occlusion (tMCAO) was induced for 1 h in wild-type (WT) or PPARα-knock-out (KO) mice. Mouse small glioma cells (BV2) microglia and primary microglia cultures were used to evaluate the direct effect of OEA on microglia. A coculture system was used to further elucidate the effect of OEA on microglial polarization and ischemic neurons' fate. OEA promoted the microglia switch from an inflammatory M1 phenotype to the protective M2 phenotype and enhanced the binding of PPARα with the arginase1 (Arg1) and Ym1 promoter in WT mice but not in KO mice after MCAO. Notably, the increased M2 microglia caused by OEA treatment were strongly linked to neuron survival after ischemic stroke. In vitro studies confirmed that OEA shifted BV2 microglia from (lipopolysaccharide) LPS-induced M1-like to M2-like phenotype through PPARα. Additionally, the activation of PPARα in primary microglia by OEA led to an M2 protective phenotype that enhanced neuronal survival against oxygen-glucose deprivation (OGD) in the coculture systems. Our findings demonstrate the novel effects of OEA in enhancing microglia M2 polarization to protect neighboring neurons by activating the PPARα signal, which is a new mechanism of OEA against cerebral ischemic injury. Therefore, OEA might be a promising therapeutic drug for stroke and targeting PPARα-mediated M2 microglia may represent a new strategy to treat ischemic stroke.

Programmed Death Ligand-1-Overexpressing Donor Exosomes Mediate Donor-Specific Immunosuppression by Delivering Co-Inhibitory Signals to Donor-Specific T Cells.

Programmed death ligand-1 (PD-L1) and donor antigens are critical for donor immature dendritic cells (DCs) targeting donor-specific T cells to induce transplant tolerance. This study aims to clarify whether DC-derived exosomes (DEX) with donor antigens (H2b) and high levels of PD-L1 expression (DEXPDL1+ ) can help to suppress graft rejection. In this study, it is demonstrated that DEXPDL1+ presents donor antigens, as well as PD-L1 co-inhibitory signals, directly or semi-directly via DCs to H2b-reactive T cells. This dual signal presentation can prolong the survival of heart grafts from B6 (H2b) mice but not from C3H (H2k) mice by inhibiting T cell activation, inducing activated T cell apoptosis, and modulating the balance of T cell differentiation from inflammatory to regulatory. Additionally, even though DEXPDL1+ treatment cannot induce tolerance after short-term treatment, this study provides a new vehicle for presenting co-inhibitory signals to donor-specific T cells. This novel strategy may facilitate the realization of donor-specific tolerance via the further optimization of drug-loading combinations and therapeutic regimens to elevate their killing ability.

Medial Buttress Plate and Allograft Bone-Assisted Cannulated Screw Fixation for Unstable Femoral Neck Fracture with Posteromedial Comminution: A Retrospective Controlled Study.

OBJECTIVE: To investigate the outcomes of open reduction and internal fixation combined with medial buttress plate (MBP) and allograft bone-assisted cannulated screw (CS) fixation for patients with unstable femoral neck fracture with comminuted posteromedial cortex. METHODS: In a retrospective study of patients operated on for unstable femoral neck fractures with comminuted posteromedial cortex from March 2016 to August 2020, the clinical and radiographic outcomes of 48 patients treated with CS + MBP were compared with the outcomes of 54 patients treated with CS only. All patients in the CS + MBP group were fixed by three CS and MBP (one-third tubular plates or reconstructive plates) with bone allografts. The surgery-related outcomes and complications were evaluated, including operative time, blood loss, union time, femoral head necrosis, femoral neck shortening, and other complications after the operation. The Harris score was evaluated at 12 months after the operation. RESULTS: All patients were followed up for 12-40 months. The average age of patients in the CS-only group (54 cases, 22 females) and CS + MBP group (48 cases, 20 females) was 48.46 ± 7.26 and 48.73 ± 6.38 years, respectively. More intraoperative blood loss was observed in the CS + MBP group than that of patients in CS-only group (153.45 ± 64.27 vs 21.86 ± 18.19 ml, t = 4.058, P = 0.015). The average operative time for patients in the CS + MBP group (75.35 ± 27.67 min) was almost double than that of patients in the CS-only group (36.87 ± 15.39 min) (t = 2.455, P < 0.001). The Garden alignment index of patients treated by CS + MBP from type I to type IV was 79%, 19%, 2%, and 0%, respectively. On the contrary, they were 31%, 43%, 24% and 2% for those in the CS-only group, respectively. The average healing times for the CS-only and CS + MBP groups were 4.34 ± 1.46 and 3.65 ± 1.85 months (t = 1.650, P = 0.102), respectively. Femoral neck shortening was better in the CS + MBP group (1.40 ± 1.73 mm, 9/19) than that in the CS-only group (4.33 ± 3.32 mm, 24/44). Significantly higher hip function was found in the CS + MBP group (85.60 ± 4.36 vs 82.47 ± 6.33, t = 1.899, P = 0.06). There was no statistical difference between femoral head necrosis (4% vs 11%, χ2  = 1.695, P = 0.193) and nonunion (6% vs 9%, χ2  = 0.318, P = 0.719). CONCLUSION: For unstable femoral neck fractures with comminuted posteromedial cortex, additional MBP combined with bone allografts showed better reduction quality and neck length control than CS fixation only, with longer operative time and more blood loss.

RGD-modified injectable hydrogel maintains islet beta-cell survival and function.

OBJECTIVE: A potential solution for islet transplantation and drug discovery vis-à-vis treating diabetes is the production of functional islets in a three-dimensional extracellular matrix. Although several scaffold materials have been reported as viable candidates, a clinically applicable one that is injectable and can maintain long-term functionality and survival of islet pancreatic beta-cells (ß-cells) is far from being established. RESULTS: In the current study, we evaluated a ready-to-use and injectable hydrogel's impact on ß-cells' function and viability, both in vitro and in vivo. We found that ß-cells in high concentration with hydrogels functionalized via Arg-Gly-Asp (RGD) demonstrated better viability and insulin secretory capacity in vitro. Moreover, it is a biocompatible hydrogel that can maintain ß-cell proliferation and vascularization without stimulating inflammation after subcutaneous injection. Meanwhile, modifying the hydrogel with RGD can maintain ß-cells' secretion of insulin, regulating the blood glucose levels of mice with streptozotocin-induced diabetes. CONCLUSIONS: Thus, these preliminary results indicate that this RGD-modified hydrogel is a potential extracellular matrix for islet transplantation at extrahepatic sites, and they also provide a reference for future tissue engineering study.

Propane-2-sulfonic acid octadec-9-enyl-amide, a novel peroxisome proliferator-activated receptors α and γ dual agonist, enhances hippocampal neurogenesis and neuroplasticity in rats with cerebral ischaemia.

Our previous studies showed that propane-2-sulfonic acid octadec-9-enyl-amide (N15), a novel peroxisome proliferator-activated receptors α and γ (PPARα/γ) dual agonist, protected against ischaemia-induced acute brain damage in mice and improved cognitive ability in the chronic phase of ischaemic stroke. It is well known that hippocampal neurogenesis is closely related to cognitive function. In the present study, we investigated the effect of N15 on hippocampal neurogenesis and neuroplasticity in a middle cerebral artery occlusion (MCAO) rat model. The middle cerebral artery of rats was blocked for 2 hours. Oral administration of 100 mg/kg N15 or vehicle was given once daily for days 2-13 after MCAO. The newly mature neurons were detected by staining. The expressions of synapse-related proteins were observed by qRT-PCR or western blotting. We found that N15-treated rats showed improved survival post-MCAO. In addition, N15 treatment markedly increased the newly mature neurons and enhanced the expression levels of growth-associated protein-43, synaptophysin, brain-derived neurotrophic factor and neurotrophin-3 in the hippocampus. Moreover, N15 promoted the activation of PPARα and PPARγ on day 7 and 14 after cerebral ischaemia. These results reveal that N15 may promote neurogenesis and neuroplasticity in MCAO rats through the activation of the PPARα/γ dual signal pathway.

The effect of dexmedetomidine on inflammatory inhibition and microglial polarization in BV-2 cells.

OBJECTIVE: Microglia have different phenotypic and functional states: M1 is associated with inflammatory responses, whereas M2 results in anti-inflammatory effects. The cellular state of microglia plays an important role in brain inflammation associating with many neuroinflammatory diseases. The purpose of this study was to detect the effect of dexmedetomidine (Dex) on inflammatory inhibition and microglial polarization in BV-2 cells. MATERIALS AND METHODS: Dex exerts anti-inflammatory effects in various experimental models. The BV-2 microglial cell line was treated with liposaccharide in the presence or absence of Dex. The M1 and M2 markers were evaluated by quantitative real-time PCR (qRT-PCR) and western blot. RESULTS: We found that Dex exerted a potent anti-inflammatory effect by reducing the expression of M1 marker genes such as tumor necrosis factor alpha (P < 0.05), interleukin-1ß (IL-1ß) (P < 0.001) and IL-6 (P < 0.001). Importantly, Dex improved the expression of microglia M2 markers arginase-1 (Arg-1) (P < 0.01), Flt3-interacting zinc finger protein 1 (Fizz-1) (P < 0.001) and CD206) (P < 0.001). Further, Dex enhanced the activation of Akt pathway. DISCUSSION: Our results indicated that Dex promotes microglia from the M1 phenotype to the M2 phenotype. Therefore, Dex may be a potential novel therapeutic drug for treating brain inflammation-associated diseases not only because of its anti-inflammatory property but also because it can remodel M1 phenotype microglia to M2 phenotype microglia.

Low dose nicotine attenuates Aß neurotoxicity through activation early growth response gene 1 pathway.

Epidemiological studies indicate that smoking is negatively correlated with the incidence and development of Alzheimer's disease (AD). Nicotine was reported to be the active factor. However, the detailed mechanisms still remain to be fully elucidated. Early growth response gene 1 (EGR-1) plays important roles in several important biological processes such as promoting cell growth, differentiation, anti oxidative stress, and apoptosis, but few in the pathogenesis of AD. In the present study, we show that nicotine can activate the MAPK/ERK/EGR-1 signaling pathway partially through α7 nAChR. In addition, the up-regulation of EGR-1 by nicotine can also increase the phosphorylation of CyclinD1 which contributes to the attenuation of amyloid-ß (Aß(25-35)) -induced neurotoxicity. Although nicotine and Aß(25-35) can activate EGR-1, the expression of EGR-1 is down-regulated following treatment with nicotine and Aß(25-35). This study demonstrates that low dose nicotine attenuates Aß(25-35)-induced neurotoxicity in vitro and in vivo through activating EGR-1 pathway.