Therapeutic hypothermia represents a highly promising approach for alleviating ischemic brain injury. However, the majority of preclinical studies predominantly rely on reperfusion-based models using young animals, which poorly reflect the clinical situation of elderly stroke patients with limited recanalization. This study sought to bridge these gaps and accelerate the clinical translation of therapeutic hypothermia while elucidating its neuroprotective mechanisms. In aged (18-20 months old) mice with permanent distal middle cerebral artery occlusion, brain-selective mild hypothermia mitigated acute F-actin stress fiber formation and junctional protein degradation in microvascular endothelial cells, thereby effectively reducing blood-brain barrier leakage and infiltration of peripheral inflammatory cells into the brain parenchyma. Hypothermia treatment induced anti-inflammatory polarization of microglia/macrophages acutely, attenuating white matter loss at both early (7 days) and chronic (35 days) stages of ischemic injury. Moreover, hypothermia treatment significantly promoted cognitive and sensorimotor recovery for at least 35 days after ischemic injury, as reflected in the electrophysiological preservation of compound action potentials in white matter tracts. Long-term behavioral recovery was strongly associated with angiogenesis and oligodendrogenesis, supporting that hypothermia-induced cell regeneration and neural tissue repair foster positive neurological outcomes. These findings underscore the potential of mild, brain-selective hypothermia for treating elderly stroke patients.

The inflammatory response after myocardial infarction (MI) is a precisely regulated process that greatly affects subsequent wound healing and remodeling. However, understanding about the process is still limited. Macrophages are critically involved in inflammation resolution after MI. Krüppel-like factor 9 (Klf9) is a C2H2 zinc finger-containing transcription factor that has been implicated in glucocorticoid regulation of macrophages. However, the contribution of Klf9 to macrophage phenotype and function in the context of MI remains unclear. Our study revealed that KLF9 deficiency resulted in higher mortality and cardiac rupture rate, as well as a considerable exacerbation in cardiac function. Single-cell RNA sequencing and flow cytometry analyses revealed that, compared with WT mice, Klf9-/- mice displayed excessive neutrophil infiltration, insufficient macrophage infiltration, and a reduced proportion of monocyte-derived CD206+ macrophages after MI. Moreover, the expression of IFN-γ/STAT1 pathway genes in Klf9-/- cardiac macrophages was dysregulated, characterized by insufficient expression at 1 day post-MI and excessive expression at day 3 post-MI. Mechanistically, Klf9 directly binds to the promoters of Stat1 gene, regulating its transcription. Overall, these findings indicate that Klf9 beneficially influences wound healing after MI by modulating macrophage recruitment and differentiation by regulating the IFN-γ/STAT1 signaling pathway.

Myasthenia gravis (MG) is an autoimmune disorder primarily caused by autoantibodies that target the acetylcholine receptor (AChR) at the neuromuscular junction (NMJ). The classical experimental autoimmune myasthenia gravis (C-EAMG) mouse model has long been used by immunizing mice with acetylcholine receptor from Torpedo fish (T-AChR), combined with complete Freund's adjuvant (CFA). This mixture is administered via subcutaneous injections into the hind footpads and back, but CFA often causes strong inflammatory reactions, including lesions at the injection sites. Our objective was to develop a new EAMG model (N-EAMG) that is more compliant with animal welfare. C57Bl/6 mice were immunized twice weekly by intraperitoneal (i.p.) injection of T-AChR with a poly(I:C) and lipopolysaccharide (LPS) adjuvant mix. Control mice were injected with either physiological saline or the adjuvant mix alone. Various doses and injection schedules were tested, and the new model was compared with C-EAMG. Clinical symptoms were scored, antibody subtypes against T-AChR and mouse AChR were measured, and NMJ morphology and functionality were evaluated. We demonstrate that the N-EAMG model is at least as effective as the C-EAMG model. Moreover, similar to the C-EAMG model, the N-EAMG model is characterized by the production of T-AChR and m-AChR antibodies. This model also exhibited alterations in transmission at the NMJ due to antibody attack, resulting in a decrease in AChR surface area and increased AChR fragmentation. Symptoms were similar in both models but appeared more rapidly in the N-EAMG model. In addition, investigating the sensitization mechanism, we showed that i.p. injections of T-AChR with the poly(I:C)/LPS adjuvant mix, led to the recruitment in monocytes and changes in the two peritoneal macrophage subpopulations that were able to phagocytose T-AChR. These observations suggest that macrophage subtypes, albeit with varying efficiency, present the T-AChR to immune cells, leading to a specific immune response and the development of anti-AChR antibodies. In conclusion, our results demonstrate that this novel EAMG model is as effective as the C-EAMG model and offers several advantages. In particular, this model is more suitable for animal welfare and can replace the classical model in preclinical and fundamental research.
Copyright © 2025 You, Lippens, Fayet, Maillard, Betemps, Grondin, Vilquin, Dragin and Le Panse.

Macrophages infiltrate all human tissues where they play key roles in innate immunity, homeostasis, and tissue function. However, extensive clinical and experimental evidence indicates that macrophages also contribute significantly to the progression of several diseases such as cancer, cardiometabolic disorders, and inflammatory and neurodegenerative conditions. Advances in single-cell omics have revealed diverse macrophage populations in both healthy and diseased tissues. However, studying their functions is challenging due to limitations in tools for targeting specific populations. The Cre-lox system, involving Cre recombinase expression driven by macrophage-specific promoters, is widely used for gene manipulation. Despite its utility, this method has drawbacks like leaky expression, variable efficiency, and potential toxicity. Moreover, genetic models are costly and can have unintended effects on immune cells, hindering comprehensive studies on macrophage function. To address this challenge, we developed an advanced lipid nanoparticle-based system for precise RNA therapeutic delivery to macrophages, either broadly or via antibody-mediated targeting of specific subsets. This versatile platform enables the administration of various RNA molecules, such as mRNA, siRNA, and sgRNA for CRISPR/Cas9 applications, in both in vitro and in vivo settings. It allows for targeted cell depletion or gene knockout, facilitating detailed functional analysis. Furthermore, the system’s flexibility and precision are enhanced by its compatibility with Cre-specific Cas9 expression, enabling comprehensive genomic and proteomic targeting of specific macrophage subsets.

In the musculoskeletal system, lymphatic vessels (LVs), which are interdigitated with blood vessels, travel and form an extensive transport network. Blood vessels in bone regulate osteogenesis and hematopoiesis, however, whether LVs in bone affect fracture healing is unclear. Here, we investigate the lymphatic draining function at the tibial fracture sites using near-infrared indocyanine green lymphatic imaging (NIR-ICG) and discover that lymphatic drainage insufficiency (LDI) starts on day one and persists for up to two weeks following the fracture in male mice. Sufficient lymphatic drainage facilitates fracture healing in male mice. Furthermore, we identify that lymphatic platelet thrombosis (LPT) blocks the draining lymphoid sinus and LVs, causes LDI, and inhibits fracture healing in male mice, which can be rescued by a blood thinner. Moreover, unblocked lymphatic drainage decreases neutrophils and increases M2-type macrophages of the hematoma niche to support osteoblast (OB) survival and bone marrow-derived mesenchymal stem cell (BMSC) proliferation via transporting damage-associated molecular patterns (DAMPs) in male rats. Lymphatic platelet thrombolysis also benefits senile fracture healing in female mice. These findings demonstrate that LPT limits bone regeneration by impeding lymphatic transporting DAMPs. Together, these findings represent a way forward in the treatment of bone repair.
© 2025. The Author(s).