NAD replenishment with nicotinamide mononucleotide protects blood-brain barrier integrity and attenuates delayed tissue plasminogen activator-induced haemorrhagic transformation after cerebral ischaemia

OBJECTIVE Tissue plasminogen activator(tPA) is the only approved pharmaco.logical therapy for acute brain ischaemia;however,a major limitation of tPA is the haemorrhagic trans.formation that follows tPA treatment.Here,we determined whether nicotinamide mononucleotide(NMN),a key intermediate of nicotinamide adenine dinucleotide biosynthesis,affects tPA-induced haemorrhagic transformation.METHODS Middle cerebral artery occlusion(MCAO) was achieved in CD1 mice by introducing a filament to the left MCA for 5 h.When the filament was removed for reperfusion,tPA was infused via the tail vein.A single dose of NMN was injected i.p.(300 mg·kg^(-1)).Mice were killed at 24 h post ischaemia,and their brains were evaluated for brain infarction,oedema,haemoglobin content,apoptosis,neuroinflammation,blood-brain barrier(BBB) permeability,the expression of tight junction proteins(TJPs) and the activity/expression of MMPs.RESULTS In the mice infused with tPA at 5 h post ischaemia,there were significant increases in mortality,brain infarction,brain oedema,brain haemoglobin level,neural apoptosis,Iba-1 staining(microglia activation) and myeloperoxidase staining(neutrophil infiltration).All these tPA-induced alterations were significantly prevented by NMN administration.Mechanistically,the delayed tPA treatment increased BBB permeability by downregulating TJPs,including claudin-1,occludin and zonula occludens-1,and enhancing the activities and protein expression of MMP9 and MMP2.Similarly,NMN administration partly blocked these tPAinduced molecular changes.CONCLUSION Our results demonstrate that NMN ameliorates tPAinduced haemorrhagic transformation in brain ischaemia by maintaining the integrity of the BBB.

Anti-stroke biologics:from recombinant proteins to stem cells and organoids

The use of biologics in various diseases has dramatically increased in recent years.Stroke,a cerebrovascular disease,is the second most common cause of death,and the leading cause of disability with high morbidity worldwide.For biologics applied in the treatment of acute ischaemic stroke,alteplase is the only thrombolytic agent.Meanwhile,current clinical trials show that two recombinant proteins,tenecteplase and non-immunogenic staphylokinase,are most promising as new thrombolytic agents for acute ischaemic stroke therapy.In addition,stem cell-based therapy,which uses stem cells or organoids for stroke treatment,has shown promising results in preclinical and early clinical studies.These strategies for acute ischaemic stroke mainly rely on the unique properties of undifferentiated cells to facilitate tissue repair and regeneration.However,there is a still considerable journey ahead before these approaches become routine clinical use.This includes optimising cell delivery methods,determining the ideal cell type and dosage,and addressing long-term safety concerns.This review introduces the current or promising recombinant proteins for thrombolysis therapy in ischaemic stroke and highlights the promise and challenges of stem cells and cerebral organoids in stroke therapy.