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Stroke remains a leading global cause of death and disability. In the last decade, the therapeutic window for mechanical thrombectomy has increased from a maximum of 6 to 24 h and beyond. While endovascular advancements have improved rates of recanalization, no post-stroke pharmacotherapeutics have been effective in enhancing neurorepair and recovery. New experimental models are needed to closer mimic the human patient. Our group has developed a model of transient 5-h occlusion in rats to mimic stroke patients undergoing thrombectomy. Our procedure was designed specifically in aged rats and was optimized based on sex in order to keep mortality and extent of injury consistent between aged male and female rats. This model uses a neurological assessment modeled after the NIH Stroke Scale. Finally, the potential for translation between our rat model of stroke and humans was assessed using comparative gene expression for key inflammatory genes. This model will be useful in the evaluation of therapeutic targets to develop adjuvant treatments for large vessel occlusion during the thrombectomy procedure., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The aim of this study was to determine whether leukemia inhibitory factor (LIF) exerts its neuroprotective effects through signal transduction of the transcription factor myeloid zinc finger-1 (MZF-1). According to the hypothesis of this study, MZF-1 mediates LIF-induced neuroprotective signaling during ELVO through increased expression and transcriptional activity. To determine the in vivo role of MZF-1 in LIF-induced neuroprotection, we used Genomatix software was used to MZF-1 sites in the promoter region of the rat superoxide dismutase 3 (SOD3) gene. Stroke was induced via middle cerebral artery occlusion, and animals were administered PBS or 125 μg/kg LIF at 6, 24, and 48 h after the injury. MZF-1 binding activity was measured using electrophoretic mobility shift assay (EMSA) and its expression/localization were determined using western blot and immunohistochemical analysis. To determine whether MZF-1 relays LIF-induced neuroprotection in vitro, primary cultured neurons were subjected to oxygen-glucose deprivation (OGD) after treatment with PBS or LIF. MZF-1 expression was measured in vitro using real time PCR and immunohistochemical staining. Transfection with siRNA was used to determine whether LIF protected cultured neurons against OGD after silencing MZF-1 expression. Four MZF-1 binding sites were identified by Genomatix, and EMSA confirmed in vivo binding activity in brain after MCAO. LIF significantly increased MZF-1 protein levels compared to PBS treatment at 72 h post-MCAO. In vivo nuclear localization of MZF-1 as well as co-localization of SOD3 and MZF-1 was observed in the cortical neurons of LIF-treated rats. Primary cultured neurons treated with LIF had significantly higher levels of MZF-1 mRNA and protein after LIF treatment compared to neurons treated with PBS. Finally, knockdown MZF-1 using siRNA counteracted the neuroprotective effects of LIF in vitro. These data demonstrate that LIF-mediated neuroprotection is dependent upon MZF-1 activity. Furthermore, these findings identify a novel neuroprotective pathway that employs MZF-1, a transcription factor associated with hematopoietic gene expression.

The first annual Stroke Translational Research Advancement Workshop (STRAW), entitled "Uncovering the Rosetta Stone: Key Elements in Translating Stroke Therapeutics from Pre-Clinical to Clinical" was held at the University of Kentucky on October 4-5, 2017. This workshop was organized by the Center for Advanced Translational Stroke Science. The workshop consisted of 2 days of activities. These included three presentations establishing the areas of research in stroke therapeutics, discussing the routes for translation from bench to bedside, and identifying successes and failures in the field. On day 2, grant funding opportunities and goals for the National Institute for Neurological Diseases and Stroke were presented. In addition, the meeting also included break-out sessions designed to connect researchers in areas of stroke, and to foster potential collaborations. Finally, the meeting concluded with an open discussion among attendees led by a panel of experts.

Competing Interests: All authors declare no conflict of interest.

Despite extensive research into stroke pathology, there have not been any major recent advancements in stroke therapeutics. Animal models of cerebral ischemia and clinical data have been used to investigate the progressive neural injury that occurs after an initial ischemic insult. This has lead researchers to focus more on the peripheral immune response that is generated as a result of cerebral ischemia. The therapies that have been developed as a result of this research thus far have proven ineffective in clinical trials. The failure of these therapeutics in clinical trials is thought to be due to the broad immunosuppression elicited as a result of the treatments and the cerebral ischemia itself. Emerging evidence indicates a more selective modulation of the immune system following stroke could be beneficial. The spleen has been shown to exacerbate neural injury following experimental stroke and would provide a strong therapeutic target. Selecting facets of the immune system to target would allow the protective and regenerative properties of the immune response to remain intact while blunting the pro-inflammatory response generated towards the injured brain.

The human umbilical cord blood (HUCB) mononuclear cell (MNC) fraction is a mixed population of cells that induces functional repair in rodent models of stroke when injected intravenously (i.v.). The transplanted cells are found in the infarcted hemisphere and the spleen. The goal of this project was to determine the nature of the interaction between the HUCB MNCs cells and splenic immune cells. Male Sprague Dawley rats underwent permanent middle cerebral artery occlusion (MCAO) and received i.v. injection of either vehicle (MCAO only), HUCB MNCs or MNCs depleted of CD14+ monocytes, CD133+ stem cells or CD19+ B cells 48 hours post-stroke. At 72 hours post-MCAO, the animals were euthanized and the spleens and blood MNCs harvested for flow cytometry and mitogen proliferation assays. All HUCB cell preparations decreased the percentage of T cells in the spleen and monocytes in the blood (p < 0.05). MNCs depleted of CD14+ and CD19+ decreased the percentage of macrophage (p < 0.001), while CD133 depleted MNCs increased the percentage of macrophage in spleen (p < 0.001); MNC did not alter the macrophage population from the level observed after MCAO. Only HUCB MNC significantly decreased Concanavalin A (ConA)-induced T cell stimulation (p < 0.05). These results suggest that the effects of HUCB MNC in the spleen are not due to a single HUCB population, but the interaction of all the subpopulations together.

Traumatic brain injury (TBI) is complex and involves multiple processes that contribute to functional decline. Progressive neuropathies result from delayed cellular death following the initial impact. Although the precise mechanisms responsible for delayed injury are unknown, numerous data implicate a role for the peripheral immune system in perpetuating neuroinflammation after TBI. A previous report demonstrated that splenic CCL20 chemokine expression was upregulated 24 h after lateral fluid percussive impact (LFPI), prior to neuronal expression but consistent with neurodegeneration. Here, we expand on those data to report increased CCL20 protein expression in white matter 48 h after LFPI and demonstrate that CCL20 is directly toxic to primary neurons and oligodendrocytes subjected to oxygen glucose deprivation. The temporal expression profile of CCL20, coupled with in vitro toxicity to primary cells, suggests that this chemokine exerts deleterious effects on cell viability following TBI. These findings warrant further investigations into the use of CCL20 as a potential biomarker and/or therapeutic target.

Delayed neuronal death associated with stroke has been increasingly linked to the immune response to the injury. Splenectomy prior to middle cerebral artery occlusion (MCAO) is neuroprotective and significantly reduces neuroinflammation. The present study investigated whether splenic signaling occurs through interferon gamma (IFNγ). IFNγ was elevated early in spleens but later in the brains of rats following MCAO. Splenectomy decreased the amount of IFNγ in the infarct post-MCAO. Systemic administration of recombinant IFNγ abolished the protective effects of splenectomy with a concurrent increase in INFγ expression in the brain. These results suggest a role for spleen-derived IFNγ in stroke pathology.

The majority of promising experimental compounds have failed in clinical trials, highlighting the need for novel approaches to treat stroke. Much research has been devoted to elucidating the signaling pathways involved in delayed neuroinflammation that can be targeted at clinically relevant time points. The field of stroke research has benefited from experiments characterizing the temporal expression profiles of candidate cytokines, chemokines, matrix metalloproteinases, and other putative pro-inflammatory molecules. Yet, these data have offered only a glimpse into the complex pathological sequelae and have not advanced the treatment of neuropathies. Upon recognition that peripheral immune cell activation is involved in penumbral expansion, the spleen has emerged as a novel target that mediates the peripheral immune response and promotes pro-inflammatory injury. Although the precise mechanisms have yet to be elucidated, accumulated evidence demonstrates that focal cerebral ischemia alters cytokine, chemokine, and immune cell profiles in the spleen. Additionally, removal of this peripheral lymphoid organ is neuroprotective, and the efficacy of several protective therapies has been linked to actions at the level of the spleen. Future experiments aimed at identifying the splenic lymphocyte populations that respond to ischemic stroke, as well as their signaling mechanisms, are critical in developing novel therapies.

Many pharmacological treatments for stroke have afforded protection in rodent models but failed to show efficacy in clinical trials. This discrepancy may be due to the lack of long-term functional studies. Previously, delayed administration of the sigma receptor agonist 1,3-di-o-tolylguanidine (DTG) reduced infarct volume after middle cerebral artery occlusion (MCAO) in rats. The present study was conducted to determine whether the protective effects of DTG lead to improvements in behavioral functioning. Rats were subjected to MCAO and administered 7.5, 1.5, or 0.75 mg/kg DTG beginning 24 h post-surgery. Histological outcomes (96 h, 2 weeks, and 5 weeks) were compared with performance on a series of behavioral tests (2 and 4 weeks). Fluoro-Jade staining and immunohistochemistry were used to assess infarct volume and immune cell recruitment. All doses significantly reduced infarct volume and perturbation of striatal white matter tracts at 96 h. These reductions were associated with decreased numbers of CD11b-positive amoeboid microglia/macrophages. Despite short-term efficacy, DTG failed to improve behavioral outcomes or reduce infarct volumes after 96 h. While DTG may prove beneficial as a short-term therapy, these data highlight the importance of long-term functional recovery when evaluating novel therapies to treat stroke.

Each year, approximately 795,000 people suffer a new or recurrent stroke. About 610,000 of these are first attacks, and 185,000 are recurrent attacks. Currently, the only FDA approved treatment for ischemic stroke is the thrombolytic recombinant tissue plasminogen activator (Alteplase), which must be given within 4.5 h of stroke onset. Beyond this time, apoptotic and inflammatory processes greatly diminish the therapeutic benefits of current treatments. While there have been many experimental treatments for stroke that showed promising preclinical efficacy, these treatments have failed to show efficacy in clinical trials. In many of these cases, the preclinical animal studies did not model the clinical setting effectively. The injury that occurs following stroke is a dynamic process. To effectively treat stroke patients at clinically relevant timepoints, it is imperative to understand both the humeral and cell-mediated phenomena that occur throughout the body in response to ischemic injury over time. Promising experimental therapeutics designed to be given 1 to 2 days following stroke require both neuroprotective and anti-inflammatory properties in order to be efficacious.