Brain repair mechanisms after cell therapy for stroke.

Cell-based therapies hold great promise for brain repair after stroke. While accumulating evidence confirms the preclinical and clinical benefits of cell therapies, the underlying mechanisms by which they promote brain repair remain unclear. Here, we briefly review endogenous mechanisms of brain repair after ischemic stroke and then focus on how different stem and progenitor cell sources can promote brain repair. Specifically, we examine how transplanted cell grafts contribute to improved functional recovery either through direct cell replacement or by stimulating endogenous repair pathways. Additionally, we discuss recently implemented preclinical refinement methods, such as preconditioning, microcarriers, genetic safety switches, and universal (immune evasive) cell transplants, as well as the therapeutic potential of these pharmacologic and genetic manipulations to further enhance the efficacy and safety of cell therapies. By gaining a deeper understanding of post-ischemic repair mechanisms, prospective clinical trials may be further refined to advance post-stroke cell therapy to the clinic.

Fluid biomarkers of the neurovascular unit in cerebrovascular disease and vascular cognitive disorders: A systematic review and meta-analysis.

Background: The disruption of the neurovascular unit (NVU), which maintains the integrity of the blood brain barrier (BBB), has been identified as a critical mechanism in the development of cerebrovascular and neurodegenerative disorders. However, the understanding of the pathophysiological mechanisms linking NVU dysfunction to the disorders is incomplete, and reliable blood biomarkers to measure NVU dysfunction are yet to be established. This systematic review and meta-analysis aimed to identify biomarkers associated with BBB dysfunction in large vessel disease, small vessel disease (SVD) and vascular cognitive disorders (VCD). Methods: A literature search was conducted in PubMed, EMBASE, Scopus and PsychINFO to identify blood biomarkers related to dysfunction of the NVU in disorders with vascular pathologies published until 20 November 2023. Studies that assayed one or more specific markers in human serum or plasma were included. Quality of studies was assessed using the Newcastle-Ottawa Quality Assessment Scale. Effects were pooled and methodological heterogeneity examined using the random effects model. Results: A total of 112 studies were included in this review. Where study numbers allowed, biomarkers were analysed using random effect meta-analysis for VCD (1 biomarker; 5 studies) and cerebrovascular disorders, including stroke and SVD (9 biomarkers; 29 studies) while all remaining biomarkers (n = 17 biomarkers; 78 studies) were examined through qualitative analysis. Results of the meta-analysis revealed that cerebrospinal fluid/serum albumin quotient (Q-Alb) reliably differentiates VCD patients from healthy controls (MD = 2.77; 95 % CI = 1.97-3.57; p < 0.0001) while commonly measured biomarkers of endothelial dysfunction (VEGF, VCAM-1, ICAM-1, vWF and E-selectin) and neuronal injury (NfL) were significantly elevated in vascular pathologies. A qualitative assessment of non-meta-analysed biomarkers revealed NSE, NfL, vWF, ICAM-1, VCAM-1, lipocalin-2, MMP-2 and MMP-9 levels to be upregulated in VCD, although these findings were not consistently replicated. Conclusions: This review identifies several promising biomarkers of NVU dysfunction which require further validation. A panel of biomarkers representing multiple pathophysiological pathways may offer greater discriminative power in distinguishing possible disease mechanisms of VCD.

Dataset on stroke infarct volume in rodents: A comparison of MRI and histological methods.

This dataset offers images of mouse brains impacted by photothrombotic stroke in the sensorimotor cortex published by Weber et al. NeuroImage (2024). Data is gathered using two primary techniques: (1) whole-brain ex-vivo magnetic resonance imaging (MRI) and (2) 40 µm thick coronal histological sections that undergo immunofluorescence staining with NeuroTrace. Infarct areas and volumes are assessed through MRI at two distinct time frames-three days (acute) and 28 days (chronic) following photothrombotic stroke induction. Subsequently, the brains are sectioned into 40 µm thick coronal slices, stained with NeuroTrace, and imaged as whole sections. The dataset holds considerable value for reuse, particularly for researchers focused on stroke volume estimation methods as well as those interested in comparing the efficacy of MRI and histological techniques.

Beneath the radar: immune-evasive cell sources for stroke therapy.

Stem cell therapy is an emerging treatment paradigm for stroke patients with remaining neurological deficits. While allogeneic cell transplants overcome the manufacturing constraints of autologous grafts, they can be rejected by the recipient's immune system, which identifies foreign cells through the human leukocyte antigen (HLA) system. The heterogeneity of HLA molecules in the human population would require a very high number of cell lines, which may still be inadequate for patients with rare genetic HLAs. Here, we outline key progress in genetic HLA engineering in pluripotent stem and derived cells to evade the host's immune system, reducing the number of allogeneic cell lines required, and examine safety measures explored in both preclinical studies and upcoming clinical trials.

A toolkit for stroke infarct volume estimation in rodents.

Stroke volume is a key determinant of infarct severity and an important metric for evaluating treatments. However, accurate estimation of stroke volume can be challenging, due to the often confined 2-dimensional nature of available data. Here, we introduce a comprehensive semi-automated toolkit to reliably estimate stroke volumes based on (1) whole brains ex-vivo magnetic resonance imaging (MRI) and (2) brain sections that underwent immunofluorescence staining. We located and quantified infarct areas from MRI three days (acute) and 28 days (chronic) after photothrombotic stroke induction in whole mouse brains. MRI results were compared with measures obtained from immunofluorescent histologic sections of the same brains. We found that infarct volume determined by post-mortem MRI was highly correlated with a deviation of only 6.6 % (acute) and 4.9 % (chronic) to the measurements as determined in the histological brain sections indicating that both methods are capable of accurately assessing brain tissue damage (Pearson r > 0.9, p < 0.001). The Dice similarity coefficient (DC) showed a high degree of coherence (DC > 0.8) between MRI-delineated regions of interest (ROIs) and ROIs obtained from histologic sections at four to six pre-defined landmarks, with histology-based delineation demonstrating higher inter-operator similarity compared to MR images. We further investigated stroke-related scarring and post-ischemic angiogenesis in cortical peri­infarct regions and described a negative correlation between GFAP+fluorescence intensity and MRI-obtained lesion size.

Nogo-A is secreted in extracellular vesicles, occurs in blood and can influence vascular permeability.

Nogo-A is a transmembrane protein with multiple functions in the central nervous system (CNS), including restriction of neurite growth and synaptic plasticity. Thus far, Nogo-A has been predominantly considered a cell contact-dependent ligand signaling via cell surface receptors. Here, we show that Nogo-A can be secreted by cultured cells of neuronal and glial origin in association with extracellular vesicles (EVs). Neuron- and oligodendrocyte-derived Nogo-A containing EVs inhibited fibroblast spreading, and this effect was partially reversed by Nogo-A receptor S1PR2 blockage. EVs purified from HEK cells only inhibited fibroblast spreading upon Nogo-A over-expression. Nogo-A-containing EVs were found in vivo in the blood of healthy mice and rats, as well as in human plasma. Blood Nogo-A concentrations were elevated after acute stroke lesions in mice and rats. Nogo-A active peptides decreased barrier integrity in an in vitro blood-brain barrier model. Stroked mice showed increased dye permeability in peripheral organs when tested 2 weeks after injury. In the Miles assay, an in vivo test to assess leakage of the skin vasculature, a Nogo-A active peptide increased dye permeability. These findings suggest that blood borne, possibly EV-associated Nogo-A could exert long-range regulatory actions on vascular permeability.

Leakage beyond the primary lesion: A temporal analysis of cerebrovascular dysregulation at sites of hippocampal secondary neurodegeneration following cortical photothrombotic stroke.

We have previously demonstrated that a cortical stroke causes persistent impairment of hippocampal-dependent cognitive tasks concomitant with secondary neurodegenerative processes such as amyloid-ß accumulation in the hippocampus, a region remote from the primary infarct. Interestingly, there is emerging evidence suggesting that deposition of amyloid-ß around cerebral vessels may lead to cerebrovascular structural changes, neurovascular dysfunction, and disruption of blood-brain barrier integrity. However, there is limited knowledge about the temporal changes of hippocampal cerebrovasculature after cortical stroke. In the current study, we aimed to characterise the spatiotemporal cerebrovascular changes after cortical stroke. This was done using the photothrombotic stroke model targeting the motor and somatosensory cortices of mice. Cerebrovascular morphology as well as the co-localisation of amyloid-ß with vasculature and blood-brain barrier integrity were assessed in the cortex and hippocampal regions at 7, 28 and 84 days post-stroke. Our findings showed transient cerebrovascular remodelling in the peri-infarct area up to 28 days post-stroke. Importantly, the cerebrovascular changes were extended beyond the peri-infarct region to the ipsilateral hippocampus and were sustained out to 84 days post-stroke. When investigating vessel diameter, we showed a decrease at 84 days in the peri-infarct and CA1 regions that were exacerbated in vessels with amyloid-ß deposition. Lastly, we showed sustained vascular leakage in the peri-infarct and ipsilateral hippocampus, indicative of a compromised blood-brain-barrier. Our findings indicate that hippocampal vasculature may represent an important therapeutic target to mitigate the progression of post-stroke cognitive impairment.

Molecular biomarkers for vascular cognitive impairment and dementia.

As disease-specific interventions for dementia are being developed, the ability to identify the underlying pathology and dementia subtypes is increasingly important. Vascular cognitive impairment and dementia (VCID) is the second most common cause of dementia after Alzheimer disease, but progress in identifying molecular biomarkers for accurate diagnosis of VCID has been relatively limited. In this Review, we examine the roles of large and small vessel disease in VCID, considering the underlying pathophysiological processes that lead to vascular brain injury, including atherosclerosis, arteriolosclerosis, ischaemic injury, haemorrhage, hypoperfusion, endothelial dysfunction, blood-brain barrier breakdown, inflammation, oxidative stress, hypoxia, and neuronal and glial degeneration. We consider the key molecules in these processes, including proteins and peptides, metabolites, lipids and circulating RNA, and consider their potential as molecular biomarkers alone and in combination. We also discuss the challenges in translating the promise of these biomarkers into clinical application.

Ischemic stroke-related gene expression profiles across species: a meta-analysis.

Stroke patients are often left with permanent disabilities with no regenerative treatment options. Unbiased RNA sequencing studies decoding the transcriptional signature of stroked tissue hold promise to identify new potential targets and pathways directed to improve treatment for stroke patients. Here, gene expression profiles of stroked tissue across different time points, species, and stroke models were compared using NCBI GEO database. In total, 34 datasets from mice, rats, humans, and primates were included, exploring gene expression differences in healthy and stroked brain tissue. Distinct changes in gene expression and pathway enrichment revealed the heterogenicity of the stroke pathology in stroke-related pathways e.g., inflammatory responses, vascular repair, remodelling and cell proliferation and adhesion but also in diverse general, stroke-unrelated pathways that have to be carefully considered when evaluating new promising therapeutic targets.

The vascular gene Apold1 is dispensable for normal development but controls angiogenesis under pathological conditions.

The molecular mechanisms of angiogenesis have been intensely studied, but many genes that control endothelial behavior and fate still need to be described. Here, we characterize the role of Apold1 (Apolipoprotein L domain containing 1) in angiogenesis in vivo and in vitro. Single-cell analyses reveal that - across tissues - the expression of Apold1 is restricted to the vasculature and that Apold1 expression in endothelial cells (ECs) is highly sensitive to environmental factors. Using Apold1-/- mice, we find that Apold1 is dispensable for development and does not affect postnatal retinal angiogenesis nor alters the vascular network in adult brain and muscle. However, when exposed to ischemic conditions following photothrombotic stroke as well as femoral artery ligation, Apold1-/- mice display dramatic impairments in recovery and revascularization. We also find that human tumor endothelial cells express strikingly higher levels of Apold1 and that Apold1 deletion in mice stunts the growth of subcutaneous B16 melanoma tumors, which have smaller and poorly perfused vessels. Mechanistically, Apold1 is activated in ECs upon growth factor stimulation as well as in hypoxia, and Apold1 intrinsically controls EC proliferation but not migration. Our data demonstrate that Apold1 is a key regulator of angiogenesis in pathological settings, whereas it does not affect developmental angiogenesis, thus making it a promising candidate for clinical investigation.

Rebooting disruptive science: Exploring the challenges and potential solutions.

There has been a decline in the number of disruptive scientific discoveries and breakthroughs. Here, reasons for the decline of disruptive science are explored including declining funding for basic research, increasing risk-aversion among scientists, pressure to publish quickly and increasing administrative workload. Solutions are proposed to reverse the trend and encourage disruptive research especially for young scientists.

Preserving stroke penumbra by targeting lipid signalling.

Pharmacological inhibition of astrocytic enzyme autotaxin rescues the stroke penumbra in mice and improves functional recovery, indicating therapeutic potential.

Editing a gateway for cell therapy across the blood-brain barrier.

Stem cell therapy has been shown to improve stroke outcomes in animal models and is currently advancing towards clinical practice. However, uncertainty remains regarding the optimal route for cell delivery to the injured brain. Local intracerebral injections are effective in precisely delivering cells into the stroke cavity but carry the risk of damaging adjacent healthy tissue. Systemic endovascular injections, meanwhile, are minimally invasive, but most injected cells do not cross CNS barriers and become mechanically trapped in peripheral organs. Although the blood-brain barrier and the blood-CSF barrier tightly limit the entrance of cells and molecules into the brain parenchyma, immune cells can cross these barriers especially under pathological conditions, such as stroke. Deciphering the cell surface signature and the molecular mechanisms underlying this pathophysiological process holds promise for improving the targeted delivery of systemic injected cells to the injured brain. In this review, we describe experimental approaches that have already been developed in which (i) cells are either engineered to express cell surface proteins mimicking infiltrating immune cells; or (ii) cell grafts are preconditioned with hypoxia or incubated with pharmacological agents or cytokines. Modified cell grafts can be complemented with strategies to temporarily increase the permeability of the blood-brain barrier. Although these approaches could significantly enhance homing of stem cells into the injured brain, cell entrapment in off-target organs remains a non-negligible risk. Recent developments in safety-switch systems, which enable the precise elimination of transplanted cells on the administration of a drug, represent a promising strategy for selectively removing stem cells stuck in untargeted organs. In sum, the techniques described in this review hold great potential to substantially improve efficacy and safety of future cell therapies in stroke and may be relevant to other brain diseases.

Molecular and anatomical roadmap of stroke pathology in immunodeficient mice.

Background: Stroke remains a leading cause of disability and death worldwide. It has become apparent that inflammation and immune mediators have a pre-dominant role in initial tissue damage and long-term recovery. Still, different immunosuppressed mouse models are necessary in stroke research e.g., to evaluate therapies using human cell grafts. Despite mounting evidence delineating the importance of inflammation in the stroke pathology, it is poorly described to what extent immune deficiency influences overall stroke outcome. Methods: Here, we assessed the stroke pathology of popular genetic immunodeficient mouse models, i.e., NOD scid gamma (NSG) and recombination activating gene 2 (Rag2-/-) mice as well as pharmacologically immunosuppressed mice and compared them to immune competent, wildtype (WT) C57BL/6J mice three weeks after injury. We performed histology, gene expression, blood serum and behavioural analysis to identify the impact of immunosuppression on stroke progression. Results: We detected changes in microglia activation/macrophage infiltration, scar-forming and vascular repair in immune-suppressed mice three weeks after injury. Transcriptomic analysis of stroked tissue revealed the strongest deviation from WT was observed in NSG mice affecting immunological and angiogenic pathways. Pharmacological immunosuppression resulted in the least variation in gene expression compared with the WT. These anatomical and genetic changes did not affect functional recovery in a time course of three weeks. To determine whether timing of immunosuppression is critical, we compared mice with acute and delayed pharmacological immunosuppression after stroke. Mice with delayed immunosuppression (7d) showed increased inflammatory and scarring responses compared to animals acutely treated with tacrolimus, thus more closely resembling WT pathology. Transplantation of human cells in the brains of immunosuppressed mice led to prolonged cell survival in all immunosuppressed mouse models, which was most consistent in NSG and Rag2-/- mice. Conclusions: We detected distinct anatomical and molecular changes in the stroke pathology between individual immunosuppressed mouse models that should be considered when selecting an appropriate mouse model for stroke research.

Deep learning-based behavioral profiling of rodent stroke recovery.

BACKGROUND: Stroke research heavily relies on rodent behavior when assessing underlying disease mechanisms and treatment efficacy. Although functional motor recovery is considered the primary targeted outcome, tests in rodents are still poorly reproducible and often unsuitable for unraveling the complex behavior after injury. RESULTS: Here, we provide a comprehensive 3D gait analysis of mice after focal cerebral ischemia based on the new deep learning-based software (DeepLabCut, DLC) that only requires basic behavioral equipment. We demonstrate a high precision 3D tracking of 10 body parts (including all relevant joints and reference landmarks) in several mouse strains. Building on this rigor motion tracking, a comprehensive post-analysis (with >100 parameters) unveils biologically relevant differences in locomotor profiles after a stroke over a time course of 3 weeks. We further refine the widely used ladder rung test using deep learning and compare its performance to human annotators. The generated DLC-assisted tests were then benchmarked to five widely used conventional behavioral set-ups (neurological scoring, rotarod, ladder rung walk, cylinder test, and single-pellet grasping) regarding sensitivity, accuracy, time use, and costs. CONCLUSIONS: We conclude that deep learning-based motion tracking with comprehensive post-analysis provides accurate and sensitive data to describe the complex recovery of rodents following a stroke. The experimental set-up and analysis can also benefit a range of other neurological injuries that affect locomotion.

Xeno-free induced pluripotent stem cell-derived neural progenitor cells for in vivo applications.

BACKGROUND: Currently, there is no regenerative therapy for patients with neurological and neurodegenerative disorders. Cell-therapies have emerged as a potential treatment for numerous brain diseases. Despite recent advances in stem cell technology, major concerns have been raised regarding the feasibility and safety of cell therapies for clinical applications. METHODS: We generated good manufacturing practice (GMP)-compatible neural progenitor cells (NPCs) from transgene- and xeno-free induced pluripotent stem cells (iPSCs) that can be smoothly adapted for clinical applications. NPCs were characterized in vitro for their differentiation potential and in vivo after transplantation into wild type as well as genetically immunosuppressed mice. RESULTS: Generated NPCs had a stable gene-expression over at least 15 passages and could be scaled for up to 1018 cells per initially seeded 106 cells. After withdrawal of growth factors in vitro, cells adapted a neural fate and mainly differentiated into active neurons. To ensure a pure NPC population for in vivo applications, we reduced the risk of iPSC contamination by applying micro RNA-switch technology as a safety checkpoint. Using lentiviral transduction with a fluorescent and bioluminescent dual-reporter construct, combined with non-invasive in vivo bioluminescent imaging, we longitudinally tracked the grafted cells in healthy wild-type and genetically immunosuppressed mice as well as in a mouse model of ischemic stroke. Long term in-depth characterization revealed that transplanted NPCs have the capability to survive and spontaneously differentiate into functional and mature neurons throughout a time course of a month, while no residual pluripotent cells were detectable. CONCLUSION: We describe the generation of transgene- and xeno-free NPCs. This simple differentiation protocol combined with the ability of in vivo cell tracking presents a valuable tool to develop safe and effective cell therapies for various brain injuries.

Stem Cell Therapy for Repair of the Injured Brain: Five Principles.

Cell therapy holds great promise for regenerative treatment of disease. Despite recent breakthroughs in clinical research, applications of cell therapies to the injured brain have not yielded the desired results. We pinpoint current limitations and suggest five principles to advance stem cell therapies for brain regeneration. While we focus on cell therapy for stroke, all principles also apply for other brain diseases.

Stimulation of the cuneiform nucleus enables training and boosts recovery after spinal cord injury.

Severe spinal cord injuries result in permanent paraparesis in spite of the frequent sparing of small portions of white matter. Spared fibre tracts are often incapable of maintaining and modulating the activity of lower spinal motor centres. Effects of rehabilitative training thus remain limited. Here, we activated spared descending brainstem fibres by electrical deep brain stimulation of the cuneiform nucleus of the mesencephalic locomotor region, the main control centre for locomotion in the brainstem, in adult female Lewis rats. We show that deep brain stimulation of the cuneiform nucleus enhances the weak remaining motor drive in highly paraparetic rats with severe, incomplete spinal cord injuries and enables high-intensity locomotor training. Stimulation of the cuneiform nucleus during rehabilitative aquatraining after subchronic (n = 8 stimulated versus n = 7 unstimulated versus n = 7 untrained rats) and chronic (n = 14 stimulated versus n = 9 unstimulated versus n = 9 untrained rats) spinal cord injury re-established substantial locomotion and improved long-term recovery of motor function. We additionally identified a safety window of stimulation parameters ensuring context-specific locomotor control in intact rats (n = 18) and illustrate the importance of timing of treatment initiation after spinal cord injury (n = 14). This study highlights stimulation of the cuneiform nucleus as a highly promising therapeutic strategy to enhance motor recovery after subchronic and chronic incomplete spinal cord injury with direct clinical applicability.