Neuroinflammation, Stem Cells, and Stroke

Abstract

Stroke remains a significant unmet clinical need with few treatment options that have a very narrow therapeutic window, thereby causing massive mortality and morbidity in the US and around the world. Accordingly, finding safe and effective novel treatments with a wider therapeutic window stands as an urgent need in stroke. The progressive inflammation that occurs centrally and peripherally after stroke serves as a unique therapeutic target to retard and even halt the secondary cell death. Stem cell therapy represents a potent approach that can diminish inflammation in both the stroke brain and periphery (e.g., spleen), advancing a paradigm-shift from a traditionally brain-focused therapy to treating stroke as a neurological disorder with a significant peripheral pathology. The purpose of this review paper is to highlight the inflammation- mediated secondary cell death that plagues both brain and spleen in stroke and to evaluate the therapeutic potential of stem cell therapy in dampening these inflammatory responses.

Stroke: An Unmet Clinical Need

Stroke is a major cause of mortality and disability in the US and around the world. Stroke is an acute episode of localized dysfunction of the brain due to blood flow interruption manifesting as a focal infarction referred to as ischemic stroke (about 85%) or as a rupture in cerebral vasculature referred to as hemorrhagic stroke (15%).¹ Fatality for ischemic stroke is around 25%, while hemorrhagic stroke carries a greater mortality at 50%.² The rampant incidence and lower mortality rate but with debilitating morbidity in stroke survivors provides the basis for preferential targeting of therapeutics to ischemic stroke over hemorrhagic stroke. Comorbidities that increase the risk of stroke include hypertension, obesity, lack of exercise, insufficient diet quality, psychosocial factors, smoking, high alcohol consumption, and diabetes mellitus.³ Currently, there are few therapeutic options available for stroke. Both tissue plasminogen activator (tPA) and mechanical thrombectomy have a very narrow therapeutic window; thus, many stroke patients cannot avail of these treatments.⁴ Accordingly, there is an urgent need to explore safe and effective novel treatments for stroke, especially those with wider therapeutic windows.

Focusing on the Secondary Cell Death of Stroke

Ischemic stroke is the impairment of blood supply to the brain secondary to an obstruction in a cerebral vessel that triggers oxygen and glucose deprivation in the brain, thereafter, producing neurovascular damage with accompanying motor and neurologic abnormalities.⁵ Multiple cell death pathways mediate ischemic stroke- induced compromise of the neurovascular unit. Among these several cell death mechanisms, excitotoxicity, oxidative stress, free radical accumulation, mitochondrial dysfunction, impaired neurogenesis, reduced angiogenesis, and abnormal vasculogenesis, and aberrant inflammation have been implicated as key mediators of ischemic stroke primary and secondary injury.^6,7

Ischemic stroke causes severe, immediate impact to the neurovascular unit and function of the central nervous system (CNS)⁸, which can be exacerbated by the body’s own immune response.^9,10 Although the damage to the neurovascular unit produced by the primary ischemic insult is most likely unsalvageable, the subsequent neuroinflammatory aberration that persists over a prolonged period provides a fertile ground for sequestration of the stroke-modulated secondary cell death.¹¹

Neuroinflammation and the Immune System

The pathological progression of stroke neuroinflammation entails the key participatory role of the immune system. The primary insult of an ischemic stroke elevates levels of damage associated molecular proteins (DAMPs), which activate the sensors of the innate immune system, such as the neutrophils and macrophage/microglia, the lectin pathway of complement activation and the toll-like receptors, altogether magnifying the inflammatory response.^12–14 In tandem, ischemic stroke also activates the adaptive immune system via the lymphocyte populations including T cells, B cells, regulatory T cells, and antigen-specific autoreactive responses.^15–17 Moreover, a neuroinflammatory response after ischemic stroke initiates downstream pathophysiological events that affect the neurovascular unit. Foremost, neuroinflammation may compromise the blood brain barrier (BBB) integrity, allowing the immune cells from the blood to penetrate the stroke brain.^18–19 Such influx of inflammation-associated cytokines and chemokines mobilizes the innate immune microglial cells in the parenchyma, allows myeloid cell infiltration, and activates the adaptive immune lymphocyte cells, altogether exacerbating stroke.^20–21 That both innate and adaptive immune systems contribute to the morbidity and mortality after stroke, highlights the critical involvement of the immune system in the evolution of inflammation and its significant contribution to ischemic stroke.²² Recognizing that neuroinflammation may define the negative outcomes of ischemic stroke indicates that targeting the inflammatory signaling pathway may prove beneficial for stroke.

Neuroinflammation Manifests Over an Extended Period Post-Stroke

Acute and chronic stroke-induced neuroinflammatory responses closely accompany secondary cell death (Figure 1). Immediately following a stroke episode, acute neuroinflammation may correspond to an endogenous repair process in response to the dead and dying cells and debris of ischemic tissue. Microglial cells, the resident macrophages of the brain, phagocytose injured brain cells in an effort to clear the brain of these damaged cells.²³ This process is crucial both for intracranial hemorrhage (ICH) and ischemic stroke, with certain distinctions. Phagocytosis of red blood cells by infiltrated macrophages is crucial for hematoma resolution in an ICH ²⁴. Treatment with IL-4 facilitates STAT6 activation in mice models of ICH, enhancing microglia and macrophage phagocytosis for increased erythrocyte clearance, and long-term functional recovery²⁵. In ischemic stroke however, both microglia and infiltrated macrophages may be involved in ferroptosis, a type of programmed cell death that is dependent on iron and lipid peroxide accumulation²⁶. Targeting ferroptosis serves as a promising approach for treating ischemic stroke, however the exact mechanisms linking ferroptosis and inflammation remain under investigation²⁷.

Figure 1. Timeline of stroke-induced inflammation.

The stages of inflammation in stroke can be delineated as acute, subacute, and chronic.

Indeed, microglial activation occurs within minutes after stroke. A defective microglial activation may increase infarction and apoptosis after stroke.^28–29 Acute neuroinflammation may confer neuroprotection by affording low transient inflammation which enhances the immune cell signals to the brain which in turn increases the expression of interleukin IL-1 cytokine, thereby delegating a surveillance task to the glial cells in the injured brain.^30–31 In parallel, the expression of IL-4 facilitates recovery and axonal regrowth following a brain insult.^32–33 Notwithstanding, the acute inflammatory stage of stroke also reveals upregulation of potent pro-inflammatory cytokines such as TNF-α, interleukin 6 (IL-6), and IL-1β in the cerebrospinal fluid (CSF) and blood in humans.⁹ Microglia 1 (M1) activated microglia locally produce TNF-α and IL-1, while IL-6 is also secreted by neurons.⁹ Moreover, ischemia-activated microglial cells express increased levels of cluster of differentiation 14 (CD14), a key pattern recognition receptor of the innate immune system, indicating that resident microglial contribute to acute ischemic inflammation.³⁴ Strategies designed to shift activated M1 microglia to the activated M2 phenotype, which secretes anti- inflammatory cytokines and neurotrophic factors, may harness an anti-inflammatory effect in stroke.⁶ On the other hand, chronic neuroinflammation may exacerbate ischemic stroke injury. Microglia-induced neuroinflammation in the chronic phase of stroke aggravates post-ischemic damage.³⁵ The chronic inflammatory stage of stroke entails BBB disruption associated with infarction of the parenchyma and cerebral vasculature.³⁶ During this extended period, invasion of immune cells and serum proteins through the compromised BBB initiates adverse pathophysiological consequences, such as worsened neuroinflammation, elevated intracranial pressure and widespread cellular death.³⁷ Thus, the primary ischemic insult is exacerbated by this secondary inflammation-plagued cell death. The aberrant neuroinflammation instigated by a dysfunctional activation of microglial cells are also seen with other immune cells. Astrocytes, another key mediator of the brain homeostasis, just like the microglia, may respond similarly to the acute pro-survival or chronic cell death switch of the immune response after stroke as astrocytes can either protect against or worsen stroke neuroinflammation.^38–39 Additionally, ischemic stroke may induce an autoimmune response to neuronal antigens that can lead to long-term neuroinflammation.⁴⁰ These observations, taken together, suggest that enhancing the neuroprotective acute inflammation, while dampening the deleterious chronic neuroinflammation may improve stroke outcomes.

This concept of the neurovascular unit stipulates a network of pathological symptoms of the disease whereby any impaired part within the neurovascular unit can subsequently affecting the whole system.⁴¹ These cellular components communicate via physical and biochemical pathways, with deficiency of one cell type potentially affecting the whole system through these interactions. The centrally located neurovascular unit is indirectly linked with peripheral systems, including key axes with the spleen as well as the gut and bone marrow.^42–46 With this in mind, a systems biology approach may be most appropriate for interrogating the pathology and treatment of stroke. Cognizant of the body’s dual inflammatory responses to the stroke insult, we refer to central inflammation to delineate the role of CNS resident cells in brain inflammation. In contrast, we refer to peripheral inflammation to distinguish the systemic immune response to inflammation after stroke.

Peripheral Immune Response to Stroke

Although the immune system is classically thought to be non-existent in the CNS, the brain exhibits its own immune system, and, together with the peripheral immune system, both contribute to the progressive neuroinflammation after stroke.^47–49 As noted above, the body’s immune response to injury determines stroke outcomes. Acting as a double-edge sword, the body’s acute inflammatory response may afford protection against infection, but may elicit deleterious effects in the chronic state, compounding the stroke primary injury. Acutely, the immune system acts to protect the body from foreign pathogens, and aids in tissue healing and regeneration when the body suffers from injury. The primary injury weakens the BBB allowing nuclear or cytosolic proteins or protein fragments containing pro-inflammatory signals called DAMPs to penetrate into the parenchyma. When recognized by pattern recognition receptors on dendritic cells, macrophages, and other cells such as vascular cells, epithelial cells, and fibroblasts, DAMPS induce a pro-inflammatory response from these cells.⁵⁰ Such induction of an immune response triggers a chronic inflammation that exacerbates the stroke injury. Moreover, BBB compromise may occur with minimal cell death. Characterized by cytoskeletal alterations in endothelial cells, dysfunctional actin polymerization, stress fiber formation, and junctional protein degradation within 30–60 minutes following ischemic/reperfusion injury, the BBB breach in the acute phase of ischemic stroke allows peripheral immune cell infiltration into the brain parenchyma.^51–52 The resulting inflammatory response engages the complement system to mobilize immune cells to the intrathecal compartment.⁵³ Next, neutrophils, monocytes and lymphocytes penetrate the BBB and under a chemoattractant process migrate to the injury site where the cells secrete free radicals, pro-inflammatory cytokines, prostaglandins and other inflammatory mediators, soliciting more immune cells and microglia to the injury site inducing cell death to the ischemic tissue.^54–55 These data indicate that the microglia acts as the key mediator of the immune response after an insult to the CNS.

In chronic neuroinflammation, both central and peripheral immune systems collaboratively mount a hyperactive inflammatory response that exacerbates the tissue injury instead of fostering brain regeneration.^47–48 Centrally, the inflammatory response involves the microglia and other immune cell mediators. Peripherally, splenic immune cells perpetuate the inflammatory response, but other immune cells from inflammatory organs, such as the thymus and cervical lymph nodes, likely also contribute to the exacerbation of stroke inflammation. Conversely, neuroinflammation also mediates pro-repair and pro-regenerative functions: leukocytes and microglia produce neurotrophic factors, microglia and macrophages clear cell debris and toxic byproducts, and macrophages promote remyelination by producing lactate, a metabolic need for axonal regrowth⁵⁶. The subsequent sections delve into the robust splenic and other peripheral inflammatory responses after stroke providing the rationale to target the spleen in sequestration of peripheral stroke inflammation.^47–48

Splenic Immune Response in Stroke

Following stroke, the spleen mounts an immune response that worsens the stroke pathology. The spleen regulates many bodily functions including serving as the main lymphatic organ, actively surveilling the body fluid circulation, filtering blood, and recycling iron from old red blood cells.^57–58 Moreover, the spleen acts as a defense system against blood-borne pathogens via mononuclear phagocytic machinery.⁵⁹

The spleen also serves as a repository of systemic immune cells with an ample supply of platelets, peripheral macrophages, and other immune cells.⁶⁰ Indeed, a large population of circulating monocytes that eventually differentiate into macrophages and dendritic cells upon extravasation and tissue entry originate from the spleen.^57,59

Body injuries cue the spleen to quickly deploy monocytes to the circulation that eventually reach the injury site.⁶¹ After myocardial infarction, splenic monocytes depart from the spleen and propagate the heart contributing to essential immunological reactions and regenerative mechanisms including wound repair.⁵⁹ Similarly in stroke, mice that received middle cerebral artery occlusion (MCAO) exhibit splenic contraction characterized by a lower number of splenic monocytes in the spleen with a concomitant elevation of monocytes in the ischemic brain.⁶⁰ Because the immunologic response to stroke (albeit chronically post-stroke) worsens the progressive brain injury from the initial insult, targeting the spleen may render therapeutic outcomes. Splenectomy two weeks prior to permanent MCAO significantly decreases cerebral infarction.⁶¹ In parallel, splenectomy before a transient MCAO reduces monocytes in the stroke brain but did not alter cerebral infarction.⁵⁷ The discrepant results of splenectomy in producing functional recovery from stroke may be due to the heterogeneous splenic monocyte populations that may exacerbate or dampen the secondary cell death. Instead of complete removal of the spleen, selective ablation of a subset of splenic monocytes, namely monocytes/macrophages or mononuclear phagocytes seems to better define the stroke outcomes.⁵⁷ Despite these inconsistent results, that splenectomy leads to the disappearance of monocytes in the spleen only to reappear in stroke suggests the close interaction of the splenic immune response with the brain insult.

Cervical Lymph Node Immune Response in Stroke

The inflammatory response accompanying stroke is highly complex and multifactorial. Stroke induces activation of peripheral immune cells which can compound ischemic brain injury⁶². Peripheral immune cells release upregulated levels of inflammatory cytokines such as IL-6 and TNF-α after stroke⁶³. One well-documented route of this peripheral inflammatory reaction after ischemic stroke entails CNS drainage to cervical lymph nodes likely via a mechanism involving the rapid release of VEGF-C into the CSF that subsequently induces lymphatic endothelial cell proliferation and macrophage activation⁶⁴. Pharmacologic blockade of VEGFR3 significantly reduces lymphatic endothelial activation, decreases proinflammatory macrophages and lessens brain tissue infarction. Furthermore, cervical lymphadenectomy partially ameliorates the deleterious effects of stroke-induced peripheral inflammation and reduces brain infarction⁶⁴. The pathophysiology of brain-lymphatic interactions after stroke represents an important mechanism of peripheral inflammation that warrants ample consideration when designing novel therapeutic targets that modulate deleterious inflammatory responses.

Skull Bone Marrow Microchannels Transport Neutrophils to Infarct

In addition to aforementioned extracerebellar contributions to stroke-induced neuroinflammation, nearby bone marrow may also contribute to the inflammatory response. Microscopic vascular channels of the skull may provide direct transport of neutrophils from the skull’s bone marrow to the ischemic area⁴³. Using murine models, an increase in skull bone marrow-derived neutrophils migrate to the neuroinflammatory site than tibial bone marrow-derived neutrophils. Interestingly, these neutrophils lodge along direct vascular channels from the marrow to the dura. Thus, targets to mitigate inflammatory cell migration from nearby bone marrow sites may provide a future therapeutic focus for stroke patients.

A Brain-Spleen Axis of Inflammation

The interaction between the brain and the spleen can be appreciated in the observed response of the immune cells in the spleen to cholinergic input in that brain injury correlates with autonomic release of pro-inflammatory cytokines from splenic macrophages. This paradigm of “brain-spleen inflammatory coupling” involves activation of autonomic input after CNS injury triggers a systemic reaction most apparent in the spleen but also detected in other peripheral organs, including the posterior hypothalamus exhibiting increased sympathetic tone, the adrenal glands displaying catecholamine release and vasoconstriction, and the liver showing an elevated number of macrophages. All of these responses are regulated by cholinergic input under an autonomic tone.⁶⁵ Downstream following a brain insult, an increased sympathetic tone coincides with the mobilization of immune cells and their inflammatory cytokines, such as TNF-α and IL-1β, from these peripheral organs, in particular the spleen, to the bloodstream and hone to the injury site.^60,66 Such pro-inflammatory response resulting from upregulated sympathetic tone elicits an equally elevated parasympathetic tone from the spleen, and likely from the other peripheral organs, affording an anti- inflammatory response.⁶⁰ Activation of the splenic macrophages under parasympathetic control mobilize to the circulation and the injured brain, dampening inflammation, specifically by suppressing TNFα^60,67 with coincident reduction in infarct size and improvement in cell survival⁶⁸ and recovery of neurological function in stroke animals.⁶⁰ That the cholinergic input to sympathetic and parasympathetic tone regulates the splenic response to the brain insult opens new avenues for developing spleen- directed stroke therapeutics.

Acute to Chronic Neurological Injury: The Role of Inflammation in Stroke

Although traditionally considered as an acute injury, stroke manifests as chronic inflammation centrally and peripherally that closely approximates the severity of the secondary cell death. Inflammation exacerbates stroke with both the brain and the periphery, especially the spleen, contributing to such detrimental inflammatory response (Table 1). Finding treatment strategies that target the spleen-mediated inflammatory response may pave the way towards identifying unexplored cell death pathways and new therapeutics in stroke. The spleen stands as a key reservoir of inflammatory cells and molecules, appealing to its pivotal active role in the peripheral inflammatory component of stroke. Equally innovative, the spleen may be selectively targeted to mount anti- inflammatory effects against stroke. Incorporating peripheral inflammation to the conventional view of CNS-focused pathology and treatment extends our understanding of the secondary cell death in stroke.

Table 1:

Brain-spleen coupling of inflammation in negative stroke outcomes

Author	Findings
Brown et al., 2020 9	Partial MHC-II constructs minimize secondary cell death as a result of splenic involvement in stroke-induced neuroinflammation
Schuhmann et al., 202112	Increased plasma levels of DAMPs within a sealed cerebral-ischemic arterial compartment modeling stroke
Selvaraj et al., 202116	After stroke, mice showed elevated leukocytes, with higher CD8+ levels correlating to worse symptoms.
Venkat et al., 2018 20	Demonstrates the use of vasculotide in T1DM rats post stroke reduces infarct volume, decreases BBB damage, decreases inflammation, and improves functionality
Palomino-Antolin et al., 2021 21	KO NLRP3 mice models demonstrate neuroprotection post stroke
Lalancette-Herbert et al., 2009 28	Demonstrates relationship between TLR2 responses in olfactory bulbs in transgenic mice models in neuroinflammation
Bohacek et al., 2012 29	TLR2 KO stroke mice models underwent reperfusion, demonstrating the role of TLR2 and prompt microglial activation in modulating post-stroke injury
Nagarajan et al., 2012 30	Chlamydia muridarum infected mice models with IL-1R or inflammasome deficiencies show the major role of IL-1 signaling in the inflammasome pathway
Yang et al., 2013 31	IL-1R KO mice demonstrate the role of IL-1 in synthesizing fibrinogen and coagulation in streptococcus pneumoniae infection
Fenn et al., 2014 32	Aged mice with inadequate IL-4/IL-4R response and KO models demonstrates the need for IL-4 in reprogramming microglia after injury
Lee et al., 2010 33	Traumatic spinal cord injury rat models demonstrate the role of IL4 in acute macrophage activation and inflammatory response
Beschorner et al., 2002 34	Ischemia-activated microglial cells express increased levels of cluster of differentiation 14 (CD14)
Al Mamun et al., 2020 35	Demonstrates the role of the IRF5-IRF4 regulatory axis in adapting stroke outcomes through M1 or M2 microglial responses
Ranjbar et al., 2019 39	Analysis of lipocalin-2 expression in stroke rat models illustrates the role of LCN2 in peripheral involvement of neuroinflammation during hypoxia
Fischer et al., 2021 38	Murine stroke models show increased release of eRNA DAMP to sensitize astrocytes towards other inflammatory signals via TLR2-NF-kB signaling
Ortega et al., 2015 40	Using cervical lymph nodes and spleen samples of murine stroke models, autoimmune responses to NR2A, MAP2 and myelin-derived peptides were observed
Fluiter et al., 2014 54	A C6 antisense oligonucleotide blocked MAC formation in the injured brain, reducing microglia, apoptosis, axonal loss, and improved functionality
Bellander et al., 2001 55	Observing 16 patients post TBI, increased C1q, C3b, and C3d, and MAC were reported
Das et al., 2011 47	Rat injury models show the role of the spleen and/or thymus in perpetuating an inflammatory response via CCL20
Kim et al., 2014 57	In stroke mice models, the spleen was contracted spleen with decreased pro-inflammatory and anti-inflammatory monocytes, while levels of monocytes the brain were increased. These monocytes decreased in the brain after a splenectomy.
Rasouli et al., 2011 60	Mice stroke models illustrate lower splenic monocytes, suggesting splenic contribution to monocyte accumulation in the brain
Ajmo et al., 2009 61	Rat stroke models underwent splenic denervation or various sympathetic catecholamine blockers. Results demonstrate the role of blood circulating catecholamines in splenic response to stroke.
Wang et al., 2014 67	Rat TBI models given puerarin showed reduced neuronal degeneration. This effect was decreased by blocking the PI3K-Akt pathway with LY294003
Muhammad et al., 2011 68	Mice stroke models infects with human influenza A demonstrate the potential of a7 nicotinic acetylcholine receptor agonists in reducing proinflammatory stroke outcomes

Neuroprotective Microglial and Neutrophil Subtypes

Stroke dictates an immense inflammatory response in the CNS including the activation and proliferation of resident microglia and infiltration of peripheral immune cells^69–70. Specifically, a heterogenous population of neutrophil subtypes enter the brain leading to hypotheses similar to the M1-vs-M2 concept for microglia that there may be both good and bad neutrophil subtypes present in the CNS after ischemic stroke injury. The pro-inflammatory M1 microglia promote secondary brain damage while the anti-inflammatory M2 microglial phenotype ameliorates CNS injury after stroke⁷¹. TLR4 (toll-like receptor 4) deficient mice with experimentally induced focal cerebral ischemia display increased levels of alternative neutrophils (N2) which express M2 phenotype markers. This elevation in alternative N2 neutrophils correlates with neuroprotective effects after stroke as opposed to the deleterious pro-inflammatory effects associated with N1 neutrophils⁶⁹. Increased frequency of alternatively activated neutrophils in ischemic brain tissue correlates with increased neuronal survival, reduced infarct size and improved clinical ⁷⁰. Therapeutics that direct immune cell phenotypes towards the neuroprotective M2 microglia and N2 neutrophils offers an important avenue that should be considered in reducing both the central and peripheral inflammation that exacerbates CNS stroke injury.

Therapeutic Targeting of Central and Peripheral Immune Systems

Both central and peripheral immune systems contribute to neuroinflammation in stroke. Traditionally, treatments focus on chronically activated microglial cells in the brain. Targeting the peripheral immune system, however, may offer a new approach to treating stroke. Intravenously grafted stem cells preferentially migrate to the spleen in stroke animal models.⁷² In parallel, splenectomies minimize stroke-induced behavioral and histological deficits.^73–76 These data suggest a close interaction between the brain and peripheral immune system, as noted above aptly called the “brain-spleen inflammatory coupling” corresponding to a cell death pathway, as well as a therapeutic target for stroke. Dampening stroke-induced inflammation not just in the brain but also in the periphery may render a better sequestration of the secondary cell death in stroke. To this end, transplantation of stem cells may harbor an anti-neuroinflammatory response that can mitigate both central and peripheral inflammatory responses thereby preserving a homeostatic neurovascular unit germane to fostering a stroke therapeutic.

As noted above, in response to injury, microglia assume various fates including the classical, pro-inflammatory M1 pathway and the M2 anti-inflammatory pathway. M1 microglia are associated with the release of pro-inflammatory cytokines including IL-6, TNF-α, and IL-1β, whereas M2 microglia surmount an anti-inflammatory and neuroprotective response^77–79. Following ischemic stroke, the spleen releases pro-inflammatory mediators, activating M1 microglia that contributes to BBB breakdown. Stem cell transplantation, probably acting via the grafted cells’ anti-inflammatory property, diverts macrophages to the anti-inflammatory M2 phenotype, thereby mitigating the splenic inflammatory response, reducing BBB damage, and creating an environment conducive to repair and regeneration⁸⁰.

Finding a novel therapy capable of simultaneously lowering the synergistic central and peripheral inflammatory responses, specifically those arising in the brain and the spleen, stands as a potent stroke therapeutic. The key involvement of the spleen in stroke secondary cell death has attracted a paradigm-shift in treating stroke from a purely brain insult to a neurological disorder with a major peripheral component, specifically splenic inflammation. Heretofore, we discuss the research progress on stem cell therapy for stroke, with emphasis on the critical role of the spleen in the inflammation pathology and treatment.

Stem Cell Therapy for Stroke

As noted above, a stroke treatment that allows for a wide therapeutic window compared to tPA and thrombectomy will likely benefit a larger stroke population. With safety and efficacy demonstrated despite a delay in treatment initiation, the targeted stroke phase entails sequestration of the secondary cell death, which neuroinflammation appears to closely approximate. To this end, stem cell therapy satisfies both prolonged treatment window and robust anti-inflammatory mechanism.

Recognizing the chronic pathological symptoms and functional deficits which plague stroke, in particular the inflammatory-mediated secondary cell death, has categorized stroke as a chronic disorder.^{7, 81–85} With the primary brain damage mostly non-rescuable, the treatment has shifted to regenerating the injured brain and mitigating secondary cell death.^7,72,86–87 Accordingly, stem cell therapy caters to regenerative medicine by targeting the chronic phase of and exerting a capacity to repair the stroke brain.

A well-defined stem cell source stands as the main criterion to ensure the quality and reproducibility of the stem cells.^88–90 Based on the tissue source, such as the fetus, embryo or adult, stem cells display varying levels of proliferation, migration, and differentiation, which may influence the treatment efficacy. A multitude of stem cells have been tested in animal models of stroke, including fetal-derived neural stem cells, embryonic stem cells, bone marrow stem cells, umbilical cord stem cells, adipose stem cells, and induced pluripotent stem cells. ^91–96 An increasing number of these cells have reached clinical trials.^97–99 The logistics in generating an ample supply of stem cells and the ethics associated with harvesting these cells remain as critical factors of consideration when contemplating translation of stem cells products from the laboratory to the clinic.

The demonstration of safety and efficacy of the transplanted cells in clinically relevant animal models represents pivotal enabling translational studies necessary prior to proceeding with any clinical application.^88–90 An insight into the mechanism mediating the stem cell-induced functional recovery will also offer guidance on further optimizing the transplant regimen. Among the many postulated mode of actions of stem cells, the two most documented regenerative pathways include the cell replacement process and the bystander effects involving stem cell secretion of therapeutic factors that lessen neuroinflammation, oxidative stress, and apoptosis, as well as those that enhance neurogenesis, angiogenesis, vasculogenesis, and mitochondrial repair.^100–102 The latter mechanism of bystander effects entails that transplanted stem cells into the stroke brain engraft poorly, yet remain effective in conferring functional recovery, suggesting that long-term survival and differentiation may not be required for repairing the stroke brain and its functions.^100–102 That stem cell engraftment is not a prerequisite for brain repair advances the notion of stem cell-derived secretome as a key source of therapeutic effects. Extracellular vesicles and exosomes harvested from stem cells release growth factors, such as vascular endothelial growth factor (VEGF), cytokines, chemokines, microRNAs, and long noncoding RNAs.^103–105 Despite the lack of a full understanding of these mechanistic underpinnings of stem cell therapy, whether stem cells induce cell replacement, bystander effects or exosomal therapeutic effects, transplanted stroke animals exhibit significant reduction of secondary cell death, especially neuroinflammation, accompanied by improved behavioral outcomes.^106–108

Splenic Targeting of Stem Cells

As discussed above, a therapy that recognizes the central and peripheral inflammation may prove effective for stroke. While stroke is considered a brain disorder, its inflammatory pathology manifests as a dual brain and peripheral insult.^{9–10,109–110} Following the primary insult, the secondary cell death involves inflammatory signals originating from both central and peripheral organs, specifically the spleen.^{9–10,109–110} The spleen acts as a key regulator of this peripheral inflammatory response in response to the primary stroke insult. Such brain-spleen coupling that propagates the inflammatory response after stroke provides the basis of targeting stem cells to the brain as well as to the spleen. Indeed, the spleen attracts peripherally transplanted stem cells in stroke.^72,111–112 Following MCAO, intravenous or intracarotid delivery of stem cells lowers brain and systemic inflammation coupled with behavioral recovery.^72,112,113 Although peripheral inflammation and its consequences in the stroke brain remain not fully understood, the observation that many peripherally transplanted stem cells migrate into the spleen suggests that direct brain engraftment may not be required for effective sequestration of inflammation.^100–102 An indirect mechanism of anti- inflammation whereby peripheral targeting of stem cells into the spleen, as opposed to the brain, offers a minimally invasive procedure that is practical and feasible in the clinic. Such peripheral administration of stem cells also circumvents the need to transport the cells across the BBB. This innovative approach of splenic targeting of stem cells may allow repeated booster cell treatments to fully abrogate the stroke-induced inflammation especially during the progressive chronic phase of the disease.

Aging Effect on Immune Response to Stroke

Aging must be considered as a comorbidity in experimental stroke studies. Aging contributes to a significant reduction in the ability of the brain to return to normal cellular and biochemical functions after ischemic stroke. Aged rats exhibit a significantly greater mortality rate from MCAO when compared to young rats¹¹⁴. Aging can result in greater oxidative DNA damage and limit neuroprotective mechanisms during ischemic stroke¹¹⁵. These mechanistic alterations in the aged brain worsen the clinical outcomes of stroke as evidenced by impaired cerebral perfusion, greater infarct sizes, white matter injury and dysregulation of pro- versus anti-inflammatory microglia^115–116. Specifically, aging exacerbates immune system dysregulation which can limit potential neuroprotective immune system effects¹¹⁵. Furthermore, aged microglia coincides with greater deleterious chronic inflammation when compared to young brains and post-ischemic aged microglia, showing less interaction with nearby neurons with reduced polarity toward the infarct region¹¹⁷. With almost 72% of stroke cases in patients 65 years or older¹¹⁸, future experimental stroke studies must account for aged related changes in the brain and immune system when developing future therapeutics.

Circadian rhythms play a role in immune system regulation as well as the regulation of adult stem cell functions. Circadian biology represents an interesting correlate to consider to maximize therapeutic effects of both immune system modulation and stem cell therapy. Regarding immune system regulation, the neutrophil proteasome showed temporal oscillations that aligned neutrophil activity with circadian cycles. The neutrophil proteasome is ultimately subject to circadian regulation which can limit neutrophil release of toxic mediators and neutrophil production of neutrophil extracellular traps¹¹⁹. Furthermore, the circadian clock and diurnal rhythmicity are linked to stem cell homeostasis and aging. Circadian dysregulation and aging ultimately leads to reduced functional capacity of stem cells¹²⁰. Incorporating aging and circadian rhythm considerations into experimental stroke studies on stem cell therapies provides innovative platforms for developing stroke therapeutics.

Strengthening the Clinical Impact of Stem Cells

While stem cell transplantation offers a novel and hopeful treatment for mitigating stroke neuroinflammation, harnessing the immune reactions between the grafted stem cells and the host requires in-depth investigations ¹²¹. Agarose hydrogel induces apoptosis of CD8+ cells that could alleviate immune-mediated cytolysis after stem cell treatment¹²². Coating stem cells in a hyaluronin/heparin sulfate/collagen mix also decreases post-stroke neuroinflammation while enhancing stem cell functional effects in mice¹²³. In addition to being immune cell targets, stem cells may also be subject to oxidative stress after implantation. When human umbilical cord mesenchymal stem cells were treated with the MG53 protein, however, these cells were less vulnerable and more functional in rat models of TBI¹²⁴. Prior to transplantation, preconditioning allows stem cells to adapt to the injurious host environment, leading to improved tolerance and therapeutic potential once grafted. Multiple methods can be employed for preconditioning, including hypoxic induction, increased acidity, nutrient deprivation, and the addition of pharmacological agents¹²⁵. For instance, harnessing the role of Hypoxia-Inducible-Factor (HIF), which activates genes implicated in hypoxia survival, supports stem cell survival in ischemic stroke^126–127. Since stem cells target an ischemic area, pre-conditioning of stem cells to low-oxygen conditions may prime these cells to exhibit tolerance and resistance to a stroke environment^128–129. Similarly, forming neurospheres (bundles of neural stem cells) prior to implantation may aid in stem cells’ extracellular matrices, proliferation, and survival rates^130–131. Beyond stroke, as in the case of Parkinson’s Disease, preconditioning stem cells with 6-OHDA is neuroprotective for dopaminergic neurons when exposed to oxidative stress^125,132.These biomaterial and engineering-based technologies may nurture the regenerative capacity of stem cells and warrant further examination.

Dampening Stroke Neuroinflammation with Stem Cells

Stroke persists as a significant unmet clinical need. An urgent need for safe and effective treatment requires a wider therapeutic window that dampens the evolving secondary cell death. That transplantation of stem cells dampen both central and peripheral inflammation in preclinical stroke models,^{72,111–113,133–134} coupled with clinical data implicating such a key role of the spleen in stroke patients,^135–136 advances its utility as a powerful stroke therapeutic. Stem cells may abrogate secondary cell death via cell replacement and bystander effects. To date, most of the stroke treatments target the brain since stroke primarily manifests as a neurological disorder. A paradigm-shift has emerged in recognition of the significant peripheral inflammation associated with the onset and progression of stroke. The spleen participates closely to the inflammatory response that approximates the deterioration of the neurovascular unit. Brain-spleen coupling entails that stroke not only manifests with a brain pathology but is equally influenced by the splenic inflammatory response. Accordingly, such dual brain and spleen pathology merits a treatment that can mitigate both central and peripheral inflammation. To this end, compelling laboratory and clinical evidence demonstrates that peripherally administered stem cells preferentially migrate to the spleen, with the cells likely attracted to the inflammatory cues presented by the spleen, but also strategically positioning themselves to abrogate this deleterious systemic inflammation (Figure 2). Directly, a few stem cells may hone to the stroke brain and secrete anti-inflammatory factors to taper neuroinflammation. Indirectly, stem cells lodged in the spleen can dampen the splenic inflammatory response, which subsequently lowers the central neuroinflammation. Altogether, both direct and indirect anti-inflammatory mechanisms rendered by transplanted stem cells can abrogate the stroke secondary cell death. Stroke presents as a neurovascular disease with brain and spleen inflammatory pathology that exacerbates the secondary cell death. Stem cell therapy confers robust anti-inflammatory response both centrally and peripherally.

Figure 2. Stem cell grafts alleviate stroke chronic inflammation in brain and spleen.

Stroke renders inflammatory responses from the brain and the spleen, creating a brain-spleen coupling of central and peripheral inflammation that exacerbates the secondary cell death. Intravenously transplanted stem cells migrate preferentially to the spleen with a few cells reaching the brain, altogether conferring direct and indirect sequestration of the inflammation-plagued secondary cell death in stroke.

Non-standard Abbreviations and Acronyms

tPA

Tissue plasminogen activator

CNS

Central nervous system

DAMPs

Damage associated molecular pattern

BBB

Blood brain barrier

ICH

Intracranial hemorrhage

IL-6

Interleukin 7

CSF

Cerebrospinal fluid

M1

Microglial 1

CD14

Cluster of differentiation 14

MCAO

Middle cerebral artery occlusion

TLR4

Toll-like receptor 4

N2

Alternative neutrophils

VEGF

Vascular endothelial growth factor

HIF

Hypoxia-Inducible-Factor