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Publicly Available Published by De Gruyter February 7, 2022

Intranasal application of stem cells and their derivatives as a new hope in the treatment of cerebral hypoxia/ischemia: a review

  • Mohammad Saied Salehi , Benjamin Jurek ORCID logo , Saeideh Karimi-Haghighi , Nahid Jashire Nezhad , Seyedeh Maryam Mousavi , Etrat Hooshmandi , Anahid Safari , Mehdi Dianatpour , Silke Haerteis , Jaleel A. Miyan , Sareh Pandamooz EMAIL logo and Afshin Borhani-Haghighi EMAIL logo
From the journal Reviews in the Neurosciences
https://doi.org/10.1515/revneuro-2021-0163
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Abstract

Intranasal delivery of stem cells and conditioned medium to target the brain has attracted major interest in the field of regenerative medicine. In pre-clinical investigations during the last ten years, several research groups focused on this strategy to treat cerebral hypoxia/ischemia in neonates as well as adults. In this review, we discuss the curative potential of stem cells, stem cell derivatives, and their delivery route via intranasal application to the hypoxic/ischemic brain. After intranasal application, stem cells migrate from the nasal cavity to the injured area and exert therapeutic effects by reducing brain tissue loss, enhancing endogenous neurogenesis, and modulating cerebral inflammation that leads to functional improvements. However, application of this administration route for delivering stem cells and/or therapeutic substances to the damaged sites requires further optimization to translate the findings of animal experiments to clinical trials.

Keywords: conditioned medium; hypoxic brain; ischemic brain; nasal delivery; secretome; stem cell

Introduction

Cerebral ischemia and hypoxic brain damage are devastating conditions that are considered as major global problems. Neonatal hypoxic–ischemic (NHI) encephalopathy can be at the root of mortality or long-lasting neurological disorders, such as epilepsy, mental retardation, and cerebral palsy, subsequently leading to long-term cognitive dysfunctions and motor deficits. The incidence of NHI encephalopathy is estimated to be 1 to 8 in thousand live births. Among the affected infants, 20–50% die during the newborn period and up to 25% of the survivors experience permanent neurological deficits (Ferriero 2004; Kurinczuk et al. 2010; Vannucci and Hagberg 2004). Besides NHI encephalopathy, one in four people over the age of 25 experience ischemic stroke, intracerebral hemorrhage or subarachnoid hemorrhage as different types of stroke during their lifetime. Hence, stroke is considered as one of the main causes of death as well as mental and physical inabilities worldwide (Borhani-Haghighi et al. 2013; Krishnamurthi et al. 2020). Although hypothermia (Tagin et al. 2012), thrombolysis/mechanical thrombectomy, or surgery (Tawil and Muir 2017) are frequently used to combat these destructive conditions, treatment failure, risk of hemorrhage, and narrow treatment time windows still hamper effective treatments.

The last two decades have witnessed a surge in studies employing stem cells as a promising therapeutic approach to treat cerebral hypoxia/ischemia. Preclinical investigations revealed the restorative potential of variety of stem cells originating from diverse sources, such as brain (Baker et al. 2019; Zhang et al. 2019), bone marrow (Kuroda 2016; Li et al. 2016), adipose tissue (Gutiérrez-Fernández et al. 2015), dental pulp (Gancheva et al. 2019; Lan et al. 2019), umbilical cord (Li et al. 2015a), uterus (Rodrigues et al. 2012; Rodrigues et al. 2016), and hair follicle (Salehi et al. 2020) to ameliorate the inhospitable character of cerebral ischemia. In addition, the protective action of other types of cells, such as induced pluripotent stem cells (Kokaia et al. 2017; Zents and Copray 2016), bone marrow mononuclear cells (Kumar et al. 2017; Vahidy et al. 2016), endothelial progenitor (Fan et al. 2010), and vascular progenitor cells (Li et al. 2014) have been evaluated in this context. Beside the source of stem cells, the route of administration is considered as one of the most important aspects of cell-based therapies. Although direct implantation of stem cells to the brain by intracerebral, intracerebroventricular or subarachnoid administration seems to represent an optimal delivery method of transplanting cells to the infarct area, the inherent tendency of stem cells for invasion may cause additional brain damage in adjacent brain regions. In addition, the necessity for neurosurgery and general anesthesia curtails the clinical applicability (Rodriguez-Frutos et al. 2016).

Hence, systemic delivery through intra-venous or intra-arterial routes is widely used methods to target the brain. Preclinical investigations employing such delivery methods have provided evidence for an enhanced outcome in the context of stroke. However, entrapment of stem cells in capillary beds of other organs like kidney, spleen, liver, and lung diminish the effectiveness of these methods. Furthermore, the necessity for the administration of stem cells in massive numbers increases the risk for adverse side-effects and augments treatment costs (Fischer et al. 2009; Mays and Savitz 2018). The intra-arterial delivery of stem cells resulted in a greater distribution and diffusion of cells into the infarct area and to adjacent regions, compared to intra-venous, intracerebral and intracerebroventricular administration (Li et al. 2010). In addition, the intra-arterial route prevents the above-described cell entrapment in other organs. Nonetheless, microembolization, as well as cerebral blood flow reduction causing a higher mortality rate, raises serious concerns for employing this route of transplantation (Walczak et al. 2008; Watanabe and Yavagal 2016). Therefore, using alternative non-neurosurgical routes that can effectively penetrate the brain with minimal systemic exposure are imperative.

The olfactory neuroepithelium of the nasal cavity provides a sufficiently absorbent surface for the uptake of biological compounds. It directly connects to the external environment and permits its accessibility for therapeutic treatments. Due to specific physiological, anatomical, and histological properties of the nasal cavity, intranasal administration of drugs has achieved a widespread interest in preclinical and clinical studies. The intranasal route has been used to deliver a wide range of biological compounds to the brain, such as viral vectors, oligonucleotides, proteins and peptides (Lochhead and Thorne 2012). Among them, oxytocin (Jurek and Neumann 2018; Salehi et al. 2017) and insulin (Chen et al. 2018; Santiago and Hallschmid 2019) are the most widely employed drugs that have been intranasally administered for different purposes.

Despite the well-studied nature of the connection between the nasal cavity and the brain, which is known for over a century, and nose-to-brain delivery of drugs which has been performed for more than three decades (Frey 1991), the entrance of cells from the nose to the brain was not expected until recently. In 2009, intranasally administered cells were reported to penetrate the brains of mice and rats and therefore represent a non-invasive pathway to bypass the blood–brain barrier (Danielyan et al. 2009). Hence, this route would eliminate or minimize the distribution of cellular grafts to peripheral organs. During the last decade, several research groups around the world employed intranasal administration of stem cells to treat brain injuries (Chapman et al. 2013; Jiang et al. 2011; Li et al. 2015b; Zhang et al. 2021). Accordingly, in this review, we summarize the therapeutic application of intranasally delivered stem cells or their derivatives to combat cerebral hypoxic/ischemia. We also discuss possible brain-entry routes for intranasally administered cells and substances.

Intranasal application of stem cells in the treatment of cerebral ischemia

Up until now, the restorative potential of intranasally administered mesenchymal stem cells and embryonic neural stem cells in animal models of cerebral hypoxia/ischemia have been extensively reported. By employing different behavioral tests, ranging from emotional assessments to motor functions and cognitive abilities (Box 1 and depicted in Figure 1) researchers evaluated the curative impact of the cell therapy as a final consequence of changes at the molecular level.

Box 1:

List of the most commonly employed behavioral tests that have been used to evaluate the behavioral performance following intranasal application of stem cells or their derivatives in animal models of cerebral hypoxia/ischemia.
Sensory/Motor Function
Behavioral test Description
Adhesive-removal This test evaluates sensorimotor functions. Adhesive tape strips are placed on the limbs or snout and the time required to sense and remove the adhesives is measured. Brain injury might increase this time (Bouet et al. 2009).
Cylinder rearing This test evaluates sensorimotor functions. The animal is placed in a clear cylinder and when rearing, the number of cylinder wall touches is measured. Brain injury might decrease the use of the contralateral paw (Magno et al. 2019).
Grid walking (foot fault) This test evaluates sensorimotor functions. The animal is placed at the center of an elevated, levelled grid with openings, and the number of “paw slipping through” the open grid is recorded. Brain injury might increase the food-fault errors (Chao et al. 2012).
Righting reflex This test evaluates sensorimotor functions. The animal is placed in a supine position on a bench pad and the time that animal requires to return to the prone position is measured. Brain injury might increase this time (Gao and Calderon 2020).
Neurological severity score This test can evaluate sensory (tactile, visual, and proprioceptive), motor, reflex, and balance performance. Neurological deficits are scored on a scale basis, with higher score representing higher neurological deficits (Bieber et al. 2019).
Rotarod This test evaluates motor coordination and balance. The animal is placed on a rotating rod, then the rod speed raises and the time that animal remains on the rod is recorded. Brain injury might decrease this time (Shiotsuki et al. 2010).
Negative geotaxis This test evaluates motor coordination. The animal is placed in a “head pointing downward position” on a 45° slope bench pad and the time that animal requires returning to face up-ward is measured. Brain injury might increase this time (Hobbs et al. 2008).
von Frey hairs This test evaluates mechanical sensitivity. Small pieces of a nylon rod, with different diameters are used to assess the sensitivity to a mechanical stimulus and mechanical thresholds (Deuis et al. 2017).
Buried food-finding This test evaluates the sense of smell. In this test, a pleasant food is hidden beneath a layer of cage bedding and the time to discover the food is recorded. Brain injury might increase this time (Machado et al. 2018).
Cognition Ability
Behavioral test Description
Morris water maze This test evaluates learning and spatial memory. After several trials, the animal is placed at four or five different positions of the water maze and must find a hidden platform in the opaque water. The time to find the platform is recorded. Brain injury might increase this time (Vorhees and Williams 2006).
Novel object recognition This test evaluates learning and memory. First, two identical objects are placed in the cage and the animal is permitted to explore them freely. Then, one of the objects is replaced with a new object, and the time that animal spends to explore the new object is recorded. Normal animal spends more time exploring the novel object (Antunes and Biala 2012).
Social discrimination This test evaluates sociability and social memory. One familiar conspecific and an unfamiliar conspecific are simultaneously presented to the experimental animal and the time that animal explores each of them is recorded. Normal animals spend more time exploring the unfamiliar conspecific (Rani et al. 2021).
Emotional Assessment
Behavioral test Description
Elevated plus maze This test evaluates anxiety-related behavior. The animal is placed in the center of the four arms of the maze and the time that animal spends in each arm is recorded. Spending more time in the closed arms is an indicator for higher anxiety-related behavior (Walf and Frye 2007).
Home cage behavior This test evaluates animal cognitive, social, emotional, and motor behaviors. The activities of animals in their home cage are continuously recorded for about 24 h and analyzed (Grieco et al. 2021).
Open field This test evaluates motor function, anxiety, and depression. The animal is placed in the center of a blank brightly lid arena and the total distance covered, as well as the time spent in the center or periphery of the arena is measured. Spending more time adjacent to the wall of the arena is an indicator for higher anxiety-related behavior (Seibenhener and Wooten 2015).
Social interaction In animals, this test evaluates social exploration including active interactions (such as social sniffing, social grooming, and chasing), passive interactions and action and reaction of an experimental animal with a new animal. The time that animals spend with each behavioral activity is recorded (Kim et al. 2019).
Sucrose preference This test evaluates anhedonia as a core symptom of depression-like behavior. The animal is allowed to choose between unsweetened water and sweet-tasting sucrose solution. Based on the animal’s natural preference for sweets, reduced preference for the sweet solution is indicative of anhedonia (Liu et al. 2018).
Figure 1: Schematics of sensory/motor, cognitive and emotional tests that are frequently used to evaluate the behavioral performance in animal models of cerebral hypoxia/ischemia.
Figure 1:

Schematics of sensory/motor, cognitive and emotional tests that are frequently used to evaluate the behavioral performance in animal models of cerebral hypoxia/ischemia.

Figure 2: Different cerebral migration and distribution of mesenchymal stem cells when administered at different time points following ischemia (Donega et al. 2013).
Figure 2:

Different cerebral migration and distribution of mesenchymal stem cells when administered at different time points following ischemia (Donega et al. 2013).

Stimulating endogenous regenerative mechanisms

In a series of experiments, Van Velthoven and colleagues (Van Velthoven et al. 2010; Van Velthoven et al. 2013; Van Velthoven et al. 2014; Van Velthoven et al. 2017) evaluated therapeutic effects of mesenchymal stem cells following nasal administration in animal models of cerebral hypoxia/ischemia. In 2010, they assessed the restorative potential of mouse bone marrow mesenchymal stem cells (BM-MSCs) on ischemic brain damage in neonatal mice (Van Velthoven et al. 2010). In this study, fluorescently labelled stem cells by PKH-26 were administered 10 days after the insult. Eighteen days after transplantation, PKH-26-labelled BM-MSCs were predominantly detected in the glomerular layer of the olfactory bulb of both hemispheres. Although many cells were present in the damaged ipsilateral hippocampus, they were absent in the contralateral part. A cylinder rearing test (see Box 1) at days 11 and 18 after cell therapy revealed a significant improvement in sensorimotor outcome, compared to the vehicle group. In addition, the cell therapy reduced gray and white matter loss, as assessed by microtubule-associated protein 2 (MAP2) and myelin basic protein (MBP) expression, 18 days after the treatment. At this time point, immunostaining against markers of neuronal (neuronal nuclei, NeuN), astrocyte (S100β), and microglia (ionized calcium binding adaptor molecule 1, Iba1) fate showed that transplanted MSCs had not yet differentiated into mature neurons, astrocytes, or microglia.

In another study by Van Velthoven et al. (2013), the research team intranasally delivered MSCs, 3 days after transient middle cerebral artery occlusion (MCAO) in neonatal rats. The cylinder rearing test revealed the improved performance of animals, 1, 8, and 15 days after cell therapy. Also, rats treated with BM-MSCs displayed significant improvements in the adhesive removal test 15 days after the treatment. To evaluate cell proliferation, rats received ethynyldeoxyuridine (EdU) at days 3–5 and bromodeoxyuridine (BrdU) at days 21–23 after ischemia. At day 28 post-stroke, the cell therapy had increased the number of EdU and BrdU-positive cells in the ipsilateral striatum, near the ischemic boundary zone, indicating an enhancement of endogenous cell proliferation. Also, Nissl, MAP2, and MBP staining showed that the stem cell therapy reduced brain tissue damage, as well as gray and white matter loss in the ipsilateral hemisphere.

Systemic delivery of genetically engineered stem cells over-expressing specific factor(s) has received considerable attention in the field of cerebral ischemia (Salehi et al. 2021). Van Velthoven et al. (2014) assessed the beneficial potential of genetically modified mouse MSCs following intranasal application in neonatal hypoxic-ischemic brain injured mice. Ten days after injury, MSCs over-expressing brain-derived neurotrophic factor (BDNF), epidermal growth factor-like protein 7 (EGFL7), persephin, or sonic hedgehog were administered. A cylinder rearing test 18 days after transplantation showed an improvement of functional recovery in experimental groups that received MSCs over-expressing BDNF and EGFL7. Naive MSCs as well as stem cells over-expressing BDNF reduced gray and white matter loss, as assessed by MAP2 and MBP staining. BDNF modified MSCs application also induced cell proliferation in the ischemic hemisphere.

In 2017 this research team evaluated therapeutic effects of rat MSCs on neonatal focal stroke in rats. Here, stem cells were administered 72 h after MCAO. The extent of brain injuries was determined using diffusion-weighted imaging and longitudinal T2-weighted magnetic resonance imaging (MRI), 1 and 2 weeks after MCAO. Based on the MRI, cell therapy was not able to significantly reduce the infarct size; however, treatment with MSCs provided protection from MBP degradation indicating attenuation of white matter loss 28 days after ischemia. At this time point, neurofilament H positive cells, as an axonal presence indicator, also increased by cell therapy. Furthermore, the cylinder rearing test revealed BM-MSC treatment decreased lateralizing behavior; hence enhanced somatosensory function (van Velthoven et al. 2017). For details on behavioral test paradigms, we refer the reader to Box 1.

Subarachnoid hemorrhage (SAH) is considered as one the major global health concerns due to the high rate of morbidity and mortality (Etminan et al. 2019). During SAH, blood flows into the subarachnoid space of the brain and results in cerebral ischemia. Nijboer et al. (2018) evaluated therapeutic effects of rat BM-MSCs on SAH in adult rats. Six days post-SAH, MSCs were delivered and the curative effects of the treatment were assessed fifteen days later. The BM-MSC therapy improved sensory functions and the mechanosensory response, assessed by adhesive removal and Frey hairs tests (see Box 1), respectively. Also, BM-MSC treatment reduced gray and white matter loss and increased mature neurons surrounding the lesion site. The BM-MSC therapy decreased astrocyte number as well as macrophage/microglia activation; therefore, reduced cerebral inflammation. Since depression is one of the long-term consequences of subarachnoid hemorrhage, the authors evaluated depression-like behavior using the sucrose preference test. MSC treatment completely restored the sucrose preference of SAH animals to the level of intact animals, indicating diminished depression-like behavior. Additionally, the cell-therapy increased ipsilateral tyrosine hydroxylase staining in cell bodies of the substantia nigra and nerve endings in the striatum, which is considered as enhanced dopamine synthesis in SAH animals.

Biosynthesis of nitric oxide is one of the key factors in the pathophysiological response of the brain to hypoxia–ischemia. Also copper content in the olfactory bulb of the rat corresponds to the level of superoxide dismutase 1 and 3. In this regard, Andrianov et al. (2020) evaluated nitric oxide and copper content in the olfactory bulbs of ischemic rat brains following the cell therapy. Ten minutes after the modeling of brain ischemia, MSCs were intranasally administered. Electron paramagnetic resonance spectroscopy revealed a significant reduction in nitric oxide production up to two days after ischemia, but no significant difference in the content of nitric oxide between transplanted and nontransplanted ischemic rats. Intranasal application of stem cells significantly increased copper content one day after ischemia, which subsequently decreased.

Based on these observations, the authors concluded that functional improvements following intranasal application of MSCs are mainly mediated by the stimulation of endogenous regenerative mechanisms, differentiation of transplanted stem cells into neurons and glial cells as well as inflammation inhibition. Following chemokine secretion at the lesion site, stem cells are able to migrate to the injured site from ipsi- and contra-lateral hemispheres. By immunomodulatory properties, MSCs suppress inflammatory processes and alter the ischemic microenvironment by releasing trophic factors, which exert neuroprotection. Furthermore, transplanted stem cells enhance cell proliferation in the subventricular zone, indicating the importance of endogenous regenerative processes after cell therapy. Lastly, intranasal administration of MSCs stimulates neurogenesis and elevate oligodendrocytes as well as myelin formation. All of these events are involved in the partial restoration of the activity of the corticospinal motor tract.

Dose and time dependency

The therapeutic effect of intranasal application of MSCs on brain damage in juvenile C57Bl/6 mice has been investigated by the group around Vanessa Donega (Donega et al. 2013; Donega et al. 2014a; Donega et al. 2014b; Donega et al. 2015). First, mouse MSCs (0.25 × 106, 0.5 × 106, 1 × 106) were administered intranasally at 10 days post hypoxia-ischemia. The doses of 0.5 × 106 and 1 × 106 MSCs significantly improved sensorimotor function 3, 4, and 5 weeks post ischemia. The treatment also reduced gray and white matter loss, 5 weeks after ischemia. To determine the therapeutic window of the treatment, researchers administered 0.5 × 106 MSCs intranasally at 3-, 10-, or 17-days post ischemia. The MSC treatment given 3- or 10-days post-injury was effective to improve motor function, 5 weeks after ischemia; however, administration of MSCs 17 days after the insult had no effect. Such a curative pattern was also observed in the reduction of gray and white matter loss. In addition, two intranasal doses of stem cells, 3 and 10 days post-ischemia, had no extra effect on sensorimotor and histological outcomes compared to the single administration either at days 3 or 10. Furthermore, cell therapy 10 days after ischemia improved cognitive function 7 weeks later (Donega et al. 2013). Also, cell therapy at different time points following ischemia resulted in different distribution of stem cells in the brain (Figure 2).

When mouse BM-MSCs were labeled with PKH-26 and administered 10 days post hypoxia/ischemia, stem cells were detected at the lesion site 2 h after administration and they reached maximum number 12 h later; afterwards, the number of labelled MSCs decreased until day 3 (Donega et al. 2014b). Two hours after administration, the presence of transplanted stem cells at the lesion site was also confirmed by ex-vivo MRI analysis of mouse brains, using MSCs labeled with micron-sized superparamagnetic iron-oxide particles. Immunostaining against Iba-1, glial fibrillary acidic protein (GFAP) and NeuN in the ipsilateral brain sections showed that transplanted stem cells are primarily surrounded by microglial cells, while astrocytes formed a boundary border around them. One to three days after the cell therapy, the number of doublecortin (DCX) positive cells in the ipsilateral subventricular zone and at the lesion site was increased (Donega et al. 2014b).

In another study by Donega et al. (2014a), human BM-MSCs (1 × 106 and 2 × 106 cell) were administered ten days after induction of ischemia. One day after the treatment, stem cells were detected in the sensorimotor and epithalamic regions of the damaged brain and no cells were found in the contralateral hemisphere. Cylinder rearing tests revealed that both doses of BM-MSCs improved the sensorimotor function at days 11 and 18 after the treatments. Although the application of 2 million BM-MSCs decreased gray and white matter damage, the lower dose of one million cells was not effective. Eighteen days after the transplantation, BM-MSCs reduced the number of GFAP and Iba-1 positive cells in the brain, indicating fewer astrocytes and microglia as well as reduced scar formation.

Lastly, Donega and her colleagues (2015) assessed the long-term safety and efficacy of mouse BM-MSC treatment for neonatal brain injury. Mouse pups received an intranasal administration of 0.5 × 106 MSCs 10 days post ischemia. The cylinder-rearing test up to 9 months after ischemia revealed an improvement in sensorimotor performance. The novel object recognition test (see Box 1) at 3 and 14 months post-ischemia showed that the cell therapy restored the cognitive performance at both time points. The BM-MSC treatment decreased both gray and white matter damage at week 5. Also, hematoxylin–eosin staining showed no evidence of neoplasia in the brain at month 14 (Donega et al. 2015).

Taken together, the authors aimed to identify optimum cell numbers, time points and frequency in order to ameliorate devastating conditions following cerebral hypoxia/ischemia. Apparently, a minimum of half a million MSCs is required to exert long-lasting beneficial effects in mice. Also, a single intranasal bolus of stem cells early after the insult is as effective as multiple boli at later time points to improve histological, cognitive, and motor outcomes. Based on these data, stem cells migrate to the injured area 2 h after intranasal application, reach a maximum number at 12 h, and dramatically decline in number at 72 h. Transplanted stem cells stimulate cascades of endogenous repairing mechanisms, which reduce hippocampal and cortical tissue loss, important areas in cognitive and motor function. Decreased lesion site size is also associated with reduced activation of microglia and astrocytes, as well as gliosis induced by immunosuppressive and anti-inflammatory properties of MSCs. Furthermore, stem cells at the injured site stimulate, through paracrine pathway, the secretion of neurotrophic factors in the subventricular zone and induce neuroblast differentiation. All of these events are necessary for long-term regeneration.

Potential benefit of hypoxic preconditioning in stem cell therapy after ischemia

In a series of studies by Ling Wei research team, Wei et al. (2013) assessed curative effects of normoxic and hypoxic preconditioned cultured rat BM-MSCs, in an adult male mouse model of focal cerebral ischemia. One day after ischemia, stem cells were administered and animals were evaluated 1.5 h or 3 days later. Both normoxic and hypoxic preconditioned stem cells were detected in the ischemic region 1.5 h after intranasal delivery; however, the pre-conditioned ones were presented in higher numbers. In the hypoxic preconditioned group, around 50% of counted stem cells in the brain were located in the ischemic region, compared to 23% in the normoxic-cultured group. Therefore, approximately 0.16% of the total number of preconditioned cells were homed in the ischemic region, compared to 0.05% of normoxic-cultured cells. Three days after the treatments, both types of stem cells reduced infarct size and cell death; however just preconditioned MSCs improved sensorimotor deficits.

Wei et al. (2015) evaluated the therapeutic potential of hypoxic preconditioned rat BM-MSCs on neonatal stroke in rats. Animals received an intranasal delivery of hypoxic preconditioned MSCs at 6 h and 3 days after stroke. Stem cell delivery at 6 h after stroke, reduced the infarct volume and blood–brain barrier leakage three days after ischemia. Seventeen days post-ischemia, angiogenic activity, evaluated by glucose transporter 1 (Glut-1) staining (marker of endothelial cell), as well as local blood flow in the peri-infarct region were higher in the stem cell transplanted group. Also, at this time point, endogenous neurogenesis dramatically increased following cell therapy. Consequently, 17 days after cerebral ischemia improved sensorimotor and olfactory function, social behaviors and home cage activities were detected in stem cell transplanted animals.

In another study, Sun et al. (2015) examined the therapeutic benefits of intranasal transplantation of hypoxic preconditioned rat BM-MSCs after hemorrhagic stroke in mice. At 3 and 7 days after ischemia, pre-labeled stem cells with Hoechst 33,342 were administered. Six hours post-transplantation, stem cells were detected around the hematoma, perivascular spaces, ipsilateral cortex, and olfactory bulbs. The cell therapy increased protein levels of BDNF, glial cell-derived neurotrophic factor, and vascular endothelial growth factor in the peri-hematoma areas that were down-regulated following ischemia. Preconditioned BM-MSCs enhanced neurogenesis, neuroblast proliferation and migration 14 days after ischemia. Also, at this time point, applied treatment improved motor function. 21 days after ischemia, Cresyl violet staining showed a reduction in brain tissue loss, and an enlargement of the ventricle cavity size (as a marker of brain atrophy) in the pre-conditioned BM-MSC-treated group.

The restorative potential of hypoxic preconditioned stem cells was also elucidated in a study by Monica J. Chau (2018). Here, delayed and repeated intranasal delivery of hypoxic preconditioned rat BM-MSCs after ischemic stroke was evaluated in mice. Hypoxic preconditioned stem cells were labeled with Hoecht 33,342, and administered at 3, 4, 5, and 6 days after stroke. Labeled MSCs were detected in the peri-infarct region 6 and 24 h after a single stem cell administration. Immunostaining against NeuN and Glut-1 in the peri infarct cortex, 21 days after stroke, revealed that the BM-MSC therapy enhanced neurogenesis and angiogenesis in the ischemic brain. The laser Doppler scanning method before, during, and 21 days after focal stroke, showed that BM-MSC transplantation increased cerebral blood flow in the peri-infarct region. The adhesive removal test, 14 days after ischemia, showed that the BM-MSC treatment reduced the removal time of the left paw.

Altogether, it has been proposed that the migration of transplanted stem cells toward the injured area, their homing and survival, can be enhanced by hypoxic preconditioning. Furthermore, delayed and repeated intranasal administration of hypoxic preconditioned stem cells substantially increases the therapeutic efficiency.

Combining hypothermia and stem cells is not recommended

Since acute hypothermia is considered as an established clinically intervention following neonatal hypoxic–ischemic cerebral damage, Herz et al. (2018) hypothesized that a combination of hypothermia and stem cell therapy may boost the regenerative potential of each treatment. To examine this hypothesis, hypoxic-ischemic brain injury was induced in neonatal mice. Immediately after ischemia, hypothermia was applied for 4 h with a rectal target temperature of 32 °C. Three days after the hypoxic-ischemic injury, murine BM-MSCs were administered. The amount of green fluorescent protein (GFP)-labelled stem cells in the brains of normothermia and hypothermia treated animals was quantified by flow cytometry, 14–16 h after nasal delivery. Similar migratory patterns of MSCs in the ipsilateral hemisphere of normal and hypothermia animals were detected, but no labeled cells were detected in the contralateral parts. Cell therapy improved exploratory and impulsive behaviors, as well as cognitive functions 32 days after the treatment. Cresyl violet staining 4 days after the treatment, showed both hypothermia and cell therapy rescued brain injuries in the hippocampus, cortex, striatum and thalamus. Although hypothermia improved axonal degeneration in the striatum and hippocampus, MSC treatment enhanced myelination. Stem cell application also ameliorated the neuronal density, decreased microglia activation and leukocyte infiltration. Hypoxia-induced brain blood vessel dilation and loss was also improved by the intervention; however, in the combination group, the results were not promising. Hence, the authors conclude that the combination of hypothermia with stem cells reversed the protective effects of each individual therapy.

Umbilical cord-derived stem cells induce a partially beneficial effect after ischemia

In addition to the therapeutic potential of BM-MSCs that has been studied so far, few studies evaluated the restorative effects of human umbilical cord MSCs on ischemic brain damage. In this regard, McDonald and colleagues (2019) induced cerebral ischemia in rat pups, then human umbilical cord MSCs were administered 24 h post-hypoxic ischemia and data was obtained 3–6 days after the treatment. The cell therapy improved short-term motor strength and control, which was evaluated by the negative geotaxis test. Ischemia decreased brain weight significantly, and the stem cell treatment partially rescued total brain, as well as ipsilateral hemisphere, tissue loss. Hypoxic-ischemic damage also reduced the number of NeuN+ neurons in the hippocampus and somatosensory cortex. Although intranasal delivery of human umbilical cord MSCs increased hippocampal neurons, somatosensory cortex neurons were not affected by the treatment. In addition, the MSC application reduced microglia and astrocyte activation in the hippocampus and somatosensory cortex.

In another investigation, hypoxic–ischemic injury was induced in neonatal rats and 30 min later, eGFP labelled human umbilical cord-derived MSCs were transplanted (Yang et al., 2020). Nissl staining revealed a remarkable neuronal cell loss in the hippocampus and cerebral cortex 3 days after injury, whereas stem cell application increased the number of surviving neurons. Hypoxic–ischemic damage also increased the number of cerebral apoptotic cells, and fewer terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) positive cells were observed in stem cell treated groups. Scattered non-differentiated stem cells were detected in the damaged brain tissue, 3 and 14 days after transplantation. To evaluate effects on long term learning and memory, the Morris water-maze test was performed and the results indicate a long-term neuroprotective effect of transplanted stem cells. These findings underline the therapeutic potential of intranasally delivered umbilical cord-derived MSCs in animal models of hypoxic-ischemic brain injury.

Beneficial potential of neural stem cells (NSCs) in cell therapy after ischemia

Although MSCs are considered the most commonly employed stem cell type for cell-based therapies in general, and the field of cerebral hypoxia/ischemia in particular, the curative potential of NSC is gaining interest. In this regard, Ji et al. (2015) employed intranasal administration of human embryonic NSCs in a neonatal rat hypoxic-ischemic model. One day after ischemia, PKH-26-labeled NSCs were delivered. Distribution of migrated cells was evaluated by in vivo bioluminescence imaging 3 h after the administration, as well as immunofluorescence staining 24 h after the cell delivery. NSCs were found in various brain regions such as hippocampus, corpus callosum, cortex, and olfactory bulb. In addition, 42 days after cell delivery, HuNu positive cells as a marker for human nuclei revealed the survival of NSCs in the injured brain, with the majority being located in the ipsilateral hemisphere. Some of the transplanted NSCs also expressed NeuN or GFAP, indicating their differentiation into the neuronal or glial lineage. In addition, neurobehavioral examinations revealed neurological improvements after cell therapy at various time points. Three days after delivery, NSC application inhibited the over-expression of nuclear factor kappa B, p-IκBα, and interleukin-1β. In addition, 42 days after cell application, reduced brain tissue loss was evident. Therefore, immunomodulatory and neuroprotective effects render intranasally delivered human NSCs a promising treatment strategy.

Altogether, these findings highlight the validity and feasibility of intranasal application of mesenchymal, as well as neural stem cells, after cerebral hypoxia/ischemia. The list of reviewed papers in this section are summarized in Table 1, which include age and sex of experimental animals, procedure of hypoxia/ischemia induction, number of transplanted cells, method of intranasal administration, and evaluated parameters in details.

Table 1:

List of summarized studies that employing intranasal administration of stem cells to treat cerebral hypoxia/ischemia.

Animal model Type of stem cell Type of ischemia Time of administration after ischemia Number of transplanted cells Enzymatic pretreatment/time before cell therapy Volume of administration/vehicle used Consciousness/Body position Evaluated parameters (employed method [time of evaluation after transplantation]) Major outcomes related to cell therapy Ref.
9-day old male and female C57Bl/6 mice Mice BM-MSCss

Mice BM-MSCs		 Permanent unilateral occlusion of common carotid artery followed by 45 min exposure to 10% oxygen in nitrogen 10 days 5 × 105 Two doses of 3 µl hyaluronidase (total 100U) in PBS to each nostril/30 min 12 µl as two doses of 3 µl to each nostril/PBS Awake

  1. Migration (PKH-26 staining [18 h])

  2. Motor function (cylinder rearing test [11d, 18d])

  3. Differentiation (NeuN, S100β and Iba-1 staining [18d])

  4. Infarct size (MAP2 and MBP staining [18d])

  1. Functional performance ↑

  2. Gray and white matter loss ↓

 Van Velthoven et al. (2010)
10-day old Sprague–Dawley rats Rat BM-MSCs MCAO (90min) 3 days 1 × 106 Two doses of 5 µl hyaluronidase in PBS to each nostril/30 min 20 µl as two doses of 5 µl to each nostril/PBS Awake

  1. Motor function (cylinder rearing test [1d, 8d, 15d])

  2. Infarct size (MAP2, MBP and cresyl violet staining [25d])

  3. Proliferation (Ki67 staining [25d])

  1. Functional performance↑

  2. Endogenous proliferation↑

  3. Gray and white matter loss↓

 Van Velthoven et al. (2013)
Postnatal day 9, C57Bl/6J male and female mice Mouse MSCs over-expressing BDNF, EGFL7, persephin and sonic hedgehog Right common carotid artery occlusion followed by exposure to 10% oxygen in nitrogen for 45 min 10 days 5 × 105 Two doses of 3 µl hyaluronidase (total 100U) in PBS to each nostril/30 min 12 µl as two doses of 3 µl to each nostril/PBS Awake

  1. Functional recovery (cylinder rearing test [11d, 18d])

  2. Infarct volume (MAP2 and MBP staining [18d])

  3. Proliferation (BrdU and Ki67 staining [18d])

  1. Functional performance↑

  2. Gray and white matter loss↓

  3. Cell proliferation↑

 Van Velthoven et al. (2014)
10-day old male and female Sprague–Dawley rats Rat MSCs MCAO (90min) 3 days 1 × 106 Two doses of 5 µl hyaluronidase in PBS to each nostril/30 min 20 µl as two doses of 5 µl to each nostril/PBS Awake

  1. Infarct size (MRI [5d, 12d])

  2. Glial activation (GFAP and isolectin-B4 staining [12d])

  3. White matter injury (MBP staining [25d])

  4. Axonal presence (neurofilament H staining [25d])

  5. Motor function (cylinder rearing test)

  1. Functional performance↑

  2. Axonal presence↑

  3. White matter loss↓

 van Velthoven et al. (2017)
Adult male Wistar rats Rat BM-MSCs Subarachnoid hemorrhage by introducing a sharpened suture into the middle cerebral artery 6 days 1.5 × 106 Two doses of 6 µl hyaluronidase (total 100U) in PBS to each nostril/30 min 24 µl as two doses of 6 µl to each nostril/PBS Awake

  1. Behavior (adhesive removal test [15d]; von Frey hairs test [15d]; sucrose preference test [15d])

  2. Infarct size (MAP2, MBP staining [15d])

  3. Glial activation (GFAP and Iba-1 staining [15d])

  4. Neurogenesis (NeuN and tyrosine hydroxylase staining)

  5. Inflammation (CD86 and CD206 staining [15d])

  1. Functional performance↑

  2. Gray and white matter loss↓

  3. Mature neurons↑

  4. Cerebral inflammation↓

  5. Depression like behavior↓

  6. Dopamine synthesis↑

 Nijboer et al. (2018)
Male Wistar rats MSCs Ligating the common carotid arteries at the bifurcation level 10 min 4 × 105 Not defined 50 µl under the mucous membrane of the nasal cavity Anesthetized Nitric oxide production and copper content (electron paramagnetic resonance spectroscopy [1d, 2d]) Copper content↑ Andrianov et al. (2020)
9-day old C57Bl/6 mice Mice MSCs Permanent unilateral occlusion of common carotid artery followed by 45 min exposure to 10% oxygen 3, 10, 17 days 0.25 × 106, 0.5 × 106, 1 × 106 3 µl hyaluronidase (100U) in PBS to each nostril/30 min 12 µl as two doses of 3 µl to each nostril/PBS Not defined

  1. Motor function (cylinder rearing test [11d, 18d, 25d])

  2. Cognitive function (social discrimination test [39d])

  3. Infarct size (MAP2 and MBP staining [25d])

  1. Functional performance↑

  2. Gray and white matter loss↓

  3. Cognitive function↑

 Donega et al. (2013)
9-day old C57Bl/6 mice Mice MSCs Permanent unilateral occlusion of common carotid artery followed by 45 min exposure to 10% oxygen 10 days 1 × 106 3 µl hyaluronidase (100U) in PBS to each nostril/30 min 12 µl as two doses of 3 µl to each nostril/PBS Not defined

  1. Infarct size (MAP2 and MBP staining [18d])

  2. Migration (PKH-26 staining [2 h, 12 h, 1d, 2d, 3d]; MRI and dragon green fluorescence staining [2 h])

  3. Endogenous neurogenesis (DCX staining [1d, 3d, 5d])

  4. Glial activation (GFAP and Iba-1 staining [1d, 5d, 18d])

  1. Gray and white matter loss↓

  2. Endogenous neurogenesis↑

  3. Mature neurons↑

 Donega et al. (2014b)
9-day old C57BL/6 mice Human BM-MSCs Permanent unilateral occlusion of common carotid artery followed by 45 min exposure to 10% oxygen 10 days 1 × 106 or 2 × 106 Two doses of 3 µl hyaluronidase (100U) in PBS to each nostril/30 min 12 µl as two doses of 3 µl to each nostril/PBS Not defined

  1. Migration (PKH-26 staining [1d])

  2. Motor function (cylinder rearing test [11d and 18d])

  3. Infarct size (MAP2 and MBP staining [18d])

  4. Gene expression (qRT-PCR analysis of 84 chemotactic factors)

  5. Glial activation (GFAP and Iba-1 staining [18d])

  1. Functional performance↑

  2. Gray and white matter loss↓

  3. Glial activation↓

 Donega et al. (2014a)
9-day old C57BL/6 mice Mice BM-MSCs Permanent unilateral occlusion of common carotid artery followed by 45 min exposure to 10% oxygen 10 days 0.5 × 106 Two doses of 3 µl hyaluronidase (100U) in PBS to each nostril/30 min 12 µl as two doses of 3 µl to each nostril/PBS Not defined

  1. Motor function (cylinder rearing test [18d, 170d, 260d])

  2. Cognitive function (novel object recognition test [110d, 410d])

  3. Infarct size (MAP2 and MBP staining [25d, 410d])

  1. Functional performance↑

  2. Cognitive function↑

  3. Gray and white matter loss↓

 Donega et al. (2015)
Adult male C57BL/6 mice Normoxic and hypoxic preconditioned (0.1–0.3% oxygen for 24 h) rat BM-MSCs Permanent unilateral occlusion of middle cerebral artery accompanied by 7-min bilateral ligation of common carotid arteries 1 day 1 × 106 100U hyaluronidase in PBS/30 min 100 µl/PBS Anesthetized/head in the upright position

  1. Migration (Hoescht staining [1.5 h])

  2. Infarct size (TTC staining [3d])

  3. Apoptosis (TUNEL staining [3d])

  4. Motor function (Adhesive- removal test [3d])

  1. Infarct size↓

  2. Apoptosis↓

  3. Functional performance↑

 Wei et al. (2013)
7-day old male Wistar rats Normoxic and hypoxic preconditioned (0.1–0.3% oxygen for 24 h) rat BM-MSCs Permanent MCAO 6 h and 3 days 1 × 106 100U hyaluronidase in PBS/30 min 100 µl/saline Awake

  1. Infarct size (TTC staining [3d])

  2. Blood-brain barrier integrity (Evans Blue administration [3d])

  3. Neurogenesis (NeuN staining [14d])

  4. Angiogenesis (glucose transporter-1 staining [14d])

  5. Behavior (adhesive removal test [14d]; olfactory function test [14d]; social interaction test [14d]; home cage behavioral tests)

 Wei et al. (2015)
Adult male C57BL/6 mice Normoxic and hypoxic preconditioned (0.1–0.3% oxygen for 24 h) rat BM-MSCs Intracerebral hemorrhage (injection of collagenase type IV into striatum) 3 and 7 days 1 × 106 100U hyaluronidase in PBS/30 min 100 µl/saline Anesthetized/head in the upright position

  1. Migration (Hoescht and propidium iodide staining [6 h after the first administration])

  2. Motor function (modified neurological severity score [7d, 14d]; rotarod test [7d, 14d]; adhesive removal test [7d, 14d]; open field test [7d])

  3. Gene expression (Western blot analysis of VEGF, BDNF, GDNF [7d])

  4. Neurogenesis (NeuN and DCX staining [7d])

  5. Brain atrophy (cresyl violet staining [14d])

  1. Functional performance↑

  2. Endogenous neurogenesis↑

  3. Brain tissue loss↓

 Sun et al. (2015)
Adult male mice Hypoxic preconditioned (0.1–0.3% oxygen for 24 h) rat BM-MSCs Permanent unilateral occlusion of middle cerebral artery accompanied by 10 min bilateral ligation of common carotid arteries 3, 4, 5 and 6 days 1 × 106 5 µl hyaluronidase (10 mg/ml) in PBS to each nostril/30 min 100 μl/cell culture media Not defined

  1. Migration (Hoescht and propidium iodide staining [6h and 24 h after the first administration])

  2. Motor function (adhesive removal test [1d and 8d after final administration])

  3. Neurogenesis (NeuN staining [15d after final administration])

  4. Angiogenesis (glucose transporter-1 staining [15d after final administration])

  5. Local cerebral blood flow (laser Doppler scanning [15d after final administration

  1. Neurogenesis↑

  2. Angiogenesis↑

  3. Local cerebral blood flow↑

  4. Functional performance↑

 Chau et al. (2018)
9-day old male and female C57BL/6J mice Mice BM-MSCs Permanent unilateral occlusion of common carotid artery followed by 60 min exposure to 10% oxygen 3 days 1 × 106 Not defined Not defined Not defined

  1. Migration (GFP labeling [14–16 h])

  2. Behavior (elevated plus maze [32d];open field test [32d]; novel object recognition test [32d])

  3. Histology (cresyl violet staining [4d])

  4. Protein expression (Western blot analysis of MAP2, MBP, Iba-1 and vascular cell adhesion molecule-1 [4d]; Immunohistochemical staining of NeuN, Oligo2, CD45 and CD31 [4d])

  5. Blood cytokine level (Luminex assay of TNFα, IL-1β, IL-6, IL-12, IL-10 [1d])

  6. Blood growth factor level (Luminex and ELISA analysis of VEGF, BDNF, EGF and IGF [1d])

  1. Functional performance↑

  2. Cognitive function↑

  3. Myelination↑

  4. Microglia activation↓

  5. Brain tissue loss

 Herz et al. (2018)
10-day old male and female Sprague–Dawley rats Human umbilical cord MSCs Permanent unilateral occlusion of common carotid artery followed by 90 min exposure to 10% oxygen 1 day 2 × 105 Two doses of 3 µl hyaluronidase (100U) in PBS to each nostril/30 min 12 µl as two doses of 3 µl to each nostril/PBS Not defined

  1. Motor function (negative geotaxis test [3d])

  2. Histology (cresyl violet and NeuN staining [6d])

  3. Glial activation (Iba-1 and GFAP staining [6d])

  4. Peripheral cytokine level (ELISA analysis of IL-1, IL-18 and monocyte chemoattractant protein-1 [6d])

  5. Gene expression (qRT-PCR analysis of BDNF, VEGF, IGF-1, claudin-5, occluding and GDNF)

  1. Functional performance↑

  2. Brain tissue loss↓

  3. Glial activation↓

 McDonald et al. (2019)
7-day old male and female Sprague–Dawley rats Human umbilical cord-derived MSCs Permanent unilateral occlusion of common carotid artery followed by 2.5 h exposure to 8% oxygen in nitrogen 30 min 1 × 106 3 µl hyaluronidase (100U) in PBS to each nostril/30 min 12 µl as two doses of 3 µl to each nostril/PBS Not defined

  1. Apoptosis (TUNEL staining [3d])

  2. Gene expression (Western blot analysis of ERK, p-ERK, JNK, p-JNK, p38 and p-p38)

  3. Histology (Nissl staining [3d])

  4. Differentiation (GFAP and neuron-specific enolase staining [3d and 14d])

  5. Learning and memory (Morris-Water maze test [29d])

  1. Neuronal loss↓

  2. Apoptosis↓

  3. Learning and memory↑

 Yang et al., (2020)
7-day old Sprague Dawley rats Human embryonic neural stem cells Permanent unilateral occlusion of common carotid artery followed by 120 min exposure to 7.8% oxygen in nitrogen 1 day 3 × 105 Not defined 12 µl as two doses of 3 µl to each nostril/saline Not defined

  1. Migration (in vivo bioluminescence imaging [3 h]; PKH-26 staining [24 h])

  2. Behavior (righting reflex and gait test [1d, 3d, 5d, 7d]; grid walking test [7d, 14d]; social choice test [28d]; Morris water maze [35d])

  3. Protein expression (Western blot analysis of IL-1β, NK-κB and IκB-α [3d])

  4. Histology (cresyl violet and MBP staining [42d])

  5. Differentiatio (NeuN and GFAP staining [42d])

  1. Brain tissue and white matter injury↓

  2. Neurological performance↑

 Ji et al. (2015)

Possible brain entry-routes of stem cells following intranasal application

Besides the intranasal application of MSCs and NSCs which is discussed here, the intranasal pathway has been used to deliver other cell types, such as glioma cells (Danielyan et al. 2009), macrophages, and microglia (Danielyan et al. 2014) to the brain. Although reports on this method of cell delivery are mainly centered around the therapeutic consequences of the cell therapy, few studies focused on the pathways by which intranasally applied cells enter the brain. As the first published article in this context, Danielyan and colleagues in 2009 reported that intranasally delivered cells to C57BL/6 mice or 10-day old Wistar rats can penetrate the brain. They employed rat BM-MSCs and human PhiYellow-T406 glioma cells, pre-labelled with carboxyfluorescein diacetate or Hoechst 33,342. Those cells were intranasally administered in conscious animals. Some animals received 100U hyaluronidase, which perforates the blood brain barrier and enables an effective penetration. Migratory routes of transplanted cells were evaluated 1 h after cell administration. The administered cells could be visualized throughout the layers of the olfactory bulb, thalamus, hippocampus, and cerebral cortex. Also, hyaluronidase application facilitates the invasion by loosening the barrier function of the nasopharyngeal mucosa and enhanced the delivery of cells to the olfactory bulb. The research team proposed that after intranasal administration, cells migrate from the nasal mucosa through the cribriform plate, along the olfactory neural pathway into the brain (parenchymal route). Also, cells may enter the cerebrospinal fluid (CSF route), moving along the surface of the cortex and penetrate into the brain parenchyma (Danielyan et al. 2009).

In another study, Galeano et al. (2018) investigated the route by which stem cells cross the cribriform plate from the nasal cavity. They intranasally administered human umbilical cord MSCs, pre-labeled with quantum dots in immunodeficient anesthetized mice, and monitored the migration of the cells 2 h later. Stem cells were detected beneath the olfactory epithelium, passing the cribriform plate near to, but separate from the nerve tracts (fila olfactoria). The authors proposed that following the intranasal transplantation, stem cells pass the olfactory epithelium and periosteum, cross the cribriform plate near the nerve tracts and enter the subarachnoid space.

In hypoxic–ischemic brain injuries, region-specific cerebral damage may determine the final destinations of applied stem cells. It has been shown that intranasally transplanted cells respond to endogenous factors released from the damaged sites (Danielyan et al. 2011). To unravel the pathway of transplanted stem cells in the hypoxic–ischemic brain, several cell-tracking techniques have been used. Apparently, the olfactory bulb, ipsilateral cortex, and hippocampus are the main destinations of stem cells, following intranasal administration in hypoxic–ischemic animals (see Box 2).

Box 2:

Major destinations of stem cells following intranasal application in the hypoxic–ischemic animals.
Type of stem cell Cell tracking agent Detection time after administration Ipsilateral Contralateral Ref.
Mice BM-MSCs PKH26 18d

  1. Olfactory bulb

  2. Subventricular zone

  3. Hippocampus

  1. Olfactory bulb

  2. Subventricular zone

 Van Velthoven et al. (2010)
Mice MSCs PKH26 1d

  1. MSCs administered 3 days after HI, were detected in the hippocampus

  2. MSCs administered 10 days after HI, were detected in the cortical layers 5 and 6, and dorsal and epithalamic regions

  3. MSCs administered 17 days after HI, were detected surrounding the lesion site in low numbers

  1. None

 Donega et al. (2013)
Normoxic cultured and hypoxic preconditioned rat BM-MSCs Hoechst 33342 1.5 h

  1. Olfactory bulb

  2. Cortex around the ischemic region

  3. Amygdala

  1. Cortex

  2. Upper midline or lower midline brain region

 Wei et al. (2013)
Mice MSCs

  1. PKH26

  2. Micron-sized superpara-magnetic iron-oxide co-labeled with the fluorescent label Dragon Green

 2, 6, 12, 24, 48, 72 h

  1. Lesion site (2 h after administration and peaked at 12 h)

  1. None

 Donega et al. (2014b)
Human BM-MSCs PKH26 1d

  1. Sensorimotor

  2. Epithalamic regions

  1. None

 Donega et al. (2014a)
Human embryonic NSCs PKH26 1d

  1. Olfactory bulb

  2. Cerebral cortex

  3. Corpus callosum

  4. Hippocampus

  1. Not define

 Ji et al. (2015)
Hypoxic preconditioned rat BM-MSCs Hoechst 33342 6 h

  1. Olfactory bulb

  2. Cortex

  3. Peri-vascular spaces

  4. Peri-hematoma regions

  1. Not define

 Sun et al. (2015)
Hypoxic preconditioned rat BM-MSCs Hoechst 33342 6 and 24 h

  1. Peri-infarct region

  1. None

 Chau et al. (2018)
Mice BM-MSCs GFP 14–16 h

  1. Injured site

  1. None

 Herz et al. (2018)

Intranasal administration of stem cell derivatives in the treatment of cerebral ischemia

Besides the therapeutic effects of transplanted stem cells in animal models of ischemia, a growing body of evidence indicates that some of the observed restorative outcomes might relate to paracrine effects, rather than engraftment. This paracrine modulatory impact derives from the secretome or conditioned medium (CM) which comprises a wide range of growth factors, chemokines, cytokines, angiogenic factors, and exosomes (Jarrige et al. 2021; Xia et al. 2019). Trophic factors, originating from stem cells, represent the most effective agent in cell therapy. These factors potently support survival, growth, and development of neuronal and glial cells. Therefore, using secreted factors by stem cells might be as effective as stem cell transplantation (Pawitan 2014). Although systemic delivery of trophic factors is preferable, several drawbacks limit their application. These drawbacks comprise sequestration by peripheral tissues and various components of the blood, and various clearance processes (rapid enzymatic inactivation), which result in short half-life. Furthermore, the blood–cerebrospinal fluid barrier and the blood–brain barrier severely reduce the central availability of delivered trophic factors (Thorne and Frey 2001).

Using stem cell-derived secretome

In order to overcome the difficulties of systemic delivery, Takanori Inoue (2013) employed the intranasal route to bypass the blood-brain barrier and decrease the systemic clearance of trophic factors. In this study, focal cerebral ischemia was induced by permanent MCAO and human exfoliated deciduous tooth (SHED)-CM as well as BM-MSC-CM was intranasally administered every day from days 3–15 post ischemia. Motor disability tests 3–15 days after the treatment revealed SHED-CM improved the motor disability, compared to BM-MSC-CM as well as control groups. Both CM administered groups showed a reduction in infarct size. Immunostaining against neurogenesis and vasculogenesis markers revealed more DCX, neurofilaments, NeuN and endothelial cell antigen positive cells in the peri-infarct area of SHED-CM treated group compared to control. Migration of neuronal progenitor cells from the subventricular zone to the peri-infarct area was also enhanced by the SHED-CM treatment.

In another study, Zhao et al. (2015) investigated whether intranasally delivered human umbilical cord MSC-CM is able to improve neurological functions and vascular remodeling in a rat model of cerebral ischemia. The MSC-CM was administered daily for 14 days, starting one day after ischemia. Footfault and modified neurological severity score tests, 7 and 14 days after MCAO, showed that intranasal administration of MSCs-CM improved the functional recovery; however, the treatment was ineffective in reducing the infarct volume. MSC-CM was also decreased blood brain barrier leakage and vascular damage. In the ischemic brain, immunostainings against ZO1, occludin, and desmin, revealed a significant increase in the functional integrity of the blood brain barrier. Assessment of angiogenesis and angiogenic factor expression in the infarct area showed that CM treatment could increase the expression of von Willebrand factor, angiopoietin-1 and angiopoietin-1 receptor.

Using stem cell-derived extracellular vesicles

Using stem cell-derived extracellular vesicles, including exosomes and microvesicles, is considered as a novel frontier in regenerative medicine. It has been reported that extracellular vesicles are involved in a variety of vital processes, such as antigen presentation, angiogenesis, inflammation, homeostatic maintenance, and immune responses (Keshtkar et al. 2018). Hence, Sisa et al. (2019) proposed that MSC-derived extracellular vesicles could have neuroprotective effects in the neonatal hypoxic-ischemic insult. Extracellular vesicles obtained from human BM-MSCs were administered immediately after the hypoxic exposure, and therapeutic effects were assessed 2 days later. Immunostainings against αMβ2 integrin revealed a significant reduction in microglia activation in several ipsilateral brain regions, including cortex, hippocampus, and striatum. This treatment also reduced cell death in the ipsilateral cortex and external capsule. Nissl staining also showed reduction of tissue loss in the pyriform cortex, thalamus, and external capsule. Furthermore, administration of MSC-derived extracellular vesicles led to improvements in behavioral outcomes, as evaluated by negative geotaxis test.

Collectively, despite all challenges and hurdles of direct stem cell transplantation in treating neurological diseases, using stem cell derivatives and/or the secretome as an alternative cell-free strategy to deliver neuroprotective substances, appears to be an effective therapeutic approach. The list of reviewed papers in this category is summarized in Table 2, which includes age and sex of experimental animals, procedure of hypoxia/ischemia induction, type of stem cell derivative, method of intranasal administration, and evaluated parameters in details. With the establishment of best practice guidelines for the intranasal application of cell derivates and the secretome, this approach might achieve a routine application in the clinical setting in the near future.

Table 2:

List of summarized studies that employing intranasal administration of stem cell derivatives to treat cerebral hypoxia/ischemia.

Animal model Stem cell derivate Type of ischemia Time of administration after ischemia Volume of administration/vehicle used Consciousness/Body position Evaluated parameters (employed method [time of evaluation after transplantation]) Major outcomes related to cell therapy Ref.
Adult male Sprague–Dawley rats Human exfoliated deciduous tooth as well as BM-MSCs derived conditioned medium Permanent MCAO Every day from days 3–15 100 µl via the olfactory pathway using a Hamilton microsyringe. 10 µl each time in alternated nostril/DMEM Anesthetized/placed on backs with necks elevated by a 4 × 4 cm roll of gauze

  1. Motor function (standardized motor disability scale [1d, 3d, 6d, 9d, 12d and 15d)]

  2. Infarct size (hematoxylin and eosin staining [16d])

  3. Neurogenesis (NeuN, neurofilament

  4. H and DCX staining [16d])

  5. Angiogenesis (RECA1 staining [16d])

  1. Functional performance ↑

  2. Infarct size ↓

  3. Neurogenesis ↑

  4. Angiogenesis ↑

 Inoue et al. (2013)
Adult male rats Human umbilical cord MSCs-conditioned medium Transient MCAO (120 min) Every day from day 1–14 1 ml/kg/day/DMEM-F12 Anesthetized/placed in a supine position, and the ventral surface of the head and neck is maintained horizontal using a small role of gauze under the dorsal neck

  1. Motor functional (Footfault and modified neurological severity score tests [7d and 14d])

  2. Infarct size (hematoxylin and eosin staining [14d])

  3. Blood-brain barrier integrity (Evans blue administration [5d]; ZO1, occludin and desmin staining [14d])

  4. Angiogenesis (vWF staining [14d])

  5. Protein expression (Angiopoietin-1, Angiopoietin-2 and angiopoietin receptor [14d])

  1. Functional performance ↑

  2. Blood brain barrier integrity ↑

  3. Angiogenesis ↑

 Zhao et al. (2015)
9-day old male and female C57/BI6 mice Extracellular vesicle obtained from human BM-MSCs Permanent unilateral occlusion of common carotid artery followed by 60 min exposure to 10% oxygen in nitrogen Immediately following hypoxic exposure 6 µl (1.25 × 109 particles)/PBS Not defined

  1. Microglial activation (αMβ2 staining [2d])

  2. Apoptosis (TUNEL staining [2d])

  3. Infarct size (Nissl staining [2d])

  4. Behavior (negative geotaxis test [2d])

  1. Glial activation ↓

  2. Apoptosis ↓

  3. Brain tissue loss ↓

  4. Functional performance ↑

 Sisa et al. (2019)

Possible brain entry-mechanisms of stem cell derivatives following intranasal application

Up until now, several pathways, including paracellular, intracellular, perineural, and perivascular routes have been proposed for the brain delivery following intranasal application of biological compounds. In the nasal cavity, owing to persistent renewal of the basal cells and neurons, tight junctions are permeable to macromolecules, which is the basis for paracellular transport. Through this route, substances could reach the brain in a matter of minutes. The primary mechanism for the intracellular brain delivery could be receptor-mediated endocytosis by the olfactory receptor neurons, as well as neuroendocytosis by the maxillary and ophthalmic branches of the trigeminal nerve. Using those pathways, drugs could enter the brain in a few hours.

The space between neurons and myelin sheaths, called perineural space, is another possible pathway for the transportation of substances from the respiratory epithelium along the trigeminal nerve or from the olfactory epithelium along the olfactory nerve. Through this pathway, compounds could reach the olfactory bulb or brain stem. After reaching the brain, perivascular pathways, which are formed between the endothelium of vessels and the surrounding glial cells, play a prominent role in the transport of the compounds to deeper areas of the brain. Therefore, all of these pathways could potentially be used for the brain-entry of stem cell derivatives (Lochhead and Davis 2019; Lochhead and Thorne 2012; Zhang et al. 2021).

Challenges and perspectives

Up until now, 18 articles have reported the curative effects of intranasally administered stem cells in mouse and rat models of cerebral ischemia/hypoxia. Almost all of these studies employed mesenchymal stem cells, predominantly harvested from bone marrow. Therefore, detailed investigations in different species are required to reveal the beneficial potential of the nasal pathway as an unspecific route for all types of stem cells. Moreover, in spite of the monumental efforts to explore the pathway of stem cells from the nasal cavity to the olfactory bulb, there are still unresolved issues regarding the mechanisms that stem cells use to reach the injured area. Furthermore, undesirable side-effects following intranasal administration of stem cells represent another challenge that should be addressed in the near future. Since the vast majority of stem cells do not reach the brain through the nasal pathway (Danielyan et al. 2011; Danielyan et al. 2014), off-target stem cells might cause considerable side-effects. Hence, new strategies to enhance the delivery of stem cells on-target are also inevitable. Lastly, it has been reported that aged animals with higher body weights required higher doses of intranasally administered radiolabeled drug than lower body weights younger animals to achieve the same drug concentration throughout the brain (Krishnan et al. 2017). Then, age, sex, and body weight of both donors and recipients of stem cells should also be considered for optimizing the efficacy of this approach.

Conclusions

During the last decade, the curative potential of intranasally delivered mesenchymal stem cells in animal models of cerebral hypoxia/ischemia has been revealed (Figure 3). This administration route, due to inherent advantages of noninvasiveness and practicability, is considered as an effective path for cell-based therapies in nervous system conditions. This entry route also facilitates the entrance of stem cell derivatives, which represents an alternative cell-free approach to deliver neuroprotective components. However, application of this administration route for delivering stem cells and/or therapeutic substances to the damaged sites requires further optimization to translate the findings of animal experiments to clinical trials.

Figure 3: Intranasal administration of stem cells or their derivatives can improve devastating conditions following cerebral hypoxia/ischemia, i.e. enhancing behavioral performances, decreasing brain insult, increasing endogenous neurogenesis, and/or modulate inflammation.
Figure 3:

Intranasal administration of stem cells or their derivatives can improve devastating conditions following cerebral hypoxia/ischemia, i.e. enhancing behavioral performances, decreasing brain insult, increasing endogenous neurogenesis, and/or modulate inflammation.

Corresponding author: Sareh Pandamooz, Stem Cells Technology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran, E-mail: ; and Afshin Borhani-Haghighi, Clinical Neurology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran, E-mail:

  1. Author contribution: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.

  2. Research funding: This study was financially supported by Shiraz University of Medical Sciences (Grant number: 23290).

  3. Conflict of interest statement: The authors declare no conflicts of interest regarding this article.

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Received: 2021-12-02
Revised: 2022-01-11
Accepted: 2022-01-13
Published Online: 2022-02-07
Published in Print: 2022-08-26

© 2022 Walter de Gruyter GmbH, Berlin/Boston

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Reviews in the Neurosciences
Reviews in the Neurosciences
Volume 33 Issue 6
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