Abstract
Translation of neuroprotective treatment effects from experimental animal models to patients with cerebral ischemia has been challenging. Since pathophysiological processes may vary across species, an experimental model to clarify human-specific neuronal pathomechanisms may help. We conducted a sco** review of the literature on human neuronal in vitro models that have been used to study neuronal responses to ischemia or hypoxia, the parts of the pathophysiological cascade that have been investigated in those models, and evidence on effects of interventions. We included 147 studies on four different human neuronal models. The majority of the studies (132/147) was conducted in SH-SY5Y cells, which is a cancerous cell line derived from a single neuroblastoma patient. Of these, 119/132 used undifferentiated SH-SY5Y cells, that lack many neuronal characteristics. Two studies used healthy human induced pluripotent stem cell derived neuronal networks. Most studies used microscopic measures and established hypoxia induced cell death, oxidative stress, or inflammation. Only one study investigated the effect of hypoxia on neuronal network functionality using micro-electrode arrays. Treatment targets included oxidative stress, inflammation, cell death, and neuronal network stimulation. We discuss (dis)advantages of the various model systems and propose future perspectives for research into human neuronal responses to ischemia or hypoxia.
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Introduction
It has been challenging to translate neuroprotective treatment effects from experimental animal models to patients with cerebral ischemia. While numerous rodent studies have shown efficacy of divergent “neuroprotective” treatment strategies, in clinical trials these treatments have failed to improve functional outcomes (Ginsberg 2008). Failure of treatments in clinical trials have been discussed extensively, with possible explanations ranging from methodological weaknesses of animal studies to differences in treatment protocols and from infarct sizes to patient selection (Ginsberg 2008). However, genetic differences between rodents and humans that may translate to neurophysiological differences are relatively undiscussed. While rodents and humans share part of their DNA, various genes and proteins are expressed differently (Mestas and Hughes 2004). Therefore, cellular responses to cerebral ischemia or hypoxia may differ between rodents and humans. Furthermore, mechanisms of action or pathways that are targeted with neuroprotective treatments can vary (Ginsberg 2008). Consequently, rodent models might not be the optimal starting point to investigate the effect of cerebral ischemia on neuronal functionality and treatment strategies.
Various experimental studies addressed responses to ischemia or hypoxia in human-derived cell models. These consist of non-neuronal and neuronal models. The most used human in vitro model to study neuronal responses to ischemia or hypoxia is the neuroblastoma-derived SH-SY5Y cell model (Liu et al. 2018). This cell model is based on a cancerous cell line with the corresponding genetic characteristics, which may affect responses to ischemia or hypoxia and treatment strategies (Biedler et al. 1973, 1978). SH-SY5Y cells can, in principle, be differentiated into neuron-like cells upon stimulation (Shipley et al. 2016). However, the majority of research with SH-SY5Y cell models is conducted in undifferentiated SH-SY5Y cells. Only a small proportion of the research made use of protocols to differentiate SH-SY5Y cells into neuron-like cells. Furthermore, most studies focused on cell viability, neglecting neuronal functionality (Liu et al. 2018).
The recent advancement of human induced pluripotent stem cell (hiPSC) technology has created new opportunities to establish human neuronal in vitro models and investigate responses to ischemia or hypoxia. HiPSCs can be derived from healthy donors or patients, capturing person-specific genetic characteristics, and differentiated into neurons to generate neuronal networks (Frega et al. 2017; Mossink et al. 2021a). This allows investigation of the effect of, for example, a genetic mutation on neuronal functionality (Mossink et al. 2021b).
With this sco** review of the literature, we aim to provide an overview of (1) human neuronal in vitro models that have been used to study neuronal responses to ischemia or hypoxia, including characteristics of the various models, (2) parts of the ischemic pathophysiological cascade that have been investigated and (3) treatment targets that have been established in those human neuronal models. We will use the results as a starting point to discuss advantages and disadvantages of the various model systems, highlight current knowledge gaps, and propose possible future perspectives for research into human neuronal responses to ischemia or hypoxia.
Methods
For this sco** review, we followed the PRISMA guidelines with regard to literature search, data collection, presentation of study characteristics and results, and discussion. However, we did not include a quality appraisal or risk of bias analysis.
To review investigated human neuronal in vitro models of cerebral ischemia or hypoxia, we applied a search in PubMed and SCOPUS databases until November 2021. We conducted the literature research with several combinations of key words and MeSH terms. We searched the literature with general terms for “human neuronal in vitro models” and the following specific search terms: “NT2-N”, “SK-N-SH”, “SH-SY5Y” or “hiPSC-derived neurons” (Liu et al. 2018). For selection of disease we used the MeSH terms “ischemic stroke” or “cerebral ischemia” and search terms “hypoxia” or “oxygen–glucose deprivation (OGD)” in combination with the different human neuronal in vitro models. One reviewer (EV) screened articles for eligibility based on the abstracts and methods. Review articles were used to screen reference lists. We only included studies with modeling of cerebral ischemia by oxygen–glucose deprivation or hypoxia. Studies were excluded when chemical simulation of ischemia was investigated. Flow chart is provided in the supplementary materials. For analyses of effects of potential neuroprotective treatments, we included studies on treatments that were applied during or after ischemia/hypoxia, and excluded studies that investigated the effect of pre-treatment (i.e. a treatment started before initiation of hypoxia or ischemia). We extracted the cell type, the level of differentiation towards neurons (if applicable), the way in which ischemia or hypoxia was modelled (including duration and recovery period), treatment strategy (if applicable), outcome measures, and results from the included studies. All results of the sco** review are presented in a descriptive way.
Results
We included 147 papers on four different human neuronal in vitro models of cerebral ischemia/hypoxia.
Model Characteristics
Model characteristics are summarized Fig. 1 and Table 1. Of the 147 included papers, 145 were on cell models derived from unhealthy donors (neuroblastoma SK-N-SH (n = 11) and SH-SY5Y (n = 132)) or carcinoma (differentiated NT2-N (n = 2)). SK-N-SH is a cell model directly derived from a neuroblastoma cell line. SH-SY5Y cell model is based on a subclone derived from the SK-N-SH cell line. These can both be differentiated into neuron-like cells by stimulation with retinoic acid (RA). However, the majority of studies on neuronal response to ischemia/hypoxia is conducted in undifferentiated cell models (SK-N-SH undifferentiated n = 9 and differentiated n = 2; SH-SY5Y undifferentiated n = 119, and differentiated n = 13). Two studies were conducted on neurons differentiated from hiPSCs of healthy donors (Juntunen et al. 2020; Pires Monteiro et al. 2021). Examples of cultured undifferentiated SH-SY5Y and hiPSC-derived neuronal networks are presented in Fig. 2, showing that undifferentiated SH-SY5Y cells have short truncated processes and do not express the neuronal markers microtubule-associated protein 2 (MAP2) and Synapsin 1/2 positive synaptic puncta. While hiPSC-derived neuronal networks show long neurites and express MAP2 and Synapsin 1/2 positive synaptic puncta.
Pie chart representing the distribution of human neuronal in vitro models used in experimental cerebral ischemia studies. The majority of the research is conducted in SH-SY5Y cell models. hiPSCs human induced pluripotent stem cells. Differentiated NT2-N = teratocarcinoma-derived Ntera2/D1 neuron-like cells
Representative images of undifferentiated SH-SY5Y cells and hiPSC-derived neuronal networks. Bright field images of undifferentiated SH-SY5Y cells (A) and hiPSC-derived neuronal networks (B) (× 4 magnification) (scalebar = 100 μm). Immunofluorescent images of undifferentiated SH-SY5Y cells (C) and hiPSC-derived neuronal networks (D) (× 60 magnification) (scalebar = 30 μm). Undifferentiated SH-SY5Y cells have short truncated processes and do not express the neuronal markers MAP2 and Synapsin 1/2 positive puncta. HiPSC-derived neuronal networks have extended neurites and express neuronal markers MAP2 and Synapsin 1/2 positive puncta. Blue = DAPI (nuclei), Green = micro-tubule associated protein 2 (MAP2), Red = Synapsin 1/2
Cerebral ischemia was modeled by oxygen–glucose deprivation (OGD; n = 142) or hypoxia (n = 5). All studies used immunocytochemical techniques to investigate cell survival, protein expression linked with cell death or inflammation, or factors related to oxidative stress. Neuronal functionality was assessed in only one study in which electrophysiological measurements were performed with micro-electrode arrays (MEAs) (Pires Monteiro et al. 2021).
Effects of Ischemia or Hypoxia on Human Cell Models
The pathophysiological cascade that follows upon cerebral ischemia/hypoxia is extensive and complex. In SH-SY5Y, SK-N-SH, NT2-N and hiPSC-derived cell models, several steps of this pathophysiological cascade have been investigated. These steps are summarized in Table 2 and explained below.
SH-SY5Y and SK-N-SH Cell Models
Calcium Homeostasis Dysregulation
Ischemia induced membrane depolarization leads to opening of calcium channels, ultimately resulting in an increase in intracellular Ca2+. With insufficient ATP production, Ca2+ extruders (e.g. Na+/Ca2+ exchanger) stop working, causing a pathological increase of the intracellular and mitochondrial Ca2+ concentration. The Na+/Ca2+ exchanger 1 (NCX1) has been investigated in one study using undifferentiated SH-SY5Y cells. The results showed that NCX1 was repressed during OGD by the RE1-silencing transcription factor (REST) (Formisano et al. 2013). In undifferentiated SK-N-SH cells increased levels of intracellular calcium was found during OGD (n = 1) (Lehane et al. 2013).
Oxidative/Nitrosative Stress
Oxidative stress results from imbalance between pro- and anti-oxidants, that in turn results in excessive formation of reactive oxygen species (ROS). Various negative effects of ROS on DNA, proteins or lipids have been established in SH-SY5Y and SK-N-SH cell models of cerebral ischemia.
The first ROS that is produced during ischemia/hypoxia is superoxide anion (O2−), which is the precursor of most other ROS. One study in undifferentiated SH-SY5Y cells found an increase in O2− (Marmol et al. 2021). Superoxide dismutases (SODs) convert superoxide to hydrogen peroxide (H2O2), which can be removed by glutathione peroxidase (GPx). GPx was decreased after ischemia/hypoxia in undifferentiated SH-SY5Y and SK-N-SH cells (Wang et al. 2020; Yin et al. 2021).
Anti-inflammatory cytokines can be induced by V-maf musculoaponeurotic fibrosarcoma oncogene homolog B. This transcriptional activator was found to be decreased after ischemia/hypoxia in one study using undifferentiated SH-SY5Y cells (Zhang et al. 2020a). The anti-inflammatory factor interleukin-10 was reduced after ischemia/hypoxia in one study with undifferentiated SH-SY5Y cells (Dong et al. 2021).
Tumor necrosis factor (TNF) is released by cells under stress and stimulates the immune response. The expression of TNF is only investigated in undifferentiated SH-SY5Y cells during and after ischemia/hypoxia (n = 15). The results show an increase in TNF expression induced by ischemia/hypoxia, which suggests increased inflammatory response (Chai et al. 2020; Dong et al. 2021; Hao et al. 2015; Huang et al. 2021; Landgraf et al. 2020; Li et al. 2019a; Li and Ma 2020; Liu et al. 2021; Meng et al. 2021; Shi et al. 2020, 2021; Yang et al. 2014; Zhao and Wang 2020; Zhi et al. 2020; Juntunen et al. 2020; Pan et al. 2020; Sriwastva et al. 2020; Xu et al. 2020a; Zhang et al. 2019b). Four studies found increased cell death rates after ischemia/hypoxia in differentiated and undifferentiated SK-N-SH cell model of cerebral ischemia (Ingrassia et al. 2012; ** et al. 2020; Yanagita et al. 2005; Yin et al. 2021).
Apoptosis is a possible pathway to cell death. Various pro- and anti-apoptotic factors have been investigated. Apoptosis can be triggered through an intrinsic pathway which is regulated by B-cell lymphoma 2 (BCL-2) protein family and is activated by internal signals. The expression of anti-apoptotic BCL-2 has been investigated in 25 studies using undifferentiated SH-SY5Y cells (Chang et al. 2017; Dong et al. 2019; Feng et al. 2021; Gao et al. 2019) and undifferentiated SH-SY5Y (n = 13) cell models of cerebral ischemia (He et al. 2014; Liu et al. 2019; Zhang et al. 2016c, 2019b; Zhao et al. 2013; Zhi et al. 2020; Lin et al. 2011; Sriwastva et al. 2020; Zeng et al. 2019; Zhang et al. 2019b), two in undifferentiated SK-N-SH cell models (Lehane et al. 2013; Soh et al. 2007), and one in hiPSC-derived neurons (Pires Monteiro et al. 2021) included treatment with a pharmacological compound. Treatment targets were neuronal activity, oxidative/nitrosative stress, inflammation, autophagy or cell death.
One study with hiPSC-derived neurons investigated the effect of neuronal network activation by the mildly excitatory hormone/neurotransmitter ghrelin (Pires Monteiro et al. 2021). The results showed partial preservation of neuronal network functioning after hypoxia (Pires Monteiro et al. 2021).
Oxidative/nitrosative stress was investigated as a treatment target in twenty studies using undifferentiated SH-SY5Y cells and one study using differentiated SH-SY5Y cells. These studies showed that various chemical compounds could reduce oxidative stress by reducing the formation of ROS or increasing anti-oxidant defense mechanisms, which led to increased cell survival (Agudo-Lopez et al. 2010; Chen et al. 2021b; Dong et al. 2021; Gao et al. 2017; Hong et al. 2017; Hsieh et al. 2021; Jimenez-Almarza et al. 2019; Landgraf et al. 2020; Li et al. 2020a; Liu et al. 1973). The wide spread use of SH-SY5Y cells indicates that pathomechanisms of human neuronal responses to ischemia or hypoxia have mainly been investigated in a cancerous cell line (Liu et al. 2018). It is known that the genetic background of a cell line affects functionality, as well as responses to interventions (Mossink et al. 2021b). Thus, a cell line with cancerous characteristics will probably respond to ischemia or hypoxia differently than healthy (neuronal) cells.
Moreover, although SH-SY5Y cells can be differentiated into neuron-like cells upon stimulation with RA (Khwanraj et al. 2015; Korecka et al. 2013; Shipley et al. 2016), the majority of studies has been conducted in undifferentiated SH-SY5Y cells. Undifferentiated and differentiated SH-SY5Y cells differ in morphology and function. Undifferentiated SH-SY5Y cells have large cell bodies with short truncated processes (Kovalevich and Langford 2013). These undifferentiated cells express immature neuronal markers, such as SOX2, and lack the expression of mature neuronal markers, such as synapsin1/2 positive puncta. Differentiated SH-SY5Y cells form and extent neuritic processes (Kovalevich and Langford 2013) and show increased expression of synaptic markers, such as SNAP25 and SYN1 (Forster et al. 2016). Differentiated SH-SY5Y cells acquired the neuronal ability to produce trains of spikes upon prolonged stimulation in current-clamp experiments (Tosetti et al. 1998) and showed spontaneous firing, bursting, and network behaviour on micro-electrode arrays (MEAs) (Yoon et al. 2020), that were not observed in undifferentiated SH-SY5Y cells. These differences may lead to divergent responses to ischemia/hypoxia between undifferentiated and differentiated SH-SY5Y cells. Thus, it is questionable whether results from studies in undifferentiated SH-SY5Y cells can be extrapolated to neurons or neuronal networks.
OGD directly models ischemic stress and allows for the exploration of divergent components of the pathophysiological cascade caused by ischemic stress. Furthermore, modelling of OGD is straightforward and can be replicated in various laboratories (Cimarosti and Henley 2008). Other proposed cerebral ischemia models are based on chemical or enzymatic modulation of specific pathophysiological pathways. These can be used to study specific components of the ischemic cascade, such as excitotoxicity or glutathione depletion. These techniques are commonly used to investigate the effect of increased glutamate release or induced oxidative stress caused by ischemic stroke, respectively (Nicholls and Ward 2000). In turn, these may lead to other downstream effects, for example calcium dysregulation (by excitotoxicity) or glutathione depletion (by oxidative stress) (Mattson et al. 1995; Mytilineou et al. 1998). However, it is important to note that these techniques probably model only parts of the ischemic stress cascade (Cimarosti and Henley 2008). Moreover, it is unclear whether chemical or enzymatic induction of downstream effects has the desired effect on undifferentiated SH-SY5Y cells, which are often used in ischemic stroke research. For example, none of the studies included in this sco** review touched upon excitotoxicity. This highlights the need for a careful assessment of the suitability of the various cell models and modes of ischemic stress induction in light of the research question at hand.
The majority of human neuronal in vitro studies on effects of ischemia/hypoxia focused on cell survival and protein expression. Expression of proteins that might have deleterious effects (such as pro-apoptotic factors, pro-inflammatory cytokines, oxidative stress factors) apparently increase and expression of proteins that might have protective effects (such as anti-apoptotic factors and anti-inflammatory factors) decrease. Various studies showed this in relationship to cell death, apoptosis, oxidative stress, or inflammation. This focus on microscopic measures of relatively downstream processes neglects early steps in the pathophysiological cascade of ischemia, such as energy failure caused by OGD (Galkin 2019), loss of cell ion homeostasis (Caplan 2009; Taoufik and Probert 2008), and depression of excitatory synaptic transmission. It also neglects effects on neuronal functionality and activity (Bolay et al. 2002; Hofmeijer and van Putten 2012; Pires Monteiro et al. 2021).
HiPSC-derived neuronal networks on MEAs are currently the only functional human neuronal cell model used to investigate neuronal network activity in cerebral ischemia (Pires Monteiro et al. 2021). HiPSCs can be derived from healthy individuals or patients and differentiated in excitatory neurons and inhibitory neurons. This allows establishment of healthy neuronal networks with physiological excitatory – inhibitory (E/I) ratio’s (Frega et al. 2017; Mossink et al. 2021a). Genetic characteristics of the donor are preserved in the model, making it possible to study patient-specific network behaviour, including responses to ischemia/hypoxia (Mossink et al. 2021b). By the use of MEAs, functionality of many neurons and synapses in the network can be studied simultaneously. Despite the lack of normal brain architecture, neuronal network functioning can be readily derived, in addition to microscopic measures. The dynamics of neuronal network failure, from reversible to irreversible damage or recovery, can be monitored continuously (Pires Monteiro et al. 2021). This allows identification of early determinants of irreversibility, including possible effects of interventions.
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