As a leading stroke research provider, Ace Therapeutics provides comprehensive brain cell death mechanism analysis services. Our team of expert utilizes cutting-edge technologies and advanced methods to study the complex process of neuronal cell death after stroke. By elucidating the underlying mechanisms, we aim to help clients identify potential targets and develop new therapeutics for stroke.

Related Services

• Cell Death Mechanism Analysis in Stroke
• Analysis of Ischemia-Triggered Glutamate Excitotoxicity

Anoxic Depolarization in Stroke

During a stroke, the loss of energy supply to neurons leads to a phenomenon known as anoxic depolarization. This event represents a rapid and catastrophic loss of the neuronal membrane potential due to the failure of ATP-dependent ion pumps, such as the Na⁺/K⁺-ATPase, which are critical for maintaining electrochemical gradients. Anoxic depolarization can be measured both in vitro (in controlled laboratory settings, such as brain slices or cultured neurons) and in vivo (in living organisms).

Ionotropic Receptors and Ion Channels in Anoxic Depolarization During Stroke

Several ionotropic receptors and ion channels have been implicated in ischemia-mediated anoxic depolarization of neurons, which in turn leads to cell death.

Fig. 1 Key ion channels that contribute to cell death signaling cascades during ischemia. (Weilinger, et al., 2013)

Ionotropic Glutamate Receptors in Ischemia-Induced Neuronal Injury

Ischemia leads to increased glutamate release. The over-stimulation of NMDA (N-methyl-D-aspartate) receptors and AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptors by glutamate results in cation influx, particularly calcium (Ca²⁺), which is a major factor driving neuronal damage and death.

NMDA Receptors (NMDARs)

NMDARs are cation-permeable, ligand-gated ion channels involved in synaptic plasticity, learning and memory. They're "coincidence detectors" — they need glutamate, glycine (or D-serine), and membrane depolarization to clear an Mg2+ block. Once activated, NMDARs direct Na⁺ and Ca²⁺ infiltration, with Ca²⁺ at the centre of both physiological and pathological processes.

NMDARs are heterotetramers of GluN1, GluN2(A-D) and GluN3(A-B) subunits that modulate Ca²⁺ permeability, Mg²⁺ affinity and neurotoxicity. GluN3A receptors are neuroprotective: they're Ca²⁺-impermeable and less Mg2+-sensitive. Through NMDARs, Ca²⁺ floods the neuronal nitric oxide synthase (nNOS) which generates nitric oxide (NO), ATP is depleted, ROS is created and the apoptotic and necrotic pathways activated. They all play a role in cell death under ischemia.

While blocking NMDARs were neuroprotective in preclinical experiments, the trials found only modest effectiveness. Such failure points to the complexities of Ca²⁺ entry routes and the functional distinction between synaptic and extrasynaptic NMDARs. Synaptic NMDARs seem neuroprotective through CREB activation, and extrasynaptic NMDARs seem neurotoxic. Medicinal interventions might be to target Ca²⁺ signalling sites in a selective fashion.

AMPA Receptors (AMPARs)

Ischemic cell death is also initiated by AMPARs, tetrameric ion channels made up of GluA1-4 subunits. AMPARs are stimulated by only glutamate, in contrast to NMDARs. Whereas GluA2-containing AMPARs can't get dissolved in Ca²⁺ (they possess a positively charged arginine (R) residue), GluA2-depleted AMPARs can get dissolved in Ca²⁺ and Zn²⁺, which makes them the excitotoxicity winners. Ischemia silences surface expression of GluA2-sensitive AMPARs, increasing Ca²⁺ flux through GluA2-deficient receptors and exacerbates neurotoxicity.

Overstimulation of AMPAR is identical to NMDAR-induced toxicity via activation of nNOS, generation of NO, and Ca²⁺-dependent calpain activity. In contrast, AMPARs also activate neuroprotective networks including CREB and BDNF. GluA2-deficient AMPARs, then, constitute Ca²⁺-dependent mechanisms for ischemic excitotoxicity.

Fig. 2 Simplified overview of excitotoxicity after ischemic stroke. (Griem-Krey, et al., 2022)

Purinergic Receptors in Ischemia-Induced Neuronal Injury

It is dual that purinergic receptors, such as adenosine-gated P1 and P2 receptors, are implicated in ischemic brain injury. P1 (metabotropic): regulates cAMP and phospholipase C; P2: ionotropic P2X and metabotropic P2Y subtypes. These receptors are ubiquitously present in the CNS, and activation by ATP or adenosine is neuroprotective or neurodegenerative depending on receptor type and context.

Ischemia lowers intracellular ATP leading to ion imbalance, glutamate and extracellular ATP release which turns on P2 receptors. For extended activation of the P2X7 receptors create pore that permits cytotoxic ATP to flow out through Panx1 channels and other channels. This bloated ATP causes even more cell death by creating a "death complex" with P2X7 and Panx1. The inhibition of P2 receptors, especially P2X7, reduces ischemic injury, as shown by shrinkage of infarct and neuroprotection in experiments.

As with adenosine, its mechanisms are also complex, in ordinary circumstances it reduces glutamate release and excitability of neurons and provides immunity. But what it does during ischemia depends on which receptor subtype. Neuroprotective effects of A2A receptor block and A3 receptor activation in ischemic models. Exonucleotidases delayed hydrolysis of ATP to adenosine imply temporally unique neurodegenerative and neuroprotective functions of purines depending on the CNS area, injury mechanism and model; they deserve additional studies.

Pannexin Channels in Ischemia-Induced Neuronal Injury

Ischemic neuronal damage and cell death are also exacerbated by pannexin channels, especially Panx1. Pannexins, discovered as vertebrate kin to invertebrate gap junctions, consist of Panx1, Panx2, and Panx3, with different distributions in the tissues. Panx1 is abundant in the brain and the immune cells, Panx2 is localised in the brain, and Panx3 is only found in osteoblasts and fibroblasts.

Panx1 channels are turned on during OGD irrespective of ligand-gated receptors (NMDA, AMPA, P2X7) by nitric oxide (NO) nitrosylation. Panx1 channels spit out ATP and other purines that exacerbate ischemic injury. They can be closed at the C-terminus by caspases (i.e., caspase-3 and -7), signalling for "find-me" messages to recruit immune cells in apoptosis.

Panx1 can be deleted or blocked pharmacologically, thus reducing ischemic damage, and is therefore an ideal target for neuroprotection. By targeting upstream activators such as NMDAR, P2X7, SFKs or caspases, Panx1 could be inhibited and damage in neurons is reduced.

Fig. 3 The Pannexin 1 channel in the plasma membrane. (Kova, et al., 2021)

Transient Receptor Potential Channels in Ischemia-Induced Neuronal Injury

Transient receptor potential (TRP) channels are tetrameric, cation-permeable channels with diverse activation mechanisms. Among TRP channel subfamilies, TRPM2 and TRPM7 play critical roles in neuronal death after ischemic insults by mediating delayed calcium dysregulation and oxidative stress-induced damage. TRPM7, in particular, is well-established as a mediator of ischemic damage in both in vitro and in vivo models, while the role of TRPM2 in vivo remains an area for future research. Both channels represent promising therapeutic targets for mitigating neuronal injury and enhancing survival following ischemic events.

Acid-Sensing Ion Channels in Ischemia-Induced Neuronal Injury

In ischemic stroke, acidosis arises from lactate due to the switch from oxidative phosphorylation to glycolysis, where pH is dropped to less than 6. This acidity causes acid-sensing ion channels (ASICs) to be switched on, allowing sodium and calcium to flow into the cells and causing ionic dysregulation and death of neurons. Ischemia activates ASIC1a by phosphorylating Ser478/Ser479 by Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) by activation of NMDAR (NR2B subunit). Enclosing NR2B or CaMKII suppresses ASIC1a-mediated calcium uptake and inhibition of neuronal death.

ASIC1a channel inhibition in the context of ischemia promises to curb neuronal death and improve function in stroke patients. ASIC1a-induced calcium influx can therefore be inhibited through its own pathway from acidosis-induced neurotoxicity.

Fig. 4 ASIC2-associated pathologies. (Sivils, et al., 2022)

1. Weilinger, N. L., et al. (2013). Ionotropic receptors and ion channels in ischemic neuronal death and dysfunction. Acta Pharmacologica Sinica, 34(1), 39-48.
2. Griem-Krey, N., et al. (2022). CaMKIIα as a Promising Drug Target for Ischemic Grey Matter. Brain Sciences, 12(12), 1639.
3. Koval, M., et al. (2021). Pannexin 1 as a driver of inflammation and ischemia–reperfusion injury. Purinergic signalling, 1-11.
4. Sivils, A., et al. (2022). Acid-sensing ion channel 2: Function and modulation. Membranes, 12(2), 113.