In Vitro and In Vivo Models of Hemorrhagic Stroke
At a glance
Hemorrhagic stroke is rare but much more fatal than ischemic stroke. Intracerebral hemorrhage (ICH) and subarachnoid haemorrhage (SAH) are the hemorrhagic strokes. With around one in 10 strokes, ICH strokes are the most prevalent haemorrhagic strokes. Unfortunately, there are no cures to optimize ICH outcomes at this time. So, new therapeutic approaches to ICH are needed now more than ever. ICH has been studied in both in vivo and in vitro models, and the pathophysiology and so the targets of therapeutics have been extensively advanced.
Ace Therapeutics provides customized animal and in vitro models of ICH to clients worldwide to help them study the underlying mechanisms of ICH-induced brain injury and discover new therapies for preclinical ICH.
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In Vitro Models of Intracerebral Hemorrhage
In vitro models of ICH study in a simplified culture system, and thus are particularly suited for high-throughput screening and mechanistic research. Current in vitro ICH models mainly target secondary injury associated with ICH, including hematoma expansion, neuronal cell death, oxidative stress, and blood-brain barrier (BBB) disruption.
Table 1. In vitro ICH models
| Experimental Models | Description | Advantages | Disadvantages |
|---|---|---|---|
| Hemoglobin | Hemoglobin is a metalloprotein in red blood cells responsible for oxygen transport. Following ICH, hemoglobin is released into the brain, where iron is oxidized, leading to inflammatory responses, blood-brain barrier disruption, and brain edema. | Consistently and accurately reflects hematoma expansion; Easy to use and suitable for high-throughput screening; Allows research on iron levels/dynamics and hemoglobin proteolysis | Unable to fully reproduce the hemotoxicity of ICH; Unable to replicate vascular damage |
| Hemin | Hemin, a byproduct of hemoglobin breakdown, is linked to hematoma expansion. It interacts with cell membranes, influencing cell survival, metabolism, and function. | Reliable and easy to use; Able to mimic several aspects of secondary ICH injury, including BBB disruption and neuroinflammation | Unable to fully reproduce the hemotoxicity of ICH; Unable to replicate vascular damage; Much lower levels of hemin are used to induce ICH in vitro; Different neural populations have different sensitivities to hemin |
| Autologous Blood | Intracerebral injection of autologous blood is a recognized in vivo model for ICH. Similarly, autologous blood is used in vitro to induce ICH, where exposure to it notably decreases action potential amplitude and cell viability in brain sections. | Able to replicate the full hemotoxicity of ICH; Relatively easy to use | Unable to replicate continuous bleeding seen in vivo; Difficult to visualize secondary ICH injury, such as hematoma expansion |
| Hydrogen Peroxide | Oxidative stress plays a critical role in the pathology of ICH, with reactive oxygen species (ROS), such as hydrogen peroxide, being produced in high levels following ICH. Consequently, hydrogen peroxide is utilized to model ICH in vitro. | Relatively easy to use; Allows detailed investigation of a specific pathway in ICH | Unable to reproduce primary ICH injury; Only replicates one aspect (oxidative stress) of secondary ICH injury |
| Glutamate | Glutamate is an excitatory neurotransmitter, and its excess is linked to increased ROS levels and excitotoxic brain injury. Due to its significant role in oxidative stress and excitotoxicity, glutamate is used to replicate ICH injury in vitro. | Relatively easy to use; Allows detailed investigation of a specific pathway in ICH; Has a longer toxicity window | Unable to reproduce primary ICH injury; Only replicates one of many mechanisms of secondary ICH injury |
Animal Models of ICH
Animal models of primary ICH have been developed using pigs, rabbits, dogs, cats, and rodents. These models have provided new insights into the pathophysiology of ICH-induced injury, including the role of mass effect and increased intracranial pressure, changes in blood flow and metabolism, and the effects of specific blood components on brain edema formation and BBB disruption. These models have also provided details of the biochemical and molecular events that cause ICH.
To decide which is the best model for each research, some points must be considered: The purpose of each model, the primary outcome of the study, considering whether hematoma is in the acute or chronic phase and molecular vs. hemodynamic analysis.
Whole Blood Injection Models
Intracerebral injection of autologous blood models enables the assessment of hemolysis-induced toxicity and subsequent immune-inflammatory responses. This technique allows researchers to evaluate interventions aimed at reducing swelling, hematoma expansion, and the effects of ICH on brain hemodynamics. First developed in the 1960s, this method is effective for treating brain parenchymal hematomas and has been applied in larger animals, such as cats, dogs, pigs, sheep, and monkeys, typically injecting blood into the frontal lobe or basal ganglia.
Variations in the technique include differences in blood source, injection volume, and injection depth. For example, some studies have infused venous blood into the deep white matter of dogs, while others injected arterial blood superficially into the frontal lobe. In smaller animals like rats and mice, blood is generally injected into the caudate nucleus, though some studies have also targeted the lateral ventricles to compare the effects of contained versus uncontained hemorrhage.
The main advantage of the autologous blood injection method is its ability to produce hematomas with consistent volumes, closely mimicking the rapid accumulation of blood seen in clinical situations. However, it does not replicate the rupture of blood vessels typical of human brain hematomas and may cause intraventricular or subarachnoid hemorrhage due to ruptures in adjacent spaces. Additionally, there is a risk of backflow along the needle track. Despite these limitations, this model is valuable for studying the biochemical and pathophysiological effects of acute ICH, as it controls the volume of injected blood, allowing for the generation of specific hematoma sizes and mass effects.
Collagenase Animal Model of ICH
The collagenase model is an experimental method used to induce ICH by injecting bacterial collagenase, a metalloproteinase that degrades collagen IV in the blood-brain barrier. This process leads to microvascular rupture and leakage at the injection site. This model involves stereotactic injection of collagenase into specific brain regions, resulting in localized cerebral parenchyma or intraventricular hemorrhage.
This model is advantageous as it effectively mimics bleeding and allows for easy manipulation and control of hematoma size by adjusting the collagenase dosage. It is commonly employed in both rodents and larger animals. However, the collagenase model does not fully replicate clinical ICH conditions; bleeding is typically slow and diffuse, stemming from small vessels and capillary beds, rather than the rapid, urgent hemorrhage resulting from the rupture of major brain vessels seen in actual ICH cases. Additionally, the use of bacterial collagenase can exacerbate inflammatory responses, making this model unsuitable for studying the immune reactions associated with ICH.
Fig.1. Schematic illustrations of injury 6 weeks after collagenase and whole blood-induced ICH. (MacLellan, et al., 2010)
Microballoon Insertion Models
The microballoon insertion model is an experimental method used to simulate the space-occupying effects of ICH. This model employs a mechanical microballoon attached to a 20-gauge venous cannula, which is inflated with saline after being inserted into the caudate nucleus of adult rats. This inflation leads to a significant increase in intracranial pressure (ICP) and a subsequent decrease in cerebral blood flow (CBF) in the ipsilateral frontal cortex and caudate nucleus.
Fig.2. Piglet brain after experiment with cortical injury produced by the intracranial balloon. (Paiva, et al., 2022)
Cerebral Blood Vessels Damage Model
The cerebral blood vessels damage model involves exposing the cortical veins of rats through a craniotomy and causing damage with a curved needle, leading to cortical hemorrhages. This model has seen limited recent use due to the variability in brain injury it produces, which affects the reliability of experimental outcomes. As an alternative, laser-induced vessel rupture can create microbleeds to assess coagulation outcomes. Scientists successfully induced intracerebral hematomas by puncturing the middle cerebral artery in 12 dogs under ultrasound guidance. However, this model has limitations, including the requirement for an open bone window, which can underestimate the effects of intracranial hypertension, and it tends to produce less severe histological damage than other models.
- MacLellan, C. L., et al. (2010). Rodent models of intracerebral hemorrhage. Stroke, 41(10_suppl_1), S95-S98.
- Paiva, W. S., et al. (2022). Animal models for the study of intracranial hematomas. Experimental and therapeutic medicine, 25(1), 20.
