Intracerebral hemorrhage (ICH) is the most devastating type of cerebral stroke caused by rupture of intracranial vessels or hemorrhagic transformation of cerebral infarction. Cerebral hemorrhage usually occurs in the basal ganglion, among which the nucleus (putamen) is the most common region (~50% of all cases of cerebral hemorrhage), followed by the thalamus (~15%), the pons (~15%), and the cerebellum (~10%) . Moderate-to-severe disability in two-thirds of surviving ICH patients also caused an impact on the socioeconomic status of the patients and their families . The prevalence of stroke in Asians is much higher than that in Westerners (20%–30% vs. 10%–15%) .
Our review focuses on the pathogenesis of ICH and briefing on the standard methods used to induce ICH brain injury in animals. Since rodents are the most used experimental models of ICH, our review revolves around the technical details of ICH induction and compare the similarities, differences, advantages, and disadvantages of the existing models. We also explain the evaluation methods, including magnetic resonance imaging (MRI), behavioral testing, and histological examination in these ICH animal models.
PATHOPHYSIOLOGY OF INTRACEREBRAL HEMORRHAGE
The pathogenesis of brain tissue damage caused by cerebral hemorrhage is divided into two stages: primary brain injury (PBI) and secondary brain injury (SBI) [14-6].
The dynamic progression and outcome of ICH are primarily influenced by hematoma expansion and perihematomal brain injury [Figure 1]. Formation of hematoma formed by hemorrhage after a cerebral blood vessel rupture is the primary source of PBI. Hematoma causes a mechanical impact on the brain parenchyma  and accelerates neurological deterioration . A hematoma usually increases over time, typically around 24–48 h . Hematoma and surrounding injuries cause brain edema formation and blood–brain barrier (BBB) disruption [8-11]: both events are important factors of brain injury after ICH . Large hematoma can cause increased ICP, reduce CPP, brain herniation, and eventually lead to death . SBI of ICH is caused by the extravasated blood trigger complex and deleterious cellular and molecular consequences, including coagulation, hemolysis, and hemoglobin breakdown, which are considered the most important factor that causes brain damage after cerebral hemorrhage . The coagulation cascades produce and activate the thrombin immediately in the brain after ICH, leading to activation of the microglia [Figure 1]. Microglia activation could induce BBB breaks down, early brain edema, and neuronal and glial apoptosis [16-18]. The hemolysis within hematoma usually lasts from hours to a few days and produces hemolysate that contains the hemoglobin and its breakdown products (e.g., iron, heme, and degraded heme products) [Figure 1]. These products increase the brain’s reactive oxygen species (ROS), resulting in delayed edema formation and neuroinflammation [19-21].
Oxidative stress and antioxidant system in intracerebral hemorrhage
Under normal physiological conditions, the mitochondrial electron transport chain mainly generates the ROS produced by cell metabolism. Structurally and functionally complete mitochondria have a good antioxidant system, including manganese superoxide dismutase, glutathione peroxidase, catalase, glutathione reductase, heme oxygenase-1, and other enzymes. These enzymes can neutralize and metabolize ROS to maintain the balance of cell physiology . Thus, cerebral hemorrhage increases ROS production and overwhelms the antioxidant defense system capacity, leading to a vicious circle that destroys mitochondrial function . Figure 1 illustrates the pathophysiology and description.
Neuroinflammation of intracerebral hemorrhage
Inflammation after ICH is a complicated response of infiltrating immune cells, from the time of injury to the stage of healing. The primary inflammatory cells participating in inflammation include resident microglia, astrocytes, blood-derived leukocytes, and macrophage. These cells are activated and accumulated within the hemorrhagic site after ICH during SBI [Figure 1] . Excessive inflammatory mediators (e.g., tumor necrosis factor-alpha, interleukin-1β [IL-1β], interferon gamma, nitric oxide synthase, intracellular adhesion molecule-1, IL-6, and matrix metalloproteinases) produced by early activation of inflammatory cells have been implicated as a possible mechanism of SBI after ICH .
Monocyte- and microglia-derived macrophage activation in intracerebral hemorrhage
“Macrophages” refer to activated myeloid cells, which can be derived either from monocytes or microglia [26-29]. Numerous studies have shown that microglia are the first responders to stroke and engage in intimate cross-talk with other intrinsic brain cells and infiltrating leukocytes that enter the brain from the peripheral through the leaky BBB [1430-33]. In ICH, activated monocyte and microglia can differentiate into macrophage, which plays a biphasic role. They may achieve a spectrum of functional phenotypes, including the “classically activated macrophage” (M1 polarization) and the “alternative activated macrophage” (M2 polarization) [Figure 1] . The M1 polarization macrophage (classical) releases a wide array of inflammatory cytokines, oxygen-free radicals, and other harmful substances during early inflammation [Figure 1]. In contrast, the M2 polarization macrophage (alternative) secretes neurotrophic substances, removes necrotic or apoptotic neuronal debris (high phagocytic activity), makes dynamic contact with neurons, and promotes the formation of glial scar tissue postinflammation [Figure 1] .
EXPERIMENTAL ANIMAL MODELS OF INTRACEREBRAL HEMORRHAGE
Animal models are necessary to help improve the existing therapeutic care and validate new interventions that might alleviate ICH consequences. Various animal models have been developed in practice for many years to resemble the clinical situations of ICH in humans.
Table 1 provides a description of the different experimental animal models available for ICH and the advantages and the disadvantages of the experimental animal models.
The collagenase injection model requires the injection of various amounts (0.01–0.1 units in 0.5–2 mL) of bacterial collagenase into the rat striatum. The significant advantage of this model is the high reproducibility by controlling the dosage of the bacterial collagenase, which can depict the PBI and the SBI with elevated intracranial pressure and hematoma formation followed by hematoma expansion . The collagenase injection model shows hematoma increases in the first 4 h. Blood from the hemorrhagic area diffuses into the brain parenchyma after collagenase injection, increasing the hematoma volume. The hematoma volume was more significant in the collagenase injection model when compared to the blood injection model at 4 and 6 weeks [Table 1] .
Due to collagenase injection, it is easy to observe the time-dependent increase in the BBB leakage . Since this model involves the formation and expansion of hematoma, toxic blood components such as free hemoglobin, heme, hemin, thrombin, and other factors affecting neurons can also be studied [Table 1]. Various studies have further probed the molecular and cellular effects caused in the brain, including neuroinflammation, neuronal death, neutrophil infiltration, and microglial activation .
However, the major limitation of the collagenase injection model is the significant inflammation caused by the exogenous bacterial collagenase and the rupturing of small blood vessels around the area of collagenase injection, which is not the case in clinical situations; hemorrhages in the human brain are usually from the arterial source . The use of collagenase induces vessels’ bursting, which results in significant bleeding even after hours of collagenase injection . Maintaining the dosage of collagenase is critical in the collagenase injection model as higher dose of collagenase might induce an extensive inflammatory response causing neurotoxic effects in the animals . The functional recovery of the animals can be monitored after 2 months of collagenase injection. In resemblance with the clinical outcomes of ICH, the functional neurological effects were diminished after 21 days postcollagenase injection [39-40]. Hence, this method can be used in the experimental setup as it is highly reproducible once the collagenase dose is stabilized.
Autologous blood injection
Autologous blood extracted from the tail vein infused into the striatum of the rats or mice with the help of an infusion system is closely similar to the clinical ICH . The significant advantage of this model over other models is the sterility as it does not introduce any exogenous substance. Like the collagenase model, the autologous blood injection model causes mechanical damage associated with the mass effect . The significant disadvantages of the blood injection model are the random blood penetration into the subarachnoid and ventricular spaces [Table 1]. This model is less reproducible when compared to the collagenase model [Table 1] .
In terms of the hematoma size, unlike the collagenase model, which shows a constantly increasing hematoma size during the first 4 h, the blood injection model demonstrates a stable hematoma size during the first 4 h. The hematoma usually resolved as early as 4–6 weeks postblood injection . The functional outcomes contrasted with the collagenase model as the animals showed complete neurological functional recovery after 21 days of blood injection. Neurological severity score and corner test showed a partial recovery from day 1 to day 28 postblood injection . The animals which underwent blood injection ICH demonstrated deceased time-dependent BBB permeability; also, the BBB permeability was bound only to the rim of the hematoma .
The first microballoon inflation model was developed in 1987 to study the mass effect caused by ICH. In this model, a microballoon was inserted stereotaxically into the right caudate nucleus of rats with the help of a guided cannula, and the coordinates are as follows; 1mm cranial and 4mm to the right of bregma; the balloon was inserted into a depth of 5 mm into the brain. The microballoon (embolization balloon) was inflated with 50 mL within 20 s and was kept for 10 min before it was deflated and removed from the rat brain [45-47]. This model effectively reduced the cerebral blood flow and increased the ICP of the animals, thereby demonstrating mass effect . The animals also showed systemic effects on cerebral perfusion pressure . The two major disadvantages of this method are the limited time window to perform balloon inflation and an extended period of balloon inflation might result in irreversible brain damage. Another disadvantage is the lack of hematoma formation. Hence, this model cannot study the SBI caused by the blood components secreted from the hematoma and perihematomal regions [Table 1] . Studies show that microballoon inflation causes cell death by apoptosis from 6 h to 24 h after deflation. Furthermore, necrotic neurons were present in the animals after 10 min or 120 min postdeflation [Table 1] .
Thrombin injection model
The model is a replica of the blood model with the only difference of injecting only thrombin and not the whole blood into the animal brain. The model differs from the blood model in that only thrombin is injected into the animal brain [Table 1]. This model’s significant advantage and disadvantage are knowing the importance of thrombin and not any other blood components involved in the SBI of ICH . However, this model showed a cognitive deficit for 5 days postthrombin injection. One further study illustrated that thrombin did not play BBB disruption . Yang et al. have also shown activated microglia and macrophages after intrastriatal thrombin injection .
Standard behavioral assessment techniques evaluate rodents’ neurological, motor, and sensorimotor functions from the neurological scores assessed clinically to conclude the stroke outcomes in human patients. Motor and sensorimotor analyses are carried out in an experimental setup to determine rodent stroke outcomes.
Table 2 summarizes the common techniques available for ICH evaluation in the experimental setup with the advantages and disadvantages of each techniques used.
The modified neurological severity score
The modified neurological severity score (mNSS) is the most commonly used neurological test in rodents . The mNSS can be performed both for ischemic stroke and ICH, including motor, sensory, and reflex functions. The higher the score, the more severe the neurological deficit. In most experimental brain injury models, mNSS is used to assess long-term outcomes. In ICH models, rats display a higher mNSS score as early as 1 day post-ICH, which indicates massive neurological impairment. The observational behavior of mNSS is based on the different parameters that are used in the test such as the hindlimb motor function, forelimb motor function, and body asymmetry test; mNSS is a useful tool to assess the long-term stroke outcome; and it can be used to evaluate the neuroprotective effects of novel treatments [Table 2]. The disadvantage of this method is the complexity, as it involves multiple neurological aspects. Hence, individual scores have to be taken into account to analyze the neurological deficits of specific brain regions .
The Garcia scale was developed to assess rats’ motor and sensorimotor functions . In this model, the animals are tested for their motor functions with spontaneous activity (0–3), symmetry of limb movement (0–3), forelimb outstretching (0–3), climbing and grip strength (0–3), climbing and grip strength (0–3), body symmetry (0–3), and sensory function of vibrissae (0–3). The collective score is represented in the Garcia scale [Table 2]. Unlike mNSS, in the Garcia scale, high scores correspond to less neurological deficits. The significant advantage of this model is its simplicity. The Garcia scale is easy to perform, providing motor and sensorimotor functions. The major disadvantage is Garcia scale focuses more on the forelimb than the hindlimb [Table 2] .
Forelimb placement test
The forelimb placement test (FPT) is based on the vibrissae-elicited forelimb placing test . Animals respond to vibrissae stimulation, making it easier to check ICH effects. When the animals are placed on the edge of the table, as soon as the vibrissae touch the table, normal animals will place the ipsilateral forelimb on the table, with ICH animals showing less or no response. FPT is easy to perform; however, the major disadvantage is that FPT is confined to the forelimb. It has to be combined with other neurological functional assays to assess the animals’ complete motor or sensorimotor functions [Table 2] .
Open field test
An open field test was developed to assess the locomotor activity of rodents . A wooden or plastic open field maze (50 cm [length] × 50 cm [width] × 38 cm [height]) is used. The animals are placed in the open field to explore for 10 min; locomotor activity is measured with the help of a video recorder. The major goal of this test is to analyze the movement of the animals placed in the chamber. With the help of video recording and automated systems, the distance, route, immobile time, and rear-up behavior can all be tracked and analyzed to evaluate the locomotor ability of the normal and ICH animals. Open field test is easy to perform and minimize human errors. The disadvantage of this method is the environmental factors that might affect the results [Table 2] .
The corner test is used to analyze the sensorimotor and postural asymmetry . The setup for the corner test is to attach two cardboards 30 cm × 20 cm at a 30° angle. Animals are analyzed based on the side they turn upon vibrissae stimulation after placing them in the middle of the two cardboards. Able to access long-term outcomes and a practical test to analyze the sensorimotor functions post-ICH [Table 2] .
The rotarod test is used to assess motor coordination. The animals are placed in a 3 cm (diameter) × 40 cm (length) rod, which rotates with the help of an electric motor. The speed of the motor increases gradually, and the latency before the animal falls is recorded. The rotarod test is sensitive as it accurately measures the motor coordination of the animals, with a major limitation of long training trials required before the ICH surgery [Table 2] .
The primary parameter measured after induction of ICH in animals is the measurement of the hematoma volume.
Morphometric measurement is the easiest method to measure the hematoma volume after ICH in the animal model . In this method, animal brains are cut coronally through the needle entry plane and serially sliced with the desired thickness. Digital photographs are taken, and the hematoma volume is measured with the help of image analysis software [Table 2]. The major disadvantage of this method is the animals need to be sacrificed at specific time points after ICH induction .
Magnetic resonance imaging for hemorrhagic volume
MRI has been the most sophisticated and accurate method to measure the hematoma volume . In this method, animals are anesthetized, and MRI images are captured inside the MRI machine. T2-weighted images are used to measure the hematoma volume based on the size of the hematoma. Specific software is used to measure the hematoma volume [Table 2]. MRI is the most efficient and precise method to calculate the hematoma volume; on the other hand, it is expensive and involves expertise and training to obtain the data [Table 2] .
Positron emission tomography/computed tomography to measure the hemorrhagic volume
The hybrid positron emission tomography/computed tomography (PET/CT) imaging can be performed to analyze the hemorrhagic volume. There are very few studies that incorporated PET/CT imagining in experimental animal models of ICH. The rats are restricted from drinking and fasting overnight before PET/CT scan in this procedure. The rats are anesthetized, and the most common radiotracer, 18F-fluorodeoxyglucose (18F-FDG), is injected via the tail vein, and the rats are allowed to rest for 30 min to ensure the uptake of 18F-FDG. The rats are then placed in the rat holder for PET imaging. Subsequently, the rats will undergo a CT scan. The hemorrhagic volume and glucose metabolism will be detected by analyzing the PET and CT images. The hybrid PET/CT is used because of the limitation of using CT, which provides us only with a detailed imagining of the brain’s tissues [Table 2].
In contrast, a hybrid PET/CT scan provides images of the tissue with abnormal metabolic activity . The advantages of this method are the cost-effective procedure to evaluate the hemorrhagic volume and other parameters, including glucose metabolism. This method is also noninvasive as MRI. Although hybrid PET/CT is under practice in clinical setup, MRI is preferred over CT or PET/CT in small animal research because of MRI’s superior anatomic and physiological details in a single examination. This also makes the usage of MRI in small animal research more common than CT scanning .
BRAIN WATER CONTENT (EDEMA)
Edema is the swelling caused by the fluid trapped in the hematoma; edema is formed after the erythrocyte lysis following hematoma formation . Edema can be represented by measuring the brain water content with the following two methods: dry/wet weight method and MRI.
Dry/wet weight method
In the dry/wet weight method, edema is quantified as a change in % brain water content determined from the difference between wet weights and dry weights divided by the wet weight. The brain samples are harvested from the animals and are weighed immediately to attain the wet weight. Then, the brain samples are dried in an oven at 100°C for 24 h and weighed to obtain the dry weight. The measurement of water content can be calculated with the simple formula ([wet weight] – [dry weight]) (wet weight)−1 × 100%. This method is simple and easy to perform with a significant disadvantage of misinterpretation of the impact of minute changes in % brain water content .
Magnetic resonance imaging for quantifying edema
MRI technique can quantify the edema or brain water content using the same approach used to determine the hematoma volume . It is noninvasive, and assessing hemispheric volumes after ICH allows us to quantify the space-occupied effects of edema formation directly. Hence, further analysis can be carried out on an experimental setup of ICH. The major disadvantage is the cost and facilities required to perform the MRI edema quantification .
BLOOD–BRAIN BARRIER DISRUPTION
BBB disruption is a phenomenon that is regulated during the PBI of ICH. BBB leakage has been analyzed to understand the severity of the brain damage [16-18]. The following two most common methods have been used to determine the permeability of BBB in the experimental ICH models [Table 2].
Microscopic assessment of BBB can be carried out with the help of two-photon or multiphoton microscopes where the animals are anesthetized, and craniectomy will be made on the desired cortical region. With fluorescent traces, microscopic images can be taken to analyze not just the BBB disruption, but cerebral blood flow, leukocyte behavior, and cell death can also be measured. The advantage of this model is the real-time imaging of the BBB disruption, with the major disadvantage being the poor tissue penetration [74-76].
Measurement of blood proteins
The most common method used to measure the BBB disruption is the traditional method by assessing the extravasated blood proteins from the desired brain region using immunohistochemistry or immunofluorescence analysis. This old yet reliable method is used to determine the albumin, fibrinogen, immunoglobulin M, and immunoglobulin G. There are no exogenous tracers such as Evans blue or fluorescein isothiocyanate (FITC) dextran used in this method, which is a major advantage. Time-point analysis cannot be measured with this method as the extravasation of endogenous tracers does not correspond to the BBB disruption at a particular time point, but rather a collective assessment of the blood protein extravasation from the time of injury [76-78].
Assessment of blood–brain barrier disruption with magnetic resonance imaging
MRI evaluation of BBB is a qualitative method used to measure the BBB disruption in both animals and humans. The evaluation of BBB disruption using MRI is based on the absence of contrast enhancement on the images taken from the brains of animals or humans. To obtain the MRI images of rats in the experimental setup, the rats are placed supine on plastic support that consists of a headlock. Then, the head is inserted into the cage-like structure, which is the imaging coil. The animal is anesthetized all through this procedure. Then, MRI images are obtained in the respective planes. BBB permeability is measured with the help of injecting two boluses, the first bolus of contrast to measure the permeability and the second bolus for comparison. The advantages of this method are the accuracy of the results, with the major disadvantage being the cost and the experience needed to handle the MRI machine .
Evans blue leakage method
Evans blue dye has been used historically to determine the BBB permeability in various animal models [Table 2] . Briefly, Evans blue dye is injected intravenously into the femoral vein of rats or mice and allowed to circulate for one hour; then, the animals undergo intracardial perfusion with normal saline. Then, the brain sections are dissected, and the fluorescence intensity of Evans blue is quantified. The major advantage of this method is its standardization, and the disadvantage is the toxicity of the Evans blue dye injection .
FITC-dextran is a standard method of choice for analyzing the BBB permeability. FITC-dextran is injected intravenously into the animals. Images are captured with fluorescence microscopy, and the brain capillaries and surrounding parenchyma with FITC-dextran can be analyzed to assess BBB integrity . The major disadvantage is the toxicity of the FITC-dextran tracer. FITC-dextran is available in various molecular weights starting from 3 kDa to 2000 kDa for vascular outlining as the high molecular FITC-dextran gets trapped in the vessels during circulation .
LIMITATIONS AND CONCLUSION
It is impossible to understand ICH’s molecular and cellular mechanism without a proper model. The major limitation in ICH research is the lack of a suitable animal model to mimic the clinical conditions. In the clinical setting, the volume of hematoma is larger, and the treatment protocol is formulated based on the size and position of the hematoma. With surgical treatment involving hematoma aspiration, it is important to note that the present animal models lack the similarity between the clinical severity of ICH and the treatment protocol.
There is no adequate remedy for ICH available to date. The dynamic and complicated pathophysiology process of ICH is not fully elucidated. This review provides an overview of the ICH pathophysiology, animal models used in ICH, and technical evaluation methods in practice, highlighting the advantages and disadvantages of the animal model used and the evaluation parameters.
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