Abstract
Background
Hemorrhagic expansion into the fourth ventricle is an independent risk factor for poor outcomes in intraventricular hemorrhage (IVH) patients. However, to date, available animal models of IVH are limited to models of supratentorial ventricular hemorrhage, and there are no specific models of fourth ventricle hemorrhage. This limitation hinders comprehensive basic research and the understanding of the pathophysiological changes that occur following fourth ventricle hemorrhage. Therefore, the development of an animal model of fourth ventricle hemorrhage is highly important.
Methods
In this study, a novel rat model of fourth ventricle hemorrhage was established via autologous blood injection through the foramen of Magendie. Anesthetized rats were positioned in a stereotaxic apparatus with their heads tilted downward at an angle of approximately 20° relative to the vertical axis. A needle was inserted through the foramen, and autologous blood obtained from the rat's heart was injected into the fourth ventricle via a microinfusion pump. Systematic evaluations of the model were conducted using small‐animal magnetic resonance imaging, histopathological analysis, and neurological function assessment.
Results
The rats developed stable and reproducible fourth ventricle hematomas and ventricular dilation. They also exhibited acute‐phase hydrocephalus and pathological features of perilesional brain tissue injury, with observed neurological deficits comparable to patients with fourth ventricle hemorrhage.
Conclusion
This model successfully recapitulates the clinicopathological and pathophysiological characteristics of patients with fourth ventricle hemorrhage and can be utilized for further investigation into the pathophysiological mechanisms underlying posthemorrhagic hydrocephalus and perilesional brainstem tissue injury.
Keywords: animal model, brainstem injury, fourth ventricle hemorrhage, hydrocephalus, intraventricular hemorrhage
In this study, a novel rat model of fourth ventricle hemorrhage was established via autologous blood injection through the foramen of Magendie. Anesthetized rats were positioned in a stereotaxic apparatus with their heads tilted downward at an angle of approximately 20° relative to the vertical axis. A needle was inserted through the foramen, and autologous blood obtained from the rat's heart was injected into the fourth ventricle via a microinfusion pump.
1. INTRODUCTION
Intraventricular hemorrhage (IVH) refers to bleeding into the intracranial ventricular system and can be classified into primary and secondary IVH. Primary IVH is typically associated with factors such as vascular malformations within the ventricular walls, aneurysm rupture, or subependymal hemorrhage. 1 In contrast, secondary IVH most commonly results from parenchymal hemorrhage extending into the ventricles, as observed in conditions such as hypertensive intracerebral hemorrhage and cerebral amyloid angiopathy. 2 , 3 Regardless of its etiology, IVH is closely associated with sudden neurological deterioration or the development of coma due to irreversible brainstem damage caused by persistent hematoma compression. 4 The incidence of intracerebral hemorrhage (ICH) complicated by IVH is as high as 50%–70%, with over 67% of IVH patients subsequently developing hydrocephalus. 5 The prognosis of IVH is poor, with a mortality rate of up to 80% among in patients with severe hydrocephalus, significant ventricular dilation, and irreversible brainstem damage. 6
Among these patients with IVH, hemorrhagic expansion into the fourth ventricle is an independent risk factor for poor prognosis. 7 , 8 The mass effect of fourth ventricle hematomas can exert significant pressure on the brainstem, resulting in damage to its nuclei and white matter tracts, thereby causing severe neurological dysfunction. Additionally, fourth ventricle hematomas may disrupt cerebrospinal fluid (CSF) circulation and absorption, leading to the development of hydrocephalus. Hydrocephalus further exacerbates intracranial pressure, further compresses brain tissue, and aggravates neurological impairments. 9 To date, most experimental animal studies have focused on supratentorial IVH (in the lateral and third ventricles), with little attention given to fourth ventricle hematomas. While supratentorial IVH is undoubtedly a significant concern, the mass effect of hematomas filling the fourth ventricle on the brainstem should not be overlooked. 8 This mass effect may lead to brainstem compression and displacement, subsequently impairing brainstem function and independently influencing the prognosis of the disease. 9
Currently, animal models of ventricular hemorrhage are limited to models of lateral ventricle hemorrhage and basal ganglia hemorrhage with intraventricular extension. 10 There is no established animal model for fourth ventricle hemorrhage, resulting in an incomplete understanding of the pathophysiological mechanisms underlying brainstem compression and injury, and no related research has been conducted. In this study, we aimed to establish a novel fourth ventricle hemorrhage rat model via autologous blood injection and to conduct systematic evaluations of this model, thereby contributing to a deeper understanding of disease mechanisms and facilitatine the development of potential therapeutic approaches.
2. MATERIALS AND METHODS
2.1. Experimental animals
Eight‐ to twelve‐week‐old adult male Sprague–Dawley rats (320–380 g, Experimental Animal Center of the Army Medical University) were used in this study. The animal care and use protocols adhered to the Guidelines for the Care and Use of Laboratory Animals and were approved by the Laboratory Animal Welfare and Ethics Committee of Third Military Medical University (AMUWEC20232125). Additionally, these procedures were reported in accordance with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines. The animals were anesthetized via an intraperitoneal injection of pentobarbital (40 mg/kg). During surgery, a heating pad was used to maintain the body temperature at 37.0 ± 1°C.
2.2. Experimental groups
In a preliminary experiment performed by our research group, the injection of 100 μL of autologous blood into the fourth ventricle did not cause short‐term neurological deficits in rats, whereas the injection of 200 μL of autologous resulted in mild neurological impairments. In contrast, the injection of 400 μL of autologous blood resulted in the formation of a fourth ventricle cast and severe neurological deficits. Therefore, in this study, the rats were divided into the following groups: the control group, the sham‐operated group (400 μL saline), the 200 μL autologous blood injection group, and the 400 μL autologous blood injection group. For model construction, the rats were injected with 400 μL of autologous blood, 200 μL of autologous blood, or 400 μL of saline into the fourth ventricle. The control group did not undergo any surgery, while the sham surgery group underwent surgical procedures and received 400 μL saline into the fourth ventricle. The experiment was divided into three steps: the first step involved survival analysis with n = 11 per group; the second step involved neurological function assessment with n = 6 per group; the third step involved histopathological examination with n = 3 per group. In the second step, two rats from each group underwent MRI on days 1, 14, and 28. Coronal and sagittal T2‐weighted magnetic resonance (MR) sequences were used to observe the presence of hematomas in the fourth ventricle and hydrocephalus in the lateral ventricles. Additionally, in the third step, 3 rats in each group were underwent perfusion fixation on postoperative day 1 for further analysis, including Nissl staining, Hematoxylin and Eosin (HE) staining, TUNEL staining, Evans Blue staining, and transmission electron microscopy (Figure 1).
FIGURE 1.
Schematic diagram of the experimental procedure.
2.3. Construction of a fourth ventricle hemorrhage model
A novel rat model of fourth ventricle hemorrhage was constructed via autologous blood injection using the posterior median foramen approach. Prior to surgery, the chest and head areas were shaved. Autologous blood was collected from the cardiac artery using a 1 mL syringe through a subxiphoid approach. Anesthetized rats were positioned on a stereotaxic apparatus, with the head secured by ear rods, and angled approximately 20° downward from the vertical. The skin was incised along the midline from behind the ears using surgical scissors, and the tissue was cut along the midline until the occipital bone was palpated. After the area was dilated and secured with a retractor, the sagittal suture, occipital protuberance, and posterior median foramen at the junction with the first cervical vertebra were clearly visible. This junction was the site of autologous blood injections. The dura mater was punctured with a sharp needle to create an entry point. A blunt needle was then attached to a 1 mL syringe and secured to a microinjection pump. The syringe was positioned so that it swung backward at a 20° angle from the vertical, aligning the needle tip with the direction of the rat's head. The blunt needle was inserted to a depth of 5.0–5.5 mm from the dura mater at a rate of 1 mm/min so that the puncture site was aligned with the cerebellum. Using the microinjection pump, 400 μL or 200 μL of heparin‐free arterial blood was slowly infused into the fourth ventricle at a rate of 20 μL/min through the posterior median foramen. In the sham‐operated group, 400 μL of saline was infused at the same rate. After autologous blood or saline injection was complete, the needle was left in place for 15 min. The needle was then withdrawn at a rate of 1 mm/min, and the muscle and skin incisions were closed with sutures (Figure 2). The rats were removed from the stereotaxic apparatus and placed on a 37°C heating pad. Once the rats recovered from anesthesia, they were returned to their cages.
FIGURE 2.
Procedure to construct the rat model of fourth ventricular hemorrhage. Photographs (A–J) and schematic illustrations (K) of the procedure used to construct the rat model of fourth ventricular hemorrhage. The rat's head was positioned downward, with ear rods inserted into the external auditory canals and adjusted so that the rat's head was centered in the stereotactic apparatus at a 20° angle to the vertical (A). After disinfection of the scalp, an incision was made along the midline to expose the midline (B, arrow). A scalpel was used to cut through the muscles along the midline, and blunt dissection with a retractor was used to expose the posterior median foramen (C, arrow). The fixator was released, and the rat was rotated to expose the chest. A 1 mL syringe was inserted at an approximately 15° angle beneath the xiphoid process until the needle tip reached a site of strong cardiac pulsation. Blood was aspirated after pulsation was observed, and pressure was applied with a sterile cotton swab to the puncture site for approximately 1 min after needle removal (D). The rat was repositioned, with the head downward at a 20° angle to the vertical. The arm of the stereotactic apparatus was swung backward to a 20° angle with the vertical, and a 1 mL syringe with a blunt needle was mounted on a microinjection pump. The blunt needle was adjusted so that the opening faced forward and was inserted through the small hole created at the posterior median foramen to a depth of 5.0–5.5 mm from the dura mater at a rate of 1 mm/min to access the fourth ventricle. Autologous blood was infused at a rate of 40 μL/min or 20 μL/min (E–H). High‐magnification image of the needle insertion site at the posterior median foramen (I, J, white circle).
2.4. Small animal MRI 7.0T
Postoperative MRI scans were conducted on days 1, 14, and 28 at the Small Animal MRI Facility of the Field Surgery Institute, Daping Hospital, Army Medical University. With the assistance of staff, cranial MRI examinations were performed to observe and measure the fourth ventricle hematoma volume and the lateral ventricle hydrocephalus volume, thereby evaluating the success, reliability, and stability of the model. Initially, the rats were anesthetized using 2% isoflurane mixed with a 70/30 ratio of nitrous oxide and oxygen. The incisors of the rats were secured to the MRI coil, and anesthesia was maintained with 1.5% isoflurane during the scan. MRI scans were performed using T2WI in both the coronal and sagittal planes, with the room temperature maintained at approximately 20°C. Following the scans, the rats were allowed to recover freely. The T2WI scan parameters were as follows: slice thickness, 1.0 mm; field of view, 35 mm × 35 mm; pixel resolution, 256 × 256; and TR/TE, 3000 ms/45 ms. Images were exported using RadiAnt DICOM Viewer software.
2.5. Calculation of the hematoma volume and the extent of hydrocephalus
In ImageJ software, the MRI images to be analyzed were opened, and the “Polygon selection” tool was selected. The fourth ventricle hematoma and regions of lateral ventricle damage were outlined, and the “Analyze” and “Measure” functions were used to obtain the corresponding area measurements. The measured areas were exported to the appropriate Excel sheet, and the areas of the fourth ventricle hematoma and regions of lateral ventricle damage were summed across all slices to obtain the total area. The following formulas were used: fourth ventricle hematoma volume = total area of the fourth ventricle hematoma × slice thickness × number of slices × (35 × 35 / 256 × 256) mm3; lateral ventricle volume = total area of both lateral ventricles × slice thickness × number of slices × (35 × 35 / 256 × 256) mm3.
2.6. Neurological assessment and survival analysis
Neurological function scores were determined for each group on days 1, 3, 7, 14, and 28 post‐modeling. The Zea–Longa scoring method 10 and modified Garcia scoring system 11 were employed to evaluate changes in neurological function. The former method is used to evaluate motor function after cerebral hemorrhage, while the latter method covers many aspects such as movement, sensation and reflex, providing a more comprehensive analysis of neurological function.
2.7. Survival analysis
The survival endpoint was defined as natural death. All animals were monitored daily for survival status and behavioral performance post‐surgery, with the observation period lasting 14 days. Survival time was calculated from the start of surgical intervention until the occurrence of an endpoint event or the end of the observation period. The following data were recorded: survival days, endpoint event status (1 = death, 0 = censored), and covariates (such as body weight). Statistical methods used Kaplan–Meier survival analysis to evaluate cumulative survival rates across groups, visualizing survival trends with stepwise curves, and comparing differences in survival curves between groups using the Log‐rank test.
2.8. Behavioral assessment
Animals in each group were subjected to the balance beam test on days 1, 3, 7, 14, and 28 post‐modeling. A balance beam measuring 80 cm in length and 2.5 cm in width was placed horizontally between two platforms, and rats in each group were allowed to walk independently across the beam. The rats were assessed on the basis of six criteria to determine whether they could use their limbs symmetrically to reach the platform, with scores ranging from 0 (normal) to 6. The score for each animal was averaged over three trials.
2.9. Histopathological examination
2.9.1. Toluidine Blue staining
Twenty‐four hours after the establishment of the model, male SD rats were anesthetized again. After perfusion and fixation with saline and 4% paraformaldehyde, the brain tissue was collected. Fresh brain tissue was fixed in 10% neutral formalin solution for 48 h, followed by routine dehydration, embedding, and deparaffinization. The tissue was then washed with distilled water and immersed in Toluidine Blue dye at 50–60°C for approximately 30 min. After staining, the tissue was gently washed with distilled water and then quickly rinsed with 70% ethanol solution, followed by differentiation with 95% ethanol. After differentiation, the tissue was dehydrated with anhydrous ethanol, cleared in xylene, and mounted with neutral resin. The process tissue was observed and photographed under an inverted microscope.
2.9.2. Nissl staining
The selected area for the model group was the brainstem tissue under the compression of hematoma. The same area was selected for both the control group and the sham operation group. The Nissl bodies were counted under a high‐power microscope at 400×. The ImageJ image analysis software was used to semi‐automatically count the number of Nissl bodies in this specific brain region. Five visual fields were randomly selected for counting, and then, the average value was calculated to represent the number of Nissl bodies in this region.
2.9.3. Evans Blue staining
Twenty‐four hours after model establishment, male SD rats were anesthetized again. Evans Blue solution (2% concentration, 4 mL/kg) was administered via the tail vein or femoral vein. After 1 h of circulation, the blood was cleared using saline. The brain was then collected and examined under a fluorescence microscope at an excitation wavelength of 540 nm to observe the distribution of cortical vessels and Evans Blue in the cortical tissue. Following gross observation, coronal brain sections were examined for Evans Blue fluorescence.
2.9.4. HE staining
Brains were perfused with ice‐cold saline and 250 mL of 4% paraformaldehyde through a 50 mL syringe until the rats' paws and liver turned white. The head was then severed, and the brain was collected. Following paraformaldehyde fixation, the tissue was dehydrated, embedded, sectioned, and stained with HE.
2.9.5. Transmission electron microscopy
Brain tissue was quickly collected on ice 24 h post‐modeling and fixed with glutaraldehyde at 4°C for 4 h. The samples were then stained in a 1% osmic acid solution at 20°C for 2 h, followed by gradient ethanol dehydration. The tissue was infiltrated with a mixture of acetone and epoxy resin (2:1) followed by acetone and epoxy resin (1:1) and finally with pure resin at 37°C. The tissue was embedded in epoxy resin, and ultrathin sections (70 nm thick) were prepared. The sections were double stained with lead citrate and uranyl acetate for 15 min, and images were collected using an electron microscope.
2.9.6. TUNEL staining
Twenty‐four hours after model establishment, brain tissue was rapidly collected on ice. Following the procedures provided by the manufacturer of the TUNEL kit, the tissue was fixed, sectioned, and washed. TUNEL working solution was added, and the sections were incubated at 37°C in the dark. After dehydration, the sections were mounted and examined, and images were processed using Image‐Pro 6.2 software to analyze the percentage of apoptotic cells in the brain tissue.
2.10. Statistical analysis
The measured values are expressed as the means ± standard deviations (x ± s), and statistical analyses were performed using SPSS software. The statistical tests used included independent samples t tests (for comparisons between two groups) and Mann–Whitney U tests. p < 0.05 was considered to indicate statistical significance.
3. RESULTS
3.1. Survival analysis and physiological parameters
By injecting varying volumes of autologous blood into the fourth ventricle, we established a fourth ventricle hemorrhage animal model and recorded the survival rate and body weight changes of the rats at fixed time points daily. The survival rates of the rats in the 400 μL saline injection group and the control group did not differ significantly (p > 0.05). However, a significant difference was observed between the control group and the 200 μL autologous blood injection group (p < 0.05), as well as between the control group and the 400 μL autologous blood injection group (p < 0.01) (Figure 3). The time point at which 50% mortality was reached in the 400 μL autologous blood injection model group was 192 h. The body weights of the rats were recorded before the procedure and at 1, 3, 7, 14, and 28 days post‐modeling. A downward trend in body weight was observed after modeling, with significant differences in body weight between the control group and the 200 and 400 μL autologous blood injection groups by the third day (p < 0.05). The lowest body weight was reached on day 7, after which the body weight gradually increased. By day 28, the body weights of rats in each group were significantly different from their body weight before modeling (p < 0. 0001) (Figure 4).
FIGURE 3.
Survival curves of different groups following the establishment of the fourth ventricular hemorrhage rat model. The survival rate of the rats in the 400 μL saline injection group did not differ significantly from that of the rats in the control group (p > 0.05). The survival rate of the 200 μL autologous blood injection group was significantly different from that of the control group (p < 0.05), and the 400 μL autologous blood injection group demonstrated a marked difference compared to the control group (p < 0.01, n = 11). (ns: p > 0.5; *p < 0.05; **p < 0.01).
FIGURE 4.
Changes in the body weights of rats in the different groups following the establishment of the fourth ventricular hemorrhage model. On the third day after modeling, significant differences in body weight were observed between the control group and the 200 μL and 400 μL autologous blood injection groups (3 days: Control group vs. 200 μL blood + IVH group, p < 0.05; control group vs. 400 μL blood + IVH group, p < 0.05). The lowest body weight was observed on the seventh day (7 days: Control group vs. 200 μL blood + IVH group, p < 0.05; control group vs. 400 μL blood + IVH group, p < 0.01), after which the body weight gradually recovered, with each group showing significant differences by the 28th day after modeling compared with before modeling (control group: 0 vs. 14, p < 0.01; 0 vs. 28, p < 0. 0001; 400 μL vehicle + IVH group, 200 μL blood + IVH group, 400 μL blood + IVH group: 0 vs. 14, p > 0.5; 0 vs. 28, p < 0.01) (ns: p > 0.5; *p < 0.05; **p < 0.01; ****p < 0. 0001).
3.2. Fourth ventricle hemorrhage volume and lateral ventricular hydrocephalus‐related changes
The injection of autologous blood to construct a fourth ventricle hemorrhage animal model resulted in clear fourth ventricle casts on T2‐weighted MR images (Figure 5). The volume of blood injected was reliably correlated with the hemorrhage volume measured by MRI at 1, 14, and 28 days post‐injection. No hemorrhage was observed in the control or sham surgery group. The hemorrhage volume in the autologous blood injection groups decreased during the follow‐up period, primarily due to the absorption of the intraventricular hemorrhagic components (Figure 5A), resulting in a significant reduction in hemorrhage volume (Figure 5C). During the follow‐up period, the lateral ventricles of the model group animals were expanded (Figure 5B). Compared with the sham surgery group, the 200 and 400 μL autologous blood injection groups presented more severe lateral ventricular hydrocephalus at all follow‐up time points. However, over time, the severity of lateral ventricular hydrocephalus in the autologous blood injection groups gradually decreased due to intraventricular absorption, although it remained greater than that in the sham surgery group (Figure 5D).
FIGURE 5.
Changes in hemorrhage volume in the fourth ventricle and hydrocephalus in the lateral ventricles. (A) Representative sagittal and coronal T2‐weighted MR images from each group during follow‐up. (B) Representative sagittal and coronal T2‐weighted MR images from each group at 1 day and 28 days post‐modeling. Compared with that in the sham surgery group, the severity of lateral ventricular hydrocephalus in the 200 μL and 400 μL autologous blood injection groups increased as the hemorrhage area expanded. (C) Measurement of hemorrhage development during follow‐up on the basis of T2‐weighted MR images. The blood volume measured by MRI at 1 day, 14, and 28 days post‐injection was reliably correlated with the volume of blood injected. No hemorrhage was observed in the control or sham surgery groups, whereas the hemorrhage volume in the autologous blood injection groups tended to decrease, primarily due to the absorption of intraventricular hemorrhage components. (D) Evolution of the severity of lateral ventricular hydrocephalus following the establishment of the fourth ventricular hemorrhage model. Significant increases in the severity of lateral ventricular hydrocephalus were observed at all follow‐up time points in the 200 μL and 400 μL autologous blood injection groups compared with the sham surgery group. Over time, the area of lateral ventricular hydrocephalus decreased.
3.3. Neurological function changes
Neurological function changes were assessed using the Zea–Longa scoring method and the modified Garcia scoring system following the establishment of the fourth ventricle hemorrhage rat model. There were no significant differences in neurological scores on days 1, 3, and 7 between the 200 μL autologous blood injection group and the control group. However, the 400 μL autologous blood injection group exhibited significant differences in neurological function at these time points. Over time, as the fourth ventricle hematoma was absorbed, the neurological function of the rats gradually improved, with no significant differences observed by day 14 (Figure 6A,B).
FIGURE 6.
Changes in neurological function following the establishment of the fourth ventricular hemorrhage rat model. (A) Modified Garcia scores for each group of rats at different follow‐up time points. Following the establishment of the fourth ventricular hemorrhage rat model, no significant differences in neurological function scores were observed between the 200 μL autologous blood injection group and the control group at 1, 3, or 7 days (p > 0.05). However, significant differences in neurological function were noted in the 400 μL autologous blood injection group (p < 0.001). As time progressed and with the absorption of the hemorrhage in the fourth ventricle, neurological function gradually improved, with no significant differences at 14 days (p > 0.05). ns: p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. (B) Zea‐Longa scores for each group of rats at different follow‐up time points. Following the construction of the fourth ventricular hemorrhage model, no significant differences in neurological function scores were found between the 200 μL autologous blood injection group and the control group at 1 or 3 days (p > 0.05). However, significant differences were observed in the 400 μL autologous blood injection group (p < 0.001). As in panel A, neurological function gradually improved over time, with no significant differences by day 14 (p > 0.05). ns: p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. (C) Balance beam scores for each group of rats at different follow‐up time points. Following the construction of the fourth ventricular hemorrhage model, no significant differences in neurological function were observed between the sham surgery group and the control group (p > 0.05). Significant differences were noted between the 200 μL and 400 μL autologous blood injection groups (p < 0.05), with the 400 μL autologous blood injection group showing more pronounced differences (p < 0.0001). Due to the absorption of the hemorrhage, the 200 μL autologous blood injection group showed no significant differences in neurological function by day 14 (p > 0.05). ns: p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
The balance beam test demonstrated that all groups had normal neurological function prior to surgery. At different time points following the establishment of the fourth ventricle hemorrhage model, there were no significant differences in neurological function between the sham‐operated group and the control group (p > 0.05). In contrast, the neurological function of the 200 and 400 μL autologous blood injection groups significantly differed from that of the control group (p < 0.05), with the 400 μL group exhibiting a more pronounced difference (p < 0.0001). Due to hematoma absorption in the model groups, the neurological function of the 200 μL autologous blood injection group did not significantly different from that of the control group by day 14. At various follow‐up time points, the 400 μL autologous blood injection group presented poorer neurological function than did the 200 μL autologous blood injection group (Figure 6C).
3.4. Pathological findings
Compared with the control group, the model group presented a significant reduction in the number of Nissl bodies (p < 0.01) and a notable increase in neuronal apoptosis (p < 0.01). The number of Nissl bodies in brain tissue was significantly lower in the 400 μL autologous blood injection group than that in the control group (p < 0.01) (Figure 7A,D). Histological analysis via HE staining indicated that the morphology of the brainstem tissue was largely preserved in the control and sham groups. However, in the 200 μL autologous blood injection group, the brainstem showed signs of nuclear pyknosis, inflammatory cell infiltration, and neuronal edema and necrosis. In the 400 μL autologous blood injection group, the choroid plexus was disrupted, and damage to the brainstem tissue was more severe than that in the 200 μL autologous blood injection group (Figure 7B). Transmission electron microscopy revealed severe mitochondrial edema in the 200 μL autologous blood injection group, whereas cellular structural damage was evident in the 400 μL autologous blood injection group (Figure 7C,E). These findings collectively indicate that the 400 μL autologous blood injection group exhibited more extensive damage to the tissue structures surrounding the fourth ventricle than did the 200 μL autologous blood injection group.
FIGURE 7.
Histological examination of the fourth ventricular hemorrhage rat model. (A, D) Nissl body quantification in the different groups. Compared with the control group, the sham surgery group presented no significant reduction in the number of Nissl bodies, whereas a significant decrease in the number of Nissl bodies was observed in the model groups (p < 0.01); this change was accompanied by a significant increase in neuronal apoptosis (p < 0.01). In the 400 μL autologous blood injection group, the number of Nissl bodies in brain tissue was significantly reduced (p < 0.01). (B) Histological examination via HE staining revealed that the morphology of brainstem tissue was generally normal in the control and sham surgery groups. In the 200 μL autologous blood injection group, brainstem tissue exhibited disruption and rupture of the ependymal membrane, shrinkage of brainstem nuclei, inflammatory cell infiltration, and neuronal edema and necrosis. The 400 μL autologous blood injection group exhibited more severe damage to brainstem tissue. (C, E) Electron microscopy results demonstrated severe mitochondrial swelling in the 200 μL autologous blood injection group. In the 400 μL autologous blood injection group, alterations in cellular morphology were evident (p < 0.01). ns: p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
The TUNEL assay results demonstrated that, compared with the control group, the 200 μL autologous blood injection group presented stronger red fluorescence signals, representing apoptotic cells, with significantly stronger signals observed in the 400 μL autologous blood injection group. Evans Blue staining indicated that the blood–brain barrier was disrupted in the 200 μL autologous blood injection group but not in the control group, with more pronounced disruption observed in the 400 μL autologous blood injection group (Figure 8A–D).
FIGURE 8.
TUNEL staining and Evans Blue extravasation of the fourth ventricular hemorrhage rat model. (A, C) TUNEL staining results revealed stronger red fluorescence signals, representing apoptotic cells, in the 200 μL autologous blood injection group than in the control group, with a significant increase in the 400 μL autologous blood injection group (p < 0.001). (B, D) Evans Blue extravasation revealed blood–brain barrier disruption in the 200 μL autologous blood injection group compared with the control group, with more pronounced disruption in the 400 μL autologous blood injection group (p < 0.001). ns: p > 0.05; **p < 0.01; ****p < 0.0001.
4. DISCUSSION
We have described a novel rat model of fourth ventricle hemorrhage. Under general anesthesia, varying volumes autologous blood were stereotaxically injected to create animal models with different degrees of fourth ventricle hemorrhage. MRI, which reliably revealed stable and reproducible fourth‐ventricle hematomas, was performed immediately after surgery. The volume of blood injected in the different groups corresponded reliably with the total hemorrhage volume measured by MRI via T2‐weighted sequences, providing strong evidence for the model's reliability and reproducibility. The neurological functional scoring results confirmed that this model effectively recapitulates the pathophysiological characteristics of clinical patients.
During the follow‐up period, the hematoma volume determined by T2WI continued to decrease in all groups, 7 which is consistent with findings from previous ICH studies utilizing autologous injection models. 10 This process is influenced by several factors. First, natural absorption of the hematoma is an important factor. The body has inherent repair and absorption mechanisms, and over time, the blood components within the hematoma are gradually broken down and absorbed. 8 Second, the inflammatory response and immune regulation may also play a role in the absorption of the hematoma. Immune cells such as macrophages can phagocytose and remove necrotic tissue and blood cells within the hematoma, facilitating its resolution. 9 Furthermore, angiogenesis and tissue remodeling may contribute to the repair and functional recovery of the surrounding tissue, thereby indirectly promoting a reduction in hematoma volume. 12 All the animals exhibited significant neurological deficits, with mortality rates increasing with increasing hemorrhage volume. Direct compression by the hematoma is a major factor contributing to neurological dysfunction. 13 Additionally, secondary injuries triggered by hematomas can exacerbate neurological impairment. For example, the brain tissue surrounding a hematoma may be subjected to ischemia, hypoxia, and inflammatory responses, leading to neuronal damage and cell death. 14 Furthermore, intraventricular hemorrhage may disrupt CSF circulation, resulting in hydrocephalus, which further elevates intracranial pressure and impairs neurological function. 15 , 16 , 17 Compared with the control and sham‐operated groups, the 400 and 200 μL autologous blood injection groups presented significant differences in neurological deficits between days 1 and 7, with the 400 μL group showing more severe neurological impairment than the 200 μL group. However, over time, as the hematoma in the fourth ventricle was absorbed, the neurological function of the 200 μL autologous blood injection group gradually improved, showing no significant difference from that of the control group by day 14, whereas the 400 μL autologous blood injection group continued to show differences. Pathological examinations further confirmed that the 400 μL autologous blood injection group had a significantly reduced number of Nissl bodies in brain tissue. HE staining revealed choroid plexus disruption, as well as more pronounced neuronal shrinkage, inflammatory cell infiltration, and edema‐necrosis in the brainstem in the 400 μL autologous blood injection group than in the 200 μL autologous blood injection group. TUNEL staining revealed more severe apoptosis of brainstem cells, and Evans Blue staining indicated more significant disruption of the blood–brain barrier in the 400 μL autologous blood injection group.
When hemorrhage in the fourth ventricle expands, it exerts pressure on the brainstem, leading to severe structural and functional consequences and resulting in a range of adverse outcomes. First, compression of the brainstem disrupts the normal function of the neural nuclei within it, affecting the regulation of vital signs such as respiration and cardiovascular activity and potentially leading to life‐threatening conditions such as respiratory rhythm disturbances and blood pressure instability. 18 Second, pressure from the hematoma can damage the brainstem's conduction pathways, impairing the transmission of neural signals and causing neurological deficits such as limb paralysis and sensory disturbances. 19 Additionally, prolonged compression may induce ischemia, hypoxia, and inflammatory responses in brainstem tissue, further exacerbating neurological damage and potentially leading to irreversible impairment. 20 , 21 Furthermore, hemorrhagic CSF can have detrimental effects on the nervous system, including disruption of the choroid plexus and the blood–brain barrier. 16 , 22 , 23 If this condition persists, it may result in decreased consciousness or even death. Therefore, fourth ventricle hemorrhage expansion is an independent risk factor for poor prognosis in ventricular hemorrhage patients, which is consistent with the findings of numerous clinical studies. However, there is currently a lack of animal models specifically of fourth ventricle hemorrhage, with most existing models simulating mainly lateral ventricular hemorrhage. 24 , 25 Thus, developing such a model is essential for understanding the pathophysiological mechanisms of fourth ventricle hemorrhage. In this study, we developed an innovative animal model of fourth ventricle hemorrhage that requires a minimally intensive anatomical approach in which the brain is accessed through the posterior median foramen without damaging the cerebral cortex for the direct injection of autologous blood. This method significantly minimizes the operation time and allows the model to accurately recapitulate the clinical imaging characteristics of fourth ventricle hemorrhage. In contrast, conventional models typically involve positioning, cranial trepanation, and vertical needle insertion for autologous blood injection, which can damage the cerebral cortex.
Using this innovative autologous blood injection method for constructing an animal model of fourth ventricle hemorrhage, we successfully and reliably simulated the pathophysiological process of fourth ventricle hemorrhage expansion. However, several limitations need to be addressed. First, the autologous blood injection method used to construct this model does not replicate the natural pathological event of cerebral vessel rupture observed in ventricular hemorrhage patients. Second, since 65% of intracerebral hemorrhages (ICHs) associated with ventricular hemorrhage occur in hypertensive individuals, future studies could be performed to develop a spontaneous fourth ventricle hemorrhage model in hypertensive rats. 11 Third, the use of SD rats in this model makes it difficult to perform genetic manipulations, so mice could be utilized for modeling in future studies, which would facilitate the exploration of pathological mechanisms and treatments. Additionally, since clinical ventricular hemorrhage is more common in elderly individuals, the development of a model in aged rats would be valuable for clinical translation purposes. 26
In summary, we established a fourth ventricle hemorrhage rat model by injecting autologous blood through the posterior median aperture, simulating the high mortality and severe neurological deficits observed in clinical cases of fourth ventricle hemorrhage. This model provides a reproducible and stable animal model for future experiments, offering the potential to mimic the pathophysiological characteristics of clinical patients and to explore the mechanisms of injury associated with fourth ventricle hemorrhage, thereby laying an experimental foundation for clinical treatment.
AUTHOR CONTRIBUTIONS
Chuhua Fu: Conceptualization; data curation; formal analysis; funding acquisition; investigation; methodology; visualization; writing – original draft. Aoqi Liu: Data curation; formal analysis; investigation; methodology; validation; visualization; writing – original draft. Yijia He: Data curation; formal analysis; investigation; methodology. Fei Li: Conceptualization; formal analysis; methodology; resources. Zhouyang Jiang: Data curation; formal analysis; investigation; methodology. Peiwen Guo: Data curation; formal analysis; funding acquisition; investigation; methodology. Zhi Chen: Conceptualization; funding acquisition; methodology; resources; supervision. Yujie Chen: Conceptualization; funding acquisition; methodology; project administration; resources; supervision; writing – review and editing. Hua Feng: Conceptualization; funding acquisition; methodology; resources; supervision.
FUNDING INFORMATION
This work was supported by the Natural Science Foundation of Hubei Province (Grant No. 2024AFB877 to Chuhua Fu), the Chongqing Medical Scientific Research Project (Joint Project of Chongqing Health Commission and Science & Technology Bureau, Grant No. 2023GGXM003 to Hua Feng), the Chongqing Municipal Health Commission (YXGD202451 to Yujie Chen), National Natural Science Foundation of China (Grant No. 82371361 to Zhi Chen), the Organization Department of Chongqing Municipal Party Committee (Grant No. cstc2024ycjh‐bgzxm0103 to Peiwen Guo), Jingmen Science and Technology Bureau (Grant No. 2024ZDYF012 to Chuhua Fu).
CONFLICT OF INTEREST STATEMENT
The authors declare that they have no competing interests.
ETHICS STATEMENT
All experiments were approved by the Laboratory Animal Welfare and Ethics Committee of Third Military Medical University (AMUWEC20232125) and reported in accordance with the ARRIVE 2.0 guidelines.
ACKNOWLEDGMENTS
We would like to thank Xudong Ding from the Xiangyang No.1 People's Hospital for cooperation.
Fu C, Liu A, He Y, et al. Establishment and evaluation of a novel rat model of the fourth ventricle hemorrhage. Anim Models Exp Med. 2025;8:1480‐1492. doi: 10.1002/ame2.70049
Chuhua Fu and Aoqi Liu contributed equally to this work.
Contributor Information
Yujie Chen, Email: chenyj@tmmu.edu.cn, Email: yujiechen6886@foxmail.com.
Hua Feng, Email: fenghua@tmmu.edu.cn, Email: fenghua8888@vip.163.com.
DATA AVAILABILITY STATEMENT
All the data generated or analyzed during this study are included in this published article. The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
All the data generated or analyzed during this study are included in this published article. The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.