Modeling of malignant middle cerebral artery infarct in rabbits

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Introduction

Malignant ischemic stroke (MIS) in the middle cerebral artery (MCA) territory is characterized by extensive (involving at least 50% of the MCA territory) hemispheric infarction [1, 2]. The resulting massive post-ischemic edema leads to brain herniation [3, 4]. Poor outcomes remain high with both conservative (up to 80%) [5, 6] and surgical (up to 55%) treatment [7, 8]. Advances in treatment and understanding of pathophysiological mechanisms depend on modeling MIS in experimental animals. Numerous MCA stroke models have been described: endovascular MCA occlusion; MCA embolization; photothrombosis; craniotomy with MCA coagulation; endothelin-1-based models; and thrombin injection into the MCA [9–11]. However, many drugs and treatments that show high efficacy in rat models have failed in human trials [12, 13]. Moreover, rats have well-developed leptomeningeal collaterals, complicating reproduction of the clinical stroke picture [14]. Existing stroke models have both advantages and limitations, and the choice of model depends on the research question. The strengths and weaknesses of established stroke models are summarized in Table 1.

 

Table 1. Advantages and disadvantages of focal ischemia models

Model	Advantages	Disadvantages
Endovascular MCA occlusion	Etiologically closest to the clinical mechanism of stroke. High reproducibility. No craniotomy required	Quality and extent of ischemia depend on correct monofilament placement in the MCA. Complete occlusion of the proximal MCA segment is not achievable. Risk of vessel wall perforation by the monofilament. Persistent retrograde collateral flow
Embolic models	Multifocal lesions. Most suitable model for studying thrombolytic therapy	Risk of spontaneous thrombus recanalization during the experiment. Relatively low reproducibility of infarct location and volume. High variability in infarct size
Photothrombotic MCA model	Simple technique; high animal survival	Requires specialized equipment and consumables. Infarct size is difficult to control
Craniotomy with MCA coagulation	Direct visualization allows the investigator to identify the target MCA segment and confirm adequate occlusion	Persistent collateral flow due to variability in cortical branch anatomy. Invasiveness of the procedure
Endothelin-1-based models	Focal cortical lesion	Variable infarct size; unpredictable spread of ischemia; transient vasospastic effect of endothelin-1. High cost of endothelin-1

 

Proper identification of the ICA and exclusion of collateral circulation are critical steps for successful modeling, as collaterals limit the full-scale malignant ischemic stroke. Therefore, identifying and characterizing the topographic-anatomical features of the ICA is essential for creating a clinically relevant MCA territory MIS model.

The study aim is to develop an experimental model of MIS in the MCA territory that closely mimics the clinical setting.

Study objectives:

• to study the topographic-anatomical variants of the ICA;
• to perform a comparative analysis of the outcomes of MIS modeling in the MCA territory.

Materials and methods

The study was conducted on 40 male California rabbits weighing 2 kg at 2 months of age. All animal procedures and housing complied with international Good Laboratory Practice guidelines and Russian methodological standards for laboratory animal care (GOST 33216-2014). Carotid territory anatomy was studied in 10 rabbits after euthanasia by anesthetic overdose. A midline skin and soft tissue incision was made on the ventral surface of the neck, and the common carotid artery (CCA) was bluntly dissected. The CCA was then punctured proximal to its bifurcation, and polymerizing colored silicone was injected. Subsequent dissection of the CCA and its branches was performed, and the brain was removed to examine the intracranial portion of the ICA.

MIS modeling in the MCA territory was performed in 30 California rabbits weighing 2 kg at 2 months of age, divided into 2 groups using simple randomization. In Group 1 (n = 15), MIS was modeled by occluding the MCA with two monofilaments. In Group 2 (n = 15), in addition to MCA occlusion with two monofilaments, decompressive craniectomy and coagulation of cortical MCA branches were performed.

Inclusion criteria:

• persistent postoperative neurological deficit consistent with the affected vascular territory;
• neurological deficit severity score of 5 or more on the Neurological Assessment Scale (NAS);
• evidence of extensive hemispheric infarction in the MCA territory confirmed by postoperative brain multislice computed tomography (MSCT);
• confirmation of two monofilaments positioned in the proximal MCA segment on post-mortem or post-euthanasia brain examination.

Exclusion criteria:

• intraoperative death;
• NAS score < 5 during follow-up;
• absence of extensive MCA territory infarction on postoperative brain MSCT;
• only one monofilament found in the proximal MCA segment on post-mortem or post-euthanasia brain examination.

Surgical technique. All procedures were performed under anesthesia using a diluted solution of Zoletil-100 (0.3 mL/kg) and xylazine (0.5 mL/kg). Surgery was conducted under sterile conditions using a 4× magnifying head loupe. The monofilament used was a 5-cm non-absorbable 6-0 Prolene suture, with the tip shaped into a ball by heating over a flame.

Surgical procedure. In Group 1 rabbits, the neck was shaved; in Group 2 animals, the scalp was additionally shaved. Surgery was performed with the rabbit in the supine position, with all four limbs secured. Blood pressure, heart rate, and oxygen saturation were continuously monitored throughout the procedure. The ventral cervical operative field was treated with antiseptic solution (0.05% chlorhexidine in alcohol) and 10% lidocaine for local anesthesia. A 6-cm midline skin and soft tissue incision was made on the ventral surface of the neck, centered at the level of an imaginary horizontal line connecting the mandibular angles. Blunt dissection was used to retract muscles and soft tissues until the CCA was visualized lateral to the trachea. After retracting the sternohyoid and sternocleidomastoid muscles, the CCA was bluntly dissected on the left side, exposing its bifurcation into the ICA and external carotid artery (ECA). To accurately identify the ICA, its course from the CCA bifurcation to the external carotid foramen was traced. The CCA was then permanently ligated 2–3 mm proximal to the ICA origin, and the ECA was ligated distally. The occipital artery, identified by its caudal course, was also ligated using 4-0 Vicryl. A temporary clip was then placed on the ICA to prevent retrograde flow. After permanent arterial ligation, an arteriotomy was made in the CCA using micro-scissors over the ICA origin to allow direct, unobstructed monofilament passage. A monofilament was then inserted into the ICA lumen using forceps while simultaneously removing the temporary clip. The filament was advanced slowly until mild resistance was felt. A second 5-cm monofilament was then inserted until it stopped advancing. After both filaments were placed, a ligature was tied around the ICA to secure them. During insertion, the filament could be visualized through the ICA wall, confirming vessel wall integrity and correct intracranial direction. The wound was then closed with a continuous 3-0 Vicryl suture. Operating time in Group 1 ranged from 20 to 37 min (mean ± SE: 30.6 ± 1.2 min). A schematic representation of monofilament placement for proximal M1 MCA occlusion is shown in Figure 1.

 

Fig. 1. Schematic of M1 segment MCA occlusion using two monofilaments.

A — basal view: 1 — CCA ligature; 2 — monofilaments in the ICA; 3 — ECA ligature; 4 — ICA ligature; 5 — tip of the first monofilament in M1; 6 — olfactory tract; 7 — lenticulostriate arteries; 8 — tip of the second monofilament in M1.

B — anterior view: 1 — CCA; 2 — tip of the first monofilament in M1; 3 — olfactory tract; 4 — lenticulostriate arteries; 5 — tip of the second monofilament in M1.

C — lateral view: 1 — CCA ligature; 2 — monofilaments in the ICA; 3 — ECA ligature; 4 — ICA ligature; 5 — lenticulostriate arteries; 6 — olfactory tract; 7 — tip of the first monofilament in M1; 8 — tip of the second monofilament in M1.

 

In Group 2 rabbits, the same procedure was performed, with the addition of decompressive craniectomy followed by coagulation of cortical arteries. A two-stage skin incision was made: first, a 6-cm incision along the sagittal suture, followed by a perpendicular incision from the midpoint of the zygomatic arch to intersect the midpoint of the first incision (Figure 2).

 

Fig. 2. Stages of skin incision.

A: 1 — midline longitudinal skin incision. B: 1 — longitudinal skin incision; 2 — second skin incision perpendicular to the first midline incision. C: 1 — skin flap elevated and reflected posteriorly and inferiorly; 2 — temporalis muscle dissected and retracted toward the skull base.

 

Next, the aponeurosis was dissected for subsequent dural repair. Cranial bones (nasal, frontal, parietal, occipital, and temporal bones down to the middle cranial fossa) were resected to create a craniectomy window with the following boundaries: no more than 5 mm lateral to the sagittal suture, 15 mm anterior to the coronal suture, and 5 mm posterior to the lambdoid suture (Figure 3). The dura mater was then opened with several radial incisions. Performed coagulation of cortical arteries. Performed coagulation of cortical arteries and plastic the dura mater with aponeurosis. The wound was closed in layers with a continuous 3-0 Vicryl suture. Operating time in Group 2 ranged from 45 to 50 min (mean ± SE: 47.4 ± 0.5 min).

 

Fig. 3. View after decompressive craniectomy and dural opening.

A: 1 — convexital arteries of the cerebral hemisphere; 2, 3 — dural flaps after opening. B: 1 — dural flap reflected caudally toward the skull base; 2 — view after coagulation of cortical arteries.

 

After surgery, the rabbit was placed in a cage with 24-hour video monitoring and access to food and water ad libitum. Hemodynamic and respiratory parameters were monitored. To prevent airway obstruction and death in the postoperative period, a 4.0-mm uncuffed endotracheal tube was inserted orally until spontaneous breathing stabilized and the animal fully recovered from anesthesia. Neurological deficit was assessed postoperatively using tests for functional impairment of the forelimbs and hindlimbs. Neurological status was evaluated using the Neurological Assessment Scale (NAS) [15] (Table 2).

 

Table 2. NAS for rabbits in the postoperative period

Test	Clinical criteria	Score
Behavior	Neck Twist	0 — normal; 1 — twist of the neck
Reflex	Righting reflex	0 — returns to baseline within 1 s; 1 — returns to baseline within 5 s; 2 — fails to return to baseline within 5 s.
Reflex stimulation	Extension reflex (forepaws)	0 — returns forepaws to baseline within 1 s; 1 — returns forepaws to baseline within 5 s; 2 — fails to return forepaws to baseline within 5 s
Reflex stimulation	Extension reflex (hindpaws)	0 — returns hindpaws to baseline within 1 s; 1 — returns hindpaws to baseline within 5 s; 2 — fails to return hindpaws to baseline within 5 s
Posture	Posture	0 — normal resistance to contralateral push; 1 — reduced resistance to contralateral push; 2 — falls with contralateral push
	Death	Maximum score in case of death: 10
Total score		10

 

On postoperative day 1, rabbits underwent MSCT using a Toshiba Aquilion 64-slice scanner and brain magnetic resonance imaging (MRI) on a Philips Achieva 1.5 T system. Infarct volume in the affected hemisphere was calculated from MSCT images using the formula:

$A\times B\times C/2$,

where A is the largest infarct diameter; B is the longest perpendicular measurement of the infarct relative to A on the axial section; and C is the total number of sections containing the infarct on the coronal view [16]. The follow-up period for rabbits in both groups was 7 days, after which the animals were euthanized. Following death or euthanasia, the brain was removed for morphological examination.

Statistical analysis

Data were analyzed using descriptive statistics, including measures of central tendency (mean ± standard error of the mean [X̄ ± SE], median [Me]), interquartile range (IQR), absolute values, and proportions (%) for categorical variables. Prior to the study, a power analysis was performed to determine the required sample size for independent groups (n): α = 0.05; power = 0.95; expected effect size = 0.90; baseline SD = 0.5. Based on this analysis, 15 rabbits were assigned to each experimental group for MIS modeling. Group comparisons were performed using the non-parametric Mann–Whitney U test (Z). Associations between categorical and continuous variables with non-normal distributions were assessed using Spearman’s rank correlation coefficient. Survival proportions between the two groups were compared using Fisher’s exact test. Statistical significance was set at p < 0.05. Statistical analysis was performed using Statistica v. 6.0 (StatSoft Inc.).

The primary endpoint was confirmation of successful MIS modeling in the MCA territory, assessed by postoperative neurological deficit changes over time.

Secondary endpoints included:

• assessment of modeling efficacy using neuroimaging;
• confirmation of adequate MIS modeling in the MCA territory by histological examination.

No correction for multiple comparisons was applied due to the exploratory nature of the analysis.

Blinding was implemented during animal care and veterinary support.

Results

Topographic-anatomical characteristics of the internal carotid artery

The CCA courses cranially, running parallel to the lateral surface of the trachea alongside the vagus nerve. In the neck, the CCA lies medial to the vagus nerve, is visibly pulsatile, and is located medial and deeper than the jugular vein — an important anatomical landmark to consider during stroke modeling when dissecting the neurovascular bundle (Figure 4).

 

Fig. 4. Jugular vein after skin incision on the ventral surface of the rabbit neck.

A — arrows indicate the jugular vein; B — spatial relationship between the CCA and jugular vein: 1 — jugular vein (superficial); 2 — CCA (deep).

 

After dissection of the neurovascular bundle, the CCA is positioned at the center of a triangle bounded by three muscles: cranially by the digastric muscle, laterally by the sternocleidomastoid muscle, and medially by the sternohyoid muscle. Up to the level of the digastric muscle posterior belly, the superior thyroid artery, the superior laryngeal artery, and the ICA arise from the CCA. The superior thyroid artery originates from the medial surface of the CCA and courses medially toward the thyroid cartilage. The superior laryngeal artery also arises from the medial surface of the CCA and courses medially toward the larynx (Figure 5). Just cranially, over a short distance, the ICA branches off, running laterally and cranially toward the external carotid foramen to enter the cranial cavity.

 

Fig. 5. Left common carotid artery and its branches.

A: 1 — right superior laryngeal artery; 2 — sternohyoid muscle; 3 — right CCA; 4 — right vagus nerve; 5 — right jugular vein; 6 — sternocleidomastoid muscle; 7 — digastric muscle. B: 1 — digastric muscle; 2 — right superior laryngeal artery; 3 — right thyroid artery; 4 — right ICA; 5 — digastric muscle.

 

In our material, the CCA diameter ranged from 2.0 to 2.1 mm (mean 2.04 mm; median 2 mm), consistent with data from other authors [17].

After the ICA branches off from the CCA at the level of the thyroid cartilage, the artery continues as the external carotid artery (ECA). The terminal branches of the ECA are covered by the posterior belly of the digastric muscle. Therefore, visualization of the terminal ECA branches requires caudal retraction of the posterior belly of the digastric muscle (Figure 6).

 

Fig. 6. Terminal branches of the ECA.

A: 1 — posterior belly of the right digastric muscle (retracted caudally); 2 — right CCA; 3 — right ICA; 4 — microsurgical dissector; 5 — hypoglossal nerve loop; 6 — terminal branches of the ECA; 7 — right ECA.

B: 1 — right CCA; 2 — muscular branch from the CCA; 3 — right ICA; 4 — right occipital artery (branch of the ECA); 5 — external carotid foramen; 6 — internal maxillotemporal artery; 7 — external maxillary artery; 8 — lingual artery; 9 — external maxillolingual artery; 10 — superior thyroid artery; 11 — thyroid cartilage.

 

The hypoglossal nerve loop crosses the ICA, CCA, and ECA with its terminal branches, then courses cranially toward the root of the tongue (Figure 7). The hypoglossal nerve loop and the posterior belly of the digastric muscle serve as landmarks for correct identification of the extracranial ICA segment.

 

Fig. 7. Relationship of the hypoglossal nerve loop to the artery of carotid system.

1 — thyroid cartilage; 2 — right CCA; 3 — branches of the right ECA; 4 — root of the tongue; 5 — hypoglossal nerve; 6 — branches of the left ECA; 7 — left ICA; 8 — left CCA.

 

The ICA arises from the lateral surface of the CCA below the level of the posterior belly of the digastric muscle and courses laterally and cranially toward the external carotid foramen. The extracranial ICA segment length on both sides ranged from 1.7 to 2.0 mm (mean 1.8 mm; median 1.8 mm), with a diameter of 1.1–1.4 mm (mean 1.3 mm; median 1.3 mm). Two ICA variants were identified based on the origin of the occipital artery.

The first and more common variant (80%) has the ICA arising from the CCA as a single trunk, coursing laterally and cranially (Figure 8), with no branches from the extracranial ICA segment. In this variant, the occipital artery arises from the ECA as a separate trunk, running caudally and laterally, crossing the ICA and forming an X-shaped configuration. The occipital artery is smaller in diameter than the ICA, facilitating its identification.

 

Fig. 8. ICA.

A: 1 — terminal branches of the ECA; 2 — ECA trunk; 3 — CCA; 4 — muscular branch; 5 — ICA; 6 — occipital artery; 7 — external carotid foramen.

B: 1 — right CCA; 2 — right ECA; 3 — right hypoglossal nerve loop; 4 — right external carotid foramen; 5 — right occipital artery; 6 — right ICA.

 

In the second variant, the ICA arises from the CCA below the posterior belly of the digastric muscle as a single trunk common to both the ICA and the occipital artery, termed the carotid-occipital trunk. This trunk courses laterally and shortly divides into two branches, forming an inverted Y-shape. One branch runs cranially toward the external carotid foramen — the ICA — while the other, smaller branch (the occipital artery) runs caudally (Figure 9).

 

Fig. 9. Second variant of the ICA.

1 — branches of the ECA; 2 — ECA trunk; 3 — CCA; 4 — vagus nerve; 5 — carotid-occipital trunk; 6 — occipital artery; 7 — ICA.

 

The occipital artery supplies the occipital muscles and may arise from the ECA as a separate trunk or as part of the carotid-occipital trunk. It gives off muscular branches and a caudal meningeal branch supplying the dura mater, which constitutes an important extracranial collateral anastomosis with the brain (Figure 10). The occipital artery diameter averages 1.0 mm.

 

Fig. 10. Anastomosis of the occipital artery.

1 — branches of the right ECA; 2 — ECA; 3 — right CCA; 4 — right vagus nerve; 5 — muscular branch from the CCA; 6 — right ICA; 7 — occipital artery; 8, 9 — muscular branch of the occipital artery; 10 — meningeal branch of the occipital artery; 11 — external carotid foramen.

 

The ICA then enters the cranial cavity through the carotid canal. Intracranially, the ICA emerges from the cavernous sinus, courses upward and laterally from the optic chiasm, and divides into its terminal branches: the middle cerebral artery (MCA) and the anterior cerebral artery (ACA) (Figure 11).

 

Fig. 11. ICA bifurcation.

1 — right MCA; 2 — right ACA; 3 — anterior communicating artery; 4 — left ACA; 5 — left MCA; 6 — left ICA; 7 — right orbital artery (branch of the ECA); 8 — optic chiasm; 9 — right ICA; 10 — right eyeball after removal of the orbital roof.

 

Before dividing into its terminal branches, the ICA gives off several intracranial branches: the ophthalmic artery, the posterior communicating artery, and the anterior choroidal artery (Figure 12). The ophthalmic artery is the first branch of the ICA, arising from its anterior surface and coursing toward the eyeball, where it supplies the retina. The posterior communicating artery arises from the posterior surface of the ICA, runs posteriorly, and connects with the posterior cerebral artery. The anterior choroidal artery arises from the posterior surface of the ICA and enters the ventricular system.

 

Fig. 12. Intracranial portion of the ICA.

1 — right trochlear nerve; 2 — pons; 3 — basilar artery; 4 — right oculomotor nerve; 5 — right ICA; 6 — right ophthalmic artery; 7 — ICA bifurcation; 8 — optic chiasm; 9 — right ACA; 10 — right MCA; 11 — anterior choroidal artery; 12 — posterior communicating artery; 13 — right posterior cerebral artery; 14 — midbrain; 15 — right superior cerebellar artery.

 

Modeling of malignant ischemic stroke in the MCA territory

MIS was successfully reproduced in 11 (73.3%) rabbits in Group 1. The maximum neurological deficit severity score on the NAS in surviving rabbits during follow-up ranged from 5 to 6 points (mean ± SE: 5.6 ± 0.2; median 6). In 4 (26.6%) animals in Group 1, only mild neurological deficit was recorded. MIS in the MCA territory manifested as depressed consciousness, reduced mobility, lethargy, contralateral forepaw and hindpaw paresis, head tilt toward the side of the stroke, facial asymmetry, circular movements toward the healthy hemisphere when attempting to walk, and loss of interest in food and water, resulting in weight loss. The dynamics of neurological deficit severity (NAS) during the postoperative period in Group 1 rabbits are shown in Figure 13.

 

Fig. 13. Dynamics of neurological deficit severity (NAS) during the postoperative period in Group 1 rabbits.

 

Brain MSCT and MRI performed on postoperative day 1 revealed signs of ischemia with extensive hemispheric involvement in the MCA territory (Figure 14).

 

Fig. 14. Brain MSCT of a rabbit on day 1 after modeling of left MCA territory MIS.

A — axial section; B — coronal section; C — sagittal section. Arrow indicates extensive hypodense area in the left cerebral hemisphere with edema formation.

 

Brain MRI confirmed extensive ischemic damage in the MCA territory with post-ischemic hemispheric edema (Figure 15). The ischemic area appeared as hyperintense signal relative to intact brain regions due to edema. Infarct volume in Group 1 rabbits ranged from 3.0 to 4.2 cm³ (mean ± SE: 3.7 ± 0.1 cm³; median 3.8 cm³; Table 3).

 

Fig. 15. Brain MRI of a rabbit in T1-weighted mode on day 2 after left MCA territory MIS.

A — axial section; B — coronal section; C — sagittal section. Arrow indicates extensive ischemic lesion with edema and midline shift.

 

Table 3. Infarct volume and neurological deficit severity (NAS) in both groups of rabbits

Parameter	Group I	Group II
	Infarct volume, cm3
Min–max	3.0–4.2	4.2–5.7
X̄ ± Se	3.7 ± 0.1	4.9 ± 0.1
Ме	3.8	5
IQR	0.2	0.2
	NAS score, points
Min–max	5–6	7–8
X̄ ± Se	5.6 ± 0.2	7.5 ± 0.1
Ме	6	7
IQR	0.0032	0.00001

 

Mortality in Group 1 rabbits was 46.6% (n = 7), with death occurring within 1–5 days postoperatively. Infarct volume in animals that died ranged from 3.8 to 4.2 cm³ (mean ± SE: 3.9 ± 0.1 cm³; median 3.9 cm³).

In Group 2 rabbits (n = 15), which additionally underwent decompressive craniectomy and cortical artery coagulation, extensive hemispheric infarction with massive post-ischemic edema was observed in 100% of cases. MIS in the MCA territory was confirmed by neurological deficit; the maximum NAS score in surviving rabbits during follow-up ranged from 7 to 8 points (mean ± SE: 7.5 ± 0.1; median 7). This was corroborated by brain MSCT (Figure 16). Infarct volume in Group 2 was larger than in Group 1, ranging from 4.2 to 5.7 cm³ (mean ± SE: 4.9 ± 0.1 cm³; median 5 cm³).

 

Fig. 16. Postoperative brain MSCT of a rabbit.

A: 1 — horizontal brain section. Edema and herniation of ischemic brain tissue through the craniectomy defect. B: 1 — 3D view of the rabbit skull showing the cranial defect after decompressive craniectomy.

 

The dynamics of neurological deficit severity (NAS) during the postoperative period in Group 2 rabbits are shown in Figure 17.

 

Fig. 17. Dynamics of neurological deficit severity (NAS) during the postoperative period in Group 2 rabbits.

 

Mortality occurred in 2 (13.3%) Group 2 rabbits, with infarct volumes of 5.2 and 5.7 cm³, within the first week after surgery. Autopsy revealed axial herniation through the foramen magnum as the cause of death (Figure 18).

 

Fig. 18. Cerebral edema and axial herniation.

A: arrows indicate edema and herniation of the cerebral hemisphere. B: arrow indicates cerebellar tonsillar herniation with brainstem compression.

 

After death or euthanasia, the rabbit brain was removed to confirm ischemic changes and verify monofilament placement in the MCA. Two monofilament placement patterns were identified (Figure 19). The first pattern — both monofilaments in the proximal MCA segment — was the most common (93.3% of cases, n = 28). In this configuration, one monofilament tip was positioned more distally, occluding the entire M1 segment, while the second was placed more proximally. In the second pattern, one monofilament was located in the ACA and the other in the MCA; this pattern was excluded from the study.

 

Fig. 19. Monofilament insertion variants via the ICA.

A: 1 — both monofilaments in the MCA. B: 1 — monofilament in the ACA; 2 — monofilament in the MCA.

 

Histological analysis and electron microscopy confirmed irreversible brain tissue changes due to ischemic injury (Figure 20).

 

Fig. 20. Histological analysis of the brain after MCA territory MIS modeling: infarct zone, hematoxylin and eosin staining.

A — infarct zone (colliquative necrosis). The lesion shows pyknosis, plasmolysis, hydropic changes, and pericellular and perivascular edema, ×200. B — infarct zone (colliquative necrosis), clearly showing necrobiotic nuclear changes: pyknosis and karyolysis, hydropic cytoplasmic changes, perivascular and pericellular edema, ×400.

 

In addition, irreversible morphological changes were confirmed by electron microscopy of the rabbit brain (Figure 21).

 

Fig. 21. Irreversible brain tissue changes: ultrastructural signs of neuropil destruction.

Neuronal processes, membranes, and organelles are destroyed. The tissue consists of extruded cellular contents, including fragments of dead organelles, in the extracellular space. CP — cell processes; V — vesicles. Electron microscopy, ×15,000.

 

Discussion

MIS in the MCA territory is the most severe form of ischemic brain injury [18]. Massive hemispheric edema results from extensive infarction, leading to depressed consciousness and cerebral and extracerebral complications. The search for new treatments and the study of pathophysiological mechanisms have become possible through experimental stroke modeling. Most experimental models use rats due to their availability, low cost, and cerebral vascular anatomy relatively similar to that of humans [19, 20]. However, many authors agree that creating a clinically relevant stroke model in rats is challenging, and results cannot be reliably translated to humans [14]. The well-developed collateral circulation in most rat strains provides substantial brain protection during single-vessel occlusion, which explains the difficulty in producing ischemic lesions with persistent neurological deficits [21]. Additionally, the rat brain is lissencephalic. For these reasons, many researchers prefer larger experimental animals, particularly rabbits [22–25], as their gyrencephalic brain and substantial white matter volume more closely resemble those of humans.

Most established MCA stroke models achieve ischemia by occluding the MCA with a monofilament or thrombus introduced via the ICA, which requires correct intraoperative identification of the ICA. We found that key anatomical landmarks for ICA identification include its location caudal to the posterior belly of the digastric muscle and its craniolateral course. For definitive confirmation, the artery should be traced to the external carotid foramen. We identified two ICA variants based on the origin of the occipital artery. In the first and more common variant, the occipital artery arises from the ECA, courses caudolaterally, and crosses the ICA in an X-shaped configuration. In the second variant, the occipital artery arises from the ICA and courses craniolaterally.

Development of MIS in the MCA territory through proximal MCA occlusion [26, 27] requires monofilament advancement along the entire M1 segment to occlude the lenticulostriate arteries.

Existing MCA stroke models typically place the monofilament only at the initial segment of the MCA, without achieving complete M1 occlusion. Craniotomy with coagulation of the proximal MCA or photothrombosis of MCA branches produces only focal cerebral ischemia [28]. Our MIS model achieves occlusion of the M1 segment and cessation of blood flow in the cortical arteries of the cerebral hemisphere. Proximal MCA occlusion is achieved by introducing two 6-0 Prolene monofilaments (diameter 0.07 mm). This diameter allows advancement through the entire M1 segment and interruption of lenticulostriate flow. The second monofilament occludes the proximal MCA, mimicking the clinical equivalent of MIS.

Coagulation of cortical arteries of the cerebral hemisphere eliminated collateral blood supply. Decompressive craniectomy in Group 2 rabbits provided access to the cortical MCA branches for coagulation. Cortical artery coagulation significantly increased infarct volume by a mean factor of 1.3 in Group 2 rabbits (mean ± SE: 4.9 ± 0.1 cm³; median 5 cm³; IQR 0.2) compared to Group 1 (mean ± SE: 3.8 ± 0.1 cm³; median 3.8 cm³; IQR 0.2) (Mann–Whitney U test: Z = –4.66; p < 0.001). Moreover, mortality in Group 2 (13.3%) was significantly lower than in Group 1 (46.6%) (Fisher’s exact test: p = 0.0135). Thus, the reduced mortality in Group 2 rabbits is attributed to decompressive craniectomy.

Spearman’s rank correlation revealed a moderate correlation between infarct volume and maximum NAS score in Group 1 rabbits (r_S = 0.71; n = 15; p = 0.0032), and a strong correlation in Group 2 rabbits (r_S = 0.88; n =15; p = 0.00001).

Maximum NAS scores differed significantly between groups (Mann–Whitney U test: p < 0.0001): 5–6 points in Group 1 (mean ± SE: 5.6 ± 0.2; median 6) and 7–8 points in Group 2 (mean ± SE: 7.5 ± 0.1; median 7).

The adequacy of MIS modeling in the MCA territory was confirmed by high neurological deficit severity on the NAS (7–8 points) and neuroimaging (MSCT and MRI) findings confirming ischemia with massive post-ischemic hemispheric edema. In addition, the adequacy of the model and the development of ischemia were confirmed by histological analysis and electron microscopy.

Study limitations. Objective limitations include the dependence of infarct quality and volume on correct monofilament placement in the MCA, and the absence of comorbid somatic pathology in experimental animals that could affect stroke course and outcome. Another limitation is the small sample size. Furthermore, the effect of cortical artery coagulation on infarct volume cannot be assessed without craniectomy.

Conclusion

We identified two ICA variants based on occipital artery origin. The first and most common variant (80%) has the occipital artery arising from the ECA. The second variant is characterized by a carotid-occipital trunk common to both the occipital artery and the ICA. Additional coagulation of cortical arteries produces a larger hemispheric infarct compared to isolated proximal MCA occlusion with two monofilaments, providing a model of MIS that closely approximates the clinical scenario.

About the authors

Salah M. Sehweil

Rostov State Medical University

Email: salahsehweil@yandex.ru
ORCID iD: 0000-0003-3942-7442

Cand. Sci. (Med.), Associate Professor, Department of Nervous Diseases and Neurosurgery

Russian Federation, Rostov-on-Don

Albert A. Sufianov

Federal Centre of Neurosurgery

Email: salahsehweil@yandex.ru
ORCID iD: 0000-0001-7580-0385

Dr. Sci. (Med.), Professor, Corr. Member of the RAS, Chief Physician

Russian Federation, Tyumen

Victor N. Prokofiev

Southern Federal University

Author for correspondence.
Email: salahsehweil@yandex.ru
ORCID iD: 0009-0004-6386-0814

Cand. Sci. (Biol.), Senior Researcher, Academy of Biology and Biomedicine

Russian Federation, Rostov-on-Don

Zoya A. Goncharova

Rostov State Medical University

Email: salahsehweil@yandex.ru
ORCID iD: 0000-0001-7093-9548

Dr. Sci. (Med.), Professor, Head, Department of Nervous Diseases and Neurosurgery

Russian Federation, Rostov-on-Don

Evgeniya Y. Kirichenko

Don State Technical University

Email: salahsehweil@yandex.ru
ORCID iD: 0000-0003-4703-1616

Dr. Sci. (Biol.), Head, Department of Bioengineering

Russian Federation, Rostov-on-Don

Alexander K. Logvinov

Southern Federal University

Email: salahsehweil@yandex.ru
ORCID iD: 0000-0002-8873-3625

Cand. Sci. (Biol.), Senior Researcher, Photochemistry Laboratory

Russian Federation, Rostov-on-Don

Victoria Y. Deribas

Rostov State Medical University

Email: salahsehweil@yandex.ru
ORCID iD: 0000-0003-4541-6707

Cand. Sci. (Med.), Head, Pathology Department

Russian Federation, Rostov-on-Don

References

Supplementary files