JP2022119624A - Method for visualizing hematoma of subarachnoid hemorrhage model due to intravascular perforation - Google Patents

Abstract

To provide a SAH model animal and its creation method which are capable of immediately, accurately and instantly evaluating success and seriousness of SAH induction in a subarachnoid hemorrhage (SAH) model animal by an intravascular perforation (EP) method and of visualizing hematoma while alive so that a sample can equalized before intervention experiment, a visualization method of hematoma distribution and an amount of hematoma in a subarachnoid cavity in the SAH model animal, and an evaluation method or the like of seriousness of subarachnoid hemorrhage in the model animal based on the result.SOLUTION: A visualization method for a hematoma distribution and an amount of hematoma in a subarachnoid cavity in a SAH model animal and includes the following: (a) a step which perforates the Willis arterial circle and inducts bleeding to the subarachnoid cavity; (b) a step which continuously administers a contrast medium into the Willis arterial circle at least after the perforation until hemostasis occurs while maintaining antegrade blood flow from the common carotid artery to the internal carotid artery; and (c) a step which captures an image of the head of the subarachnoid hemorrhage model by computerized tomography (CT).SELECTED DRAWING: None

Description

TECHNICAL FIELD The present invention relates to a method for preparing an animal model of subarachnoid hemorrhage, a method for visualizing hematoma distribution in the subarachnoid space and the amount of hematoma in a subject (excluding humans), and the like.

Aneurysmal subarachnoid hemorrhage accounts for approximately 5% of stroke cases affecting more than 600,000 patients annually worldwide. It is still a disease with poor functional prognosis as well as life prognosis, and it is still actively used to establish treatment strategies for delayed cerebral ischemia (DCI) and early brain injury (EBI). Basic research is being conducted. In the above basic research, a rodent subarachnoid hemorrhage (SAH) model and the like are used. Methods such as endovascular perforation (EP) of the annulus have been reported. Among them, the EP method has become an important method frequently used in rodent SAH models in recent years, especially in research aimed at investigating the pathogenesis of EBI. Although the SAH model by the EP method was first reported in 1995, the clinical pathology after cerebral aneurysm rupture, such as delayed cerebral vasospasm, neurological dysfunction, cerebral edema formation, and high mortality, was well documented. It is highly evaluated as a model that imitates (Non-Patent Document 1).

On the other hand, in the model, the success rate and severity of SAH induction varied greatly from the beginning, and there were many criticisms such as differences in experimental methods and results between facilities, so we aimed to refine and standardize the protocol. Research was also active. The most serious problem is the variation in severity, and when conducting intervention experiments using this model, it is necessary to make the severity between groups as uniform as possible. On the premise of this, a method is used in which the severity is retrospectively evaluated based on the macroscopic findings of the excised brain, and then the samples are distributed. It has been pointed out that it is difficult to avoid bias because the sample size is adjusted at the time of data analysis, and the exclusion criteria are ambiguous. Furthermore, the animals must be sacrificed within the period when the hematoma remains in the basilar cistern (approximately within 3 days), which limits the follow-up period and makes it impossible to compare long-term prognosis.

If we can visualize the hematoma at the time of SAH induction and evaluate its severity in the same way as clinically, it should contribute to reducing bias and enable observation of long-term prognosis. In recent years, methods for estimating the severity of SAH model animals by the EP method by CT and MRI have been reported (Non-Patent Documents 2, 3), but visualization methods that can accurately reflect the hematoma volume immediately have not been reported. not

Sugawara T, Ayer R, Jadhav V, et al. A new grading system evaluating bleeding scale in filament perforation subarachnoid hemorrhage rat model. J Neurosci Methods 2008;167:327-334. Weyer V, Maros ME, Kronfeld A, et al. Longitudinal imaging and evaluation of SAH-associated cerebral large artery vasospasm in mice using micro-CT and angiography. J Cereb Blood Flow Metab 40: 2265-2277, 2020 Shishido H, Egashira Y, Okubo S, et al. A magnetic resonance imaging grading system for subarachnoid hemorrhage severity in a rat model. J Neurosci Methods 243: 115-119, 2015

The problem of the present invention is to be able to evaluate the success and severity of SAH induction immediately and accurately in an SAH model animal by the EP method, and to make it possible to homogenize samples before intervention experiments. SAH model animal that can visualize the SAH model animal and its preparation method, a method for visualizing hematoma distribution in the subarachnoid space in the SAH model animal, and the amount of hematoma, and a method for evaluating the severity of subarachnoid hemorrhage in the model animal based on the results etc. is to be provided.

The present inventors have focused on computed tomography (micro-CT), which has rapidly spread in recent years in animal experiments, as a means of visualizing hematoma. However, because there is little difference in density and X-ray absorption between different soft tissue tissue types, visualization of intracranial structures requires the use of X-ray absorbing contrast agents. Since the hematoma is an image of the contrast medium that has leaked into the subarachnoid space, the amount of In order to keep the contrast medium concentration and the cerebral perfusion pressure at the puncture site constant, it is important not to block the antegrade blood flow in the internal carotid artery. Because a puncture device was inserted, another route for inserting the catheter required for administration of the contrast medium could not be secured. Therefore, as a result of extensive studies, the present inventors have found that by inserting an instrument for puncture from the pterygopalatine artery (PPA), which is a branch of the internal carotid artery, contrast agent is administered to the external carotid artery. Securing a catheter for hemorrhage as an insertion route and continuously injecting the contrast agent from before SAH induction to spontaneous hemostasis, the contrast agent can be injected without blocking the blood flow from the common carotid artery to the internal carotid artery. extravasation of the blood vessels. In SAH model animals prepared by the above method, hematoma was visualized immediately after induction of SAH using micro-CT, and the severity was evaluated. Based on these findings, the present inventors have found that in SAH model animals by the EP method, by combining continuous injection of a contrast agent and microCT, the success and severity of immediate SAH induction can be evaluated with high accuracy. and completed the present invention.

That is, the present invention relates to the following.
[Section 1]
A method for creating an animal model of subarachnoid hemorrhage, comprising:
(A) puncturing the circle of Willis to induce bleeding into the subarachnoid space; and (B) maintaining antegrade blood flow from the common carotid artery to the internal carotid artery, at least from said puncture to hemostasis. a step of continuously administering a contrast medium into the circle of Willis for a period of time, thereby enabling visualization of the hematoma by computed tomography immediately after the bleeding is induced.
[Section 2]
Item 2. The method according to Item 1, wherein the instrument for puncturing is inserted from the pterygopalatine artery, and the contrast medium is administered from the external carotid artery into the internal carotid artery.
[Section 3]
Item 3. The preparation method according to item 1 or 2, wherein the puncture site of the circle of Willis is from the internal carotid artery to the anterior cerebral artery.
[Section 4]
4. The preparation method according to any one of Items 1 to 3, wherein the continuous administration of the contrast agent is performed from before the puncture to 3 minutes after the puncture.
[Section 5]
A method for visualizing hematoma distribution and hematoma volume in the subarachnoid space in an animal model of subarachnoid hemorrhage, comprising the following:
(a) puncturing the circle of Willis to induce bleeding into the subarachnoid space;
(b) maintaining antegrade blood flow from the common carotid artery to the internal carotid artery, continuously administering a contrast agent into the circle of Willis for at least the period from the puncture to hemostasis; and (c) the above A visualization method comprising the step of imaging the head of a subarachnoid hemorrhage model animal by computed tomography (CT).
[Section 6]
Item 6. The visualization method according to Item 5, wherein the instrument for puncturing is inserted from the pterygopalatine artery, and the contrast medium is administered from the external carotid artery into the internal carotid artery.
[Section 7]
Item 7. The visualization method according to item 5 or 6, wherein the CT is micro-CT.
[Item 8]
A method for evaluating the severity of subarachnoid hemorrhage in an animal model of subarachnoid hemorrhage, comprising:
(a) puncturing the circle of Willis to induce bleeding into the subarachnoid space;
(b) maintaining antegrade blood flow from the common carotid artery to the internal carotid artery, continuously administering a contrast agent into the circle of Willis at least from the puncture to hemostasis;
(c) imaging the head of the subarachnoid hemorrhage model animal by computed tomography (CT); and (d) evaluating the severity of subarachnoid hemorrhage in the subarachnoid hemorrhage model animal based on the imaging. Evaluation method, including process.
[Item 9]
Item 9. The evaluation method according to Item 8, wherein the instrument for puncturing is inserted from the pterygopalatine artery, and the contrast medium is administered from the external carotid artery into the internal carotid artery.
[Item 10]
Item 10. The evaluation method according to Item 8 or 9, wherein the CT is microCT.

INDUSTRIAL APPLICABILITY According to the present invention, a method for preparing an animal model of subarachnoid hemorrhage can be provided. Further, according to the present invention, a method for visualizing the distribution of hematoma in the subarachnoid space and the amount of hematoma in an animal model of subarachnoid hemorrhage, a method for evaluating the severity of subarachnoid hemorrhage in an animal model for subarachnoid hemorrhage, etc. are provided. can do. The production method of the present invention can produce a subarachnoid hemorrhage model animal suitable for visualizing SAH distribution (hematoma distribution and hematoma volume in the subarachnoid space) and evaluating the severity of SAH. The visualization method and severity evaluation method of the present invention can immediately visualize the hematoma distribution and hematoma volume of SAH in detail, and the results obtained by the visualization method can be used for macroscopic grading of SAH. It is a particularly good method for visualizing and assessing the severity of SAH because it does not require animal sacrifice for visualization. By using the evaluation method of the present invention, it is possible to homogenize the severity between groups before conducting an intervention experiment using an animal model of subarachnoid hemorrhage. Furthermore, long-term prognosis can be observed because animals do not need to be sacrificed for visualization.
In addition, even with MRI and the like, which are considered to be able to diagnose bleeding in the subarachnoid space to some extent, immediate and quantitative evaluation of hematoma distribution and hematoma volume is extremely difficult, and the method of the present invention Considering the fact that it is very expensive compared to the microCT used and the maintenance cost is also much higher, the method of the present invention is extremely superior to existing methods for visualization of subarachnoid hemorrhage.・It can be said that it is an evaluation method.

FIG. 1 is a diagram showing a comparison of techniques for preparing SAH model animals between the conventional EP method (A) and the preparation method of the present invention (B). In the figure, ICA is the internal carotid artery, ECA is the external carotid artery, PPA is the pterygopalatine artery, and CCA is the common carotid artery. FIG. 2 is a diagram showing a method of SAH induction by the EP method for visualizing SAH with a contrast medium using microCT. (A) Scheme of SAH induction by continuous intra-arterial injection of contrast agent. (B) Surgical view after cannulation of the ECA with a 24-gauge catheter (arrow) and introduction of a wired tube (arrowhead) through the PPA. A tube was advanced through the ICA, provided that all temporary occluding vascular clips were removed and antegrade blood flow was resumed. (c) Experimental setup for microCT imaging. General anesthesia was maintained using a ventilator (arrow) and inhaled anesthetic (arrowhead) to reduce motion artifacts. FIG. 3 is a diagram showing the correlation between CT results and severity of SAH. The upper panel shows the grade of SAH in the photographs: Sham (A), Mild (B), Moderate (C) and Severe (D). The lower left panel shows a sagittal CT image at the midline and the lower right panel shows an axial CT image at the fundus. The CT images show that microCT with contrast could visualize the extent of SAH immediately after induction. A sham-operated CT image (bottom panel of A) shows that injection of the contrast medium has no effect on the visualization of the intracranial structures themselves. FIG. 4 shows CT images depicting the severity of SAH. (A) Shows the division of the basal cistern into six segments in photographs and CT images. The left is a photograph of SAH, and the right is a CT image of SAH. SAH was assessed for each of these segments and assigned a score of 0-3 as described below. SAH grading was assessed by dividing the basal cistern into 6 segments in the photographs, and cSAH scores were assessed by the same segments in axial and sagittal CT. (B) Shows the correlation between cSAH score and SAH grade in each segment. There was a significant correlation between cSAH score and SAH grade in 4 out of 6 segments. (c) Overall correlation between cSAH score and SAH grade. There was a significant linear correlation between SAH grade and cSAH score. FIG. 5 is a diagram showing a comparison between CT images and pathological findings. (A) shows that the distribution of SAH could be visualized in CT images. Images were obtained as a photograph (left), micro-CT (middle), and H&E (hematoxylin and eosin) staining (right) of the same slice of brain in the EP model, respectively. (B) shows that intracerebral hemorrhage (ICH) could be visualized in CT images. Arrows indicate findings suggestive of ICH on CT images. Formation of ICH was observed in the H&E-stained specimen in the part corresponding to the lesion. Scale bars indicate 2 mm (white bars) and 500 μm (black bars), respectively.

1. Method for preparing an animal model of subarachnoid hemorrhage of the present invention The present invention provides the following:
A method for creating an animal model of subarachnoid hemorrhage, comprising:
(A) puncturing the circle of Willis to induce bleeding into the subarachnoid space; and (B) maintaining antegrade blood flow from the common carotid artery to the internal carotid artery, at least from said puncture to hemostasis. Provided is a method for preparing an animal model, comprising a step of continuously administering a contrast agent into the circle of Willis during the induction of bleeding, thereby enabling visualization of the hematoma by computed tomography immediately after the bleeding is induced.

Animals to be subjected to the production method of the present invention include, for example, mammals. Mammals include, for example, rodents such as mice, rats, hamsters and guinea pigs, laboratory animals such as rabbits, domestic animals such as pigs, cows, goats, horses, sheep and minks, pets such as dogs and cats, humans, Primates such as monkeys, cynomolgus monkeys, rhesus monkeys, marmosets, orangutans, chimpanzees, and the like can include, but are not limited to, these.

In the step (A) of the preparation method of the present invention, bleeding due to arterial perforation can be induced in the subarachnoid space of an animal subjected to the preparation method of the present invention, and the circle of Willis (in particular, the internal carotid artery) can be anterograded. The method of puncturing (or perforating) the circle of Willis is not particularly limited as long as the blood flow can be maintained. Specific examples include the filament method using nylon threads and the wire/tubing technique. The puncture (or perforation) may be performed under anesthesia (eg, isoflurane, sevoflurane, ketamine hydrochloride, propofol, etc.), and anesthesia may be performed by a method known per se, such as inhalation or injection. Anesthesia may be continuously applied throughout the entire process of the production method of the present invention, or may be stopped or interrupted as appropriate.

As for the insertion route of any of the puncture devices described above for delivering the device into the circle of Willis, antegrade blood flow from the common carotid artery to the internal carotid artery is maintained, and a contrast agent is continuously administered. There is no particular limitation as long as another catheter insertion route (e.g., the external carotid artery) is secured for the pterygopalatine artery (PPA). ).

The site to be punctured (or perforated) in the step (A) of the preparation method of the present invention is the circle of Willis, preferably from the internal carotid artery to the anterior cerebral artery in the circle of Willis, specifically the tip of the internal carotid artery. from the cerebral artery to the anterior cerebral artery origin, more preferably around the anterior cerebral artery origin. It is desirable that the puncture instrument is quickly pulled back from the circle of Willis to the origin of the insertion route (preferably PPA) after puncture so as not to block blood flow in the internal carotid artery.

In step (B) of the preparation method of the present invention, a desired amount of contrast agent can be continuously administered into the circle of Willis at least from the start of bleeding by puncture in step (A) to spontaneous hemostasis, and The method of continuous administration of the contrast agent is not particularly limited as long as the concentration of the contrast agent contained in the blood flowing through the puncture site in step (A) can be kept constant during the period. Continuous dosing can be used.

Examples of the method of administering the contrast agent in the step (B) of the preparation method of the present invention include administration into the internal carotid artery using a catheter inserted into the external carotid artery. In this case, it is desirable to insert and fix the tip of the catheter to such an extent that it does not protrude from the bifurcation of the external carotid artery to the internal carotid artery side so as not to impede blood flow in the internal carotid artery.

The contrast agent is not particularly limited as long as the hematoma distribution in the subarachnoid space and the amount of hematoma can be imaged by computed tomography (CT), which will be described later. iodic acid, iodoform, triiodophenol, tetraiodoethylene, iohexol, etc.). Preferred contrast agents include iohexol (300 mg/ml), which has been proven not to cause neuropathy and can be intrathecally administered to the human body. The concentration of the contrast agent is not particularly limited as long as the hematoma distribution in the subarachnoid space and the amount of hematoma can be visualized, and can be appropriately selected within the concentration range usually used for head CT image diagnosis. Examples include 100 mg/ml to 300 mg/ml, preferably 200 mg/ml to 300 mg/ml.

The contrast agent is capable of maintaining a constant concentration of the contrast agent contained in the blood flowing through the puncture site at least from the start of bleeding due to puncture in step (A) to spontaneous hemostasis, and is capable of maintaining a constant concentration of the contrast agent contained in the blood flowing through the puncture site. Therefore, in order to image the hematoma distribution and hematoma volume in the subarachnoid space, it is necessary to continuously administer a constant amount at a constant rate from before puncture until spontaneous hemostasis is completed. For example, the administration of the contrast agent is performed before the puncture, specifically during the first puncture, so that the blood contrast agent concentration is already kept constant during the puncture of the circle of Willis in step (A). Dosing should start ~5 minutes before. Considering the period from puncture to the circle of Willis until spontaneous hemostasis is completed, administration of the contrast medium is continued, for example, for 1 to 10 minutes after puncture, preferably for about 3 minutes after puncture.

In step (b) of the production method of the present invention, the contrast agent is continuously administered (that is, continuously administered) into the internal carotid artery for the period described above. The dosage and administration rate of the contrast agent are not particularly limited, but for example, the amount of the contrast agent solution is 0.5 ml to 0.8 ml, preferably 0.6 ml to 0.7 ml, and the amount is 6 ml/h to 10 ml/h, preferably 7 ml/h. It can be administered at a rate of h-9 ml/h.

The present invention also provides an SAH model animal by the EP method, which can visualize hematoma (e.g., hematoma distribution, hematoma) by CT immediately after induction of SAH and in a live state immediately after induction of SAH. do. In the SAH model animal, the anterograde blood flow in the internal carotid artery is not blocked, and the contrast medium is continuously administered through a route other than the puncture device, so from the moment of puncture to spontaneous hemostasis Since the blood contrast medium concentration in the internal carotid artery and the cerebral perfusion pressure were maintained constant during the period, a high correlation was shown between the severity of hemorrhage and the amount of contrast medium leaking into the subarachnoid space. It is possible to evaluate the severity of model animals without performing SAH grading in isolated brains.

2. Hematoma distribution in the subarachnoid space of the present invention and method for visualizing the amount of hematoma The present invention provides the following:
A method for visualizing hematoma distribution and hematoma volume in the subarachnoid space in an animal model of subarachnoid hemorrhage, comprising the following:
(a) puncturing the circle of Willis to induce bleeding into the subarachnoid space;
(b) maintaining antegrade blood flow from the common carotid artery to the internal carotid artery, continuously administering a contrast agent into the circle of Willis for at least the period from the puncture to hemostasis; and (c) the above Provided is a visualization method comprising the step of imaging the head of a subarachnoid hemorrhage model animal by computed tomography (CT).

Regarding the method for visualizing the distribution of hematoma in the subarachnoid space and the amount of hematoma of the present invention, the steps (a) and (b) of the visualization method are the same as in the above-mentioned “1. shall refer to the entire contents of

In the step (c) of the visualization method of the present invention, the hematoma distribution and hematoma volume in the subarachnoid space of the head of the subarachnoid hemorrhage model animal are evaluated at least visually using the conventional six-fold method. There are no particular restrictions on the setting of computed tomography (CT), etc., and a method known per se may be used as long as it can be visualized to the extent that evaluation equivalent to severity evaluation (SAH grading) can be performed. As for computed tomography (CT), it is preferable to use microCT from the viewpoint of resolution. Based on the head CT image taken in step (c), the hematoma distribution and hematoma volume can be determined by a method known per se.

3. A method for evaluating the severity of subarachnoid hemorrhage in an animal model of subarachnoid hemorrhage of the present invention The present invention provides the following:
A method for evaluating the severity of subarachnoid hemorrhage in an animal model of subarachnoid hemorrhage, comprising:
(a) puncturing the circle of Willis to induce bleeding into the subarachnoid space;
(b) maintaining antegrade blood flow from the common carotid artery to the internal carotid artery, continuously administering a contrast agent into the circle of Willis at least from the puncture to hemostasis;
(c) imaging the head of the subarachnoid hemorrhage model animal by computed tomography (CT); and (d) evaluating the severity of subarachnoid hemorrhage in the subarachnoid hemorrhage model animal based on the imaging. An evaluation method is provided, including steps.

Regarding the method for evaluating the severity of subarachnoid hemorrhage in an animal model of subarachnoid hemorrhage of the present invention, the steps (a) to (c) of the evaluation method are the same as in the above-mentioned “1. and "2. Method for visualizing hematoma distribution in subarachnoid space and hematoma volume of the present invention".

In step (d) of the evaluation method of the present invention, if the severity of subarachnoid hemorrhage in the subarachnoid hemorrhage model animal can be appropriately evaluated based on the head CT image taken in step (c), evaluation The method is not particularly limited. As an evaluation method, for example, the severity of CT images may be evaluated based on the known SAH grading system (Sugawara et al. 2008) classification, etc., or, for example, SAH for hematoma distribution, hematoma amount, etc. The evaluation may be performed after independently setting the evaluation criteria. For example, according to the cSAH scoring system classification proposed by the present inventors, SAH can be classified as shown in Table 1 based on the head CT image taken in step (c).

When performing the evaluation method of the present invention, when considering the possibility of bleeding with different phases (e.g., delayed rerupture), for example, neurobehavioral tests at 24 hours after the onset of SAH before the experiment When used as a screening test, cases in which neurological symptoms significantly deviate from expected neurological symptoms based on the visualized amount of hematoma may be further screened, such as excluding them from the sample.

Since the evaluation method of the present invention can immediately evaluate the severity of SAH in subarachnoid hemorrhage model animals, for example, in sample division of model animals for intervention experiments, in order to make the severity uniform among samples The results of the evaluation method can be used for Therefore, the present invention also provides a subarachnoid hemorrhage model animal population homogenized in severity using the evaluation method. The population is useful as a population of SAH model animals for intervention experiments.

Micro CT System All imaging in the examples was performed using Cosmo Scan GX (Rigaku). Scanning was performed immediately after induction of SAH. MicroCT data were acquired at an X-ray tube voltage of 50 kVp and 160 μA (for CT acquisition). For high-resolution CT acquisition, a nominal resolution of 90 μm and an exposure time of 4 minutes were performed.

Experimental Animals As experimental animals, 16-week-old male Sprague-Dawley (SD) rats (Japan SLC Co., Ltd.) (weighing 295-340 g) were used. Experimental animals were always anesthetized during CT imaging to control their behavior and escape. All of the above animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Occupational and Environmental Health. In this experiment, anesthesia is usually applied with animals inhaling isoflurane through an intubated tube. Continuous infusion of contrast agent (iohexol 300 mg/ml) is applied by inserting a 24-gauge catheter through the ipsilateral external carotid artery and connecting it to a mechanical syringe pump.

Creation of EP method SAH model and administration of contrast agent in internal carotid artery
An endovascular perforation (EP) SAH rat model was created using a microtube and a tungsten wire based on a known method with some modifications. Specifically, by inserting the microtube and tungsten wire required for puncturing the rat through the pterygopalatine artery (PPA) instead of the external carotid artery as in the conventional method, administration of the contrast agent The necessary catheter can be inserted from the external carotid artery (Fig. 1), and the combined use of arterial puncture and continuous infusion into the internal carotid artery causes extravasation of the contrast agent and indirectly affects the distribution of SAH. vividly depicted.

Briefly, it was prepared as follows (see also Figure 2).
(1) General anesthesia was induced with 3% isoflurane, a 16G catheter was intubated, and inhalation anesthesia with 1-3% isoflurane was maintained and continued. Forced ventilation was started using a ventilator.
(2) The left common carotid artery (CCA), the internal carotid artery (ICA), and the external carotid artery (ECA) were exposed from the carotid trigone through a median neck incision, separated from the surrounding tissue, and the ECA secured.
(3) Secure the pterygopalatine artery (PPA) at the distal part of the left ICA, place an 8-0 silk thread at the bifurcation with the ICA, temporarily block the CCA and ICA beyond the PPA bifurcation with a vascular clip, and then PPA was ligated off distally.
(4) Polytetrafluoroethylene (PTFE) tubing (Braintree Scientific, SUBL-120, ID: 0.006 inch; OD: 0.012 inch) and tungsten wire (Scientific Instruments Services, Inc., catalog number W91, Diameter length: 0.076 mm; length: 47 mm), the bifurcation was ligated with 8-0 silk thread to secure the tube to the insertion site.
(5) The left ICA and CCA were unblocked and anterograde blood flow was resumed (blockage time is generally within 1 to 2 minutes). Subsequently, the ECA bifurcation was temporarily blocked with a vascular clip.
(6) A 24G catheter was inserted through the left ECA, ligated with two 8-0 silk threads, the blockage was released, and continuous infusion of iohexol (300 mg/ml) was started at 8 ml/h using a syringe pump. did.
(7) The microtube was advanced distally to the ICA first, inserted 18 mm from the PPA bifurcation, and then tungsten was advanced.
(8) The tungsten wire was advanced so as to deviate 1.5 mm from the tip of the tube and was punctured, and then the tip of the microtube was immediately pulled back to the vicinity of the bifurcation of the PPA so as not to interfere with antegrade blood flow.
(9) 3 minutes after puncture, continuous administration of the contrast agent was terminated. To prevent bleeding, the ICA was temporarily blocked again with a vascular clip, the microtube and 24G catheter were removed from the PPA and ECA, respectively, and the stumps were ligated and then blocked.
(10) Inhalation anesthesia with isoflurane and mandatory ventilation were continued, and micro-CT was taken within 30 minutes while wearing a ventilator.
(11) The anesthesia was terminated in stages, and after spontaneous respiration had stabilized, the ventilator was terminated. The patient was extubated and the wound was sutured closed to complete the operation.

Image analysis of CT imaging
The imaging ability of CT imaging was evaluated by comparing it with the macroscopic findings of the brain removed immediately after imaging. We evaluated the correlation between CT image findings and SAH grading for hematoma thickness in 6 segments evaluated by SAH grading (bil. ICA terminal, bil. IC-Pcom bifurcation, BA pons, BA medullary portion periphery).
Each segment was assigned a CT grade of 0-3, depending on the amount of subarachnoid thrombus within the segment, as shown in Table 1 below.

Pathological evaluation
After gross assessment of SAH grading, pathological assessment was performed using brains fixed in 10% neutral buffered formalin. It was embedded in paraffin, sectioned, and evaluated by H&E. We evaluated the correlation between the distribution of hematoma in the subarachnoid space and the presence or absence of intracerebral hemorrhage in areas that are difficult to evaluate by gross observation from the base of the brain, especially in the intraventricular and interhemispheric fissures, and the correlation with CT findings.

Data analysis A simple regression analysis and Spearman's correlation coefficient with rank test were performed to assess the correlation between SAH grade and CT grade and between each investigator's CT grade. Data are presented as mean±standard error (SEM). Statistical differences between various groups were assessed with one-way ANOVA using Holm-Sidak post hoc analysis. An unpaired t test was used for comparisons between two groups. A value of P < 0.05 is considered statistically significant. A value of r > 0.4 is considered a significant correlation and r > 0.7 a strong correlation. Statistical analysis was performed using StatView version 5.0 for Windows.

Result 1 (Visualization of SAH immediately after induction by microCT (immediacy))
A comparison of a head CT image taken immediately after induction of SAH and a photograph of an excised brain is shown (Fig. 3). We succeeded in visualizing the hematoma distribution of SAH immediately after induction by the EP method by continuously administering a contrast agent transarterially. Successful induction of SAH could be confirmed immediately, and the hematoma volume and puncture site could be inferred from the distribution of the hematoma (Fig. 4).

Result 2 (Evaluation of bleeding scale using microCT: comparison with SAH grading (imaging ability))
Visualization of the hematoma distribution in axial and sagittal CT imaging (Table 1 above) enabled classification of severity similar to SAH grading using the conventional sextuple method (Table 2). A comparison of microCT grading and SAH grading of isolated brains is shown (Figs. 3 and 4). As can be seen from the results, correlation was demonstrated (P=0.0109, r=0.657).

Result 3 (SAH distribution and complication of intracerebral hemorrhage (diagnostic ability))
Micro-CT showed that the contrast agent was distributed not only in the basilar cistern but also in the interhemispheric fissure, the ventricle, and the posterior fossa. When the hematoma distribution in the subarachnoid space was diagnosed in the histological section of the excised brain, it was confirmed that the hematoma distribution was consistent with the imaging findings (Fig. 5A). In addition, accumulation of the contrast medium was observed in the brain parenchyma, and there were multiple cases that could be diagnosed as intracerebral hemorrhage (n=3). In a case diagnosed as intracerebral hematoma by CT image, it was also confirmed to be intracerebral hematoma by pathological diagnosis (Fig. 5B). In addition to intracerebral hematoma, there are cases in which SAH is widely distributed in the ventricles, interhemispheric fissures, and posterior cranial fossa, which were not evaluated by conventional SAH grading. It has been suggested.

Points to Note In the above example, it is important not to disturb the anterograde blood flow of the ICA in order to keep the contrast agent concentration in the blood and the cerebral perfusion pressure at the puncture site constant. , The tip of the catheter inserted into the ECA should be inserted and fixed so that it does not protrude from the ECA bifurcation to the ICA side. In addition, when puncturing, it is necessary to quickly insert the microtube and tungsten wire and pull them back until the tip reaches the PPA origin immediately after puncturing.

A new evaluation of severity by conventional SAH grading and micro-CT When the imaging ability of the method used in the above example was verified by comparing it with SAH grading, the correlation between image findings and macroscopic findings was confirmed. I was able to Therefore, by using this method, which can faithfully reproduce the severity of SAH, the success and severity of induction can be evaluated immediately when the SAH model is created, and homogenization between samples can be achieved before intervention experiments. become. By replacing the widely used macroscopic SAH grading with CT imaging, retrospective assessment of severity becomes unnecessary and long-term prognosis can be observed. It is thought that it can contribute to the development of SAH research.

Quantitative and qualitative diagnostic capabilities of CT imaging (comparison with pathological findings)
In the above example, when the SAH model was actually created and visualized, not only the severity but also the distribution of the hematoma in the subarachnoid space varied from case to case. It was also found that complication of internal hematoma may occur.
In cases of intracerebral hematoma formation, as a matter of course, local focal symptoms such as paralysis appear, and the functional prognosis is inevitably poor, so it is desirable to exclude them from experimental samples. In the past, cerebral blood flow was confirmed by laser Doppler, and intracranial pressure was monitored to indirectly determine whether SAH was successfully induced, and in some cases to consider whether to perform a second puncture. However, with this method, it was difficult to distinguish between cases where only ICH was produced by puncturing in the direction of the brain parenchyma and cases where the cerebral vessels were not perforated, and there were cases in which the conventional method had problems.
In comparison with the pathological findings in this example, this method is capable of diagnosing very small intracerebral hemorrhages and visualizing small amounts of subarachnoid hemorrhages. It was also useful for delineating hematomas distributed in the room and interhemispheric fissures. It will be possible to confirm the entire bleeding situation in detail and immediately, and it is thought that future SAH research on the relationship between bleeding patterns and treatment effects will progress dramatically. It may also help to establish a puncture method that easily induces SAH with moderate severity.

Examination of Effects of Contrast Agent Since iohexol (300 mg/ml) used in this example is also clinically used in encephalomyelography, it should not affect nerve function. However, just in case, we investigated the effects of leakage of the contrast medium into the spinal cord on brain damage and neurological symptoms by injecting the contrast medium into the cerebral cisterna, and found no effects on nerves. (Fig. 5).

The preparation method of the present invention is useful because it enables visualization of SAH distribution (hematoma distribution and hematoma volume in the subarachnoid space) and preparation of an animal model of subarachnoid hemorrhage suitable for evaluating the severity of SAH. . In addition, the visualization method of the present invention can immediately visualize the SAH hematoma distribution and hematoma volume in detail, and the results obtained by the visualization method are correlated with the macroscopic SAH grading. It is useful because it has excellent visualization ability and does not require animal slaughter for visualization. Furthermore, by using the evaluation method of the present invention, it is possible to evaluate the severity of SAH before an intervention experiment, which is useful because bias can be avoided.

Claims (10)

A method for creating an animal model of subarachnoid hemorrhage, comprising:
(A) puncturing the circle of Willis to induce bleeding into the subarachnoid space; and (B) maintaining antegrade blood flow from the common carotid artery to the internal carotid artery, at least from said puncture to hemostasis. a step of continuously administering a contrast medium into the circle of Willis for a period of time, thereby enabling visualization of the hematoma by computed tomography immediately after the bleeding is induced. 2. The method of claim 1, wherein the instrument for puncture is inserted through the pterygopalatine artery and the contrast medium is administered through the external carotid artery into the internal carotid artery. 3. The preparation method according to claim 1, wherein the puncture site of the circle of Willis is from the internal carotid artery to the anterior cerebral artery. 4. The preparation method according to any one of claims 1 to 3, wherein the continuous administration of the contrast agent is performed from before the puncture to 3 minutes after the puncture. A method for visualizing hematoma distribution and hematoma volume in the subarachnoid space in an animal model of subarachnoid hemorrhage, comprising the following:
(a) puncturing the circle of Willis to induce bleeding into the subarachnoid space;
(b) maintaining antegrade blood flow from the common carotid artery to the internal carotid artery, continuously administering a contrast agent into the circle of Willis for at least the period from the puncture to hemostasis; and (c) the above A visualization method comprising the step of imaging the head of a subarachnoid hemorrhage model animal by computed tomography (CT). 6. The visualization method according to claim 5, wherein the instrument for puncturing is inserted from the pterygopalatine artery, and the contrast medium is administered from the external carotid artery into the internal carotid artery. 7. The visualization method according to claim 5 or 6, wherein the CT is microCT. A method for evaluating the severity of subarachnoid hemorrhage in an animal model of subarachnoid hemorrhage, comprising:
(a) puncturing the circle of Willis to induce bleeding into the subarachnoid space;
(b) maintaining antegrade blood flow from the common carotid artery to the internal carotid artery, continuously administering a contrast agent into the circle of Willis at least from the puncture to hemostasis;
(c) imaging the head of the subarachnoid hemorrhage model animal by computed tomography (CT); and (d) evaluating the severity of subarachnoid hemorrhage in the subarachnoid hemorrhage model animal based on the imaging. Evaluation method, including process. 9. The evaluation method according to claim 8, wherein the instrument for puncturing is inserted from the pterygopalatine artery, and the contrast medium is administered from the external carotid artery into the internal carotid artery. 10. The evaluation method according to claim 8 or 9, wherein the CT is microCT.

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