A method for visualizing hematomas in a subarachnoid hemorrhage model caused by endovascular perforation

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

[0001] The present invention relates to a method for creating a subarachnoid hemorrhage model animal, and a method for visualizing the hematoma distribution and hematoma volume in the subarachnoid space in a subject (excluding humans).

Background Art

[0002] Aneurysmal subarachnoid hemorrhage accounts for approximately 5% of stroke cases that affect more than 600,000 patients worldwide every year. It is still a disease with a poor prognosis not only for life but also for function, and basic research is still actively being conducted to establish treatment strategies for delayed cerebral ischemia (DCI) and early brain injury (EBI). In the above basic research, a subarachnoid hemorrhage (SAH) model of rodents is used, and as a method for creating the rodent SAH model, methods such as an autologous blood injection method, an intracerebral venous opening method, and an endovascular perforation (EP) method of the circle of Willis have been reported. Among them, the EP method has recently become an important technique frequently used in rodent SAH models in research aiming at elucidating the pathophysiology of EBI. Although an SAH model by the EP method was first reported in 1995, it is highly evaluated as a model that well mimics clinical pathologies after cerebral aneurysm rupture, such as delayed cerebral vasospasm, neurological dysfunction, brain edema formation, and high mortality (Non-Patent Document 1).

[0003] However, this model has been criticized from the beginning for its wide variability in the success rate and severity of SAH induction, as well as for differences in experimental methods and results between institutions. Research aimed at refining and standardizing the protocol has been active. The most problematic issue is the variability in severity. When conducting interventional experiments using this model, it is necessary to ensure uniform severity between groups. Currently, a strict randomization and blinded protocol is used, and the severity is assessed retrospectively based on macroscopic findings in the excised brain. This method involves retrospectively assessing severity based on macroscopic findings in the excised brain and then allocating samples. However, it has been pointed out that this method has the potential for bias, such as the need to adjust sample size during data analysis, which can lead to unclear exclusion criteria. Furthermore, the need to sacrifice animals while the hematoma remains in the basal cistern (generally within 3 days) limits the follow-up period, making it impossible to compare long-term outcomes.

[0004] Visualizing hematomas during SAH induction and assessing their severity, similar to clinical practice, would help reduce bias and enable long-term prognosis. While methods for estimating severity using CT and MRI in SAH animal models using the EP method have been reported in recent years (Non-Patent Documents 2 and 3), no visualization method has been reported that can accurately reflect hematoma volume in real time. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] 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. [Non-patent document 2] 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 [Non-Patent Document 3] 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 [Summary of the Invention] [Problems to be Solved by the Invention]

[0006] An object of the present invention is to provide a SAH model animal capable of visualizing a hematoma while alive so that the success and severity of SAH induction can be accurately evaluated immediately in a SAH model animal by the EP method, and the samples can be homogenized before an intervention experiment, a method for creating the same, a method for visualizing the hematoma distribution and the hematoma volume in the subarachnoid space in the SAH model animal, and a method for evaluating the severity of subarachnoid hemorrhage in the model animal based on the results, etc. [Means for Solving the Problems]

[0007] We focused on computed tomography (microCT), which has become increasingly popular in animal experiments in recent years, as a means of visualizing hematomas. However, because there is little difference in density and X-ray absorption between different soft tissue types, the use of X-ray-absorbing contrast agents is necessary for visualization of intracranial structures. Because hematomas are images of contrast agents leaked into the subarachnoid space, maintaining a constant blood contrast agent concentration and cerebral perfusion pressure at the puncture site is crucial to ensure uninterrupted antegrade blood flow through the internal carotid artery. However, conventional EP techniques require insertion of a puncture device through the external carotid artery, which precludes the use of an alternative route for inserting a catheter for contrast agent administration. Therefore, after extensive research, the inventors have developed a method for inserting a puncture device through the pterygopalatine artery (PPA), a branch of the internal carotid artery, to secure the external carotid artery as an insertion route for a catheter for contrast agent administration. By continuously injecting contrast agent from before SAH induction until spontaneous hemostasis occurs, extravasation of contrast agent from the common carotid artery to the internal carotid artery is possible without blocking blood flow from the common carotid artery to the internal carotid artery. In an SAH model animal created using the above method, hematomas were visualized and their severity was assessed using micro-CT immediately after SAH induction. This demonstrated a high correlation with the SAH grade assessment of the excised brain. Based on these findings, the inventors have successfully combined continuous infusion of contrast agent with micro-CT in an SAH model animal using the EP method, thereby enabling accurate real-time assessment of the success and severity of SAH induction. This has led to the completion of the present invention.

[0008] That is, the present invention relates to the following. [Section 1] A method for producing a subarachnoid hemorrhage model animal, comprising: (A) A step of puncturing the circle of Willis and inducing bleeding into the subarachnoid space; and (B) a step of continuously administering a contrast agent into the circle of Willis at least from the puncture to hemostasis while maintaining antegrade blood flow from the common carotid artery to the internal carotid artery. A method for creating the model animal, which includes [the above steps], enabling visualization of the hematoma by computed tomography immediately after induction of the hemorrhage. [Item 2] The method according to Item 1, wherein the instrument for the puncture is inserted from the pterygopalatine artery, and the contrast agent is administered from the external carotid artery into the internal carotid artery. [Item 3] The creation 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. [Item 4] The creation method according to any one of Items 1 to 3, wherein the continuous administration of the contrast agent is performed from before the puncture until 3 minutes after the puncture. [Item 5] A method for visualizing the hematoma distribution and the amount of hematoma in the subarachnoid space in a subarachnoid hemorrhage model animal, comprising the following: (a) A step of puncturing the circle of Willis to induce hemorrhage into the subarachnoid space; (b) A step of continuously administering a contrast agent into the circle of Willis while maintaining the anterograde blood flow from the common carotid artery to the internal carotid artery, at least from the puncture until hemostasis; and (c) A step of imaging the head of the subarachnoid hemorrhage model animal by computed tomography (CT). The visualization method comprising the above steps. [Item 6] The visualization method according to Item 5, wherein the instrument for the puncture is inserted from the pterygopalatine artery, and the contrast agent is administered from the external carotid artery into the internal carotid artery. [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 a subarachnoid hemorrhage model animal, comprising the following: (a) A step of puncturing the circle of Willis to induce hemorrhage into the subarachnoid space; (b) A step of continuously administering a contrast agent into the circle of Willis while maintaining the anterograde blood flow from the common carotid artery to the internal carotid artery, at least from the puncture until hemostasis; (c) A step of 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 methods, including: [Section 9] Item 9. The evaluation method according to Item 8, wherein the puncture instrument is inserted from the pterygopalatine artery, and the contrast agent is administered from the external carotid artery into the internal carotid artery. [Section 10] Item 10. The evaluation method according to Item 8 or 9, wherein the CT is a micro-CT. [Effects of the Invention]

[0009] The present invention provides a method for producing a subarachnoid hemorrhage animal model. The present invention also provides a method for visualizing hematoma distribution and hematoma volume in the subarachnoid space of a subarachnoid hemorrhage animal model, and a method for evaluating the severity of subarachnoid hemorrhage in a subarachnoid hemorrhage animal model. The production method of the present invention makes it possible to produce a subarachnoid hemorrhage animal model 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 enable immediate detailed visualization of SAH hematoma distribution and hematoma volume. The results obtained by this visualization method correlate with macroscopic SAH grading, and since there is no need to sacrifice animals for visualization, they are particularly excellent methods for visualizing and evaluating the severity of SAH. The evaluation method of the present invention makes it possible to standardize the severity between groups before conducting interventional experiments using a subarachnoid hemorrhage animal model. Furthermore, since there is no need to sacrifice animals for visualization, long-term prognosis observation is also possible. Furthermore, even with MRI and other methods that are thought to be able to diagnose subarachnoid hemorrhage to some extent, it is extremely difficult to make instantaneous and quantitative assessments of hematoma distribution and volume, and they are extremely expensive compared to the micro-CT used in the method of the present invention, and their maintenance costs are also much higher. Considering these factors, the method of the present invention can be said to be an extremely superior method for visualizing and evaluating subarachnoid hemorrhage compared to existing methods. [Brief explanation of the drawings]

[0010]

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Mode for Carrying Out the Invention

[0011] 1. Method for Creating a Subarachnoid Hemorrhage Model Animal of the Present Invention The present invention is as follows: A method for creating a subarachnoid hemorrhage model animal, comprising the following: (A) a step of puncturing the circle of Willis to induce bleeding into the subarachnoid space, and (B) a step of continuously administering a contrast agent into the circle of Willis while maintaining the antegrade blood flow from the common carotid artery to the internal carotid artery, at least from the puncture until hemostasis. A method for producing the model animal, which includes [a certain component], thereby enabling visualization of a hematoma by computed tomography immediately after induction of the hemorrhage. is provided.

[0012] Examples of the animal used in the production method of the present invention include mammals. Examples of mammals include rodents such as mice, rats, hamsters, guinea pigs, experimental animals such as rabbits, domestic animals such as pigs, cows, goats, horses, sheep, minks, pets such as dogs and cats, humans, primates such as monkeys, cynomolgus monkeys, rhesus monkeys, marmosets, orangutans, chimpanzees, etc., but are not limited thereto.

[0013] In step (A) of the production method of the present invention, as long as bleeding by arterial perforation can be induced in the subarachnoid space of the animal used in the production method of the present invention and the anterograde blood flow of the circle of Willis (particularly, the internal carotid artery) can be maintained, the method of puncturing (or perforating) the circle of Willis is not particularly limited. Specifically, for example, the filament method using a nylon thread or the wire / tubing technique can be mentioned. The puncture (or perforation) may be performed under anesthesia (e.g., isoflurane, sevoflurane, ketamine hydrochloride, propofol, etc.), and the anesthesia can be performed by a method known per se, for example, inhalation, injection, etc. The anesthesia may be continuously performed throughout all steps of the production method of the present invention, or may be appropriately stopped or interrupted.

[0014] As the insertion route of the instrument for delivering any of the above-mentioned puncturing instruments into the circle of Willis, there is no particular limitation as long as the anterograde blood flow from the common carotid artery to the internal carotid artery is maintained and another catheter insertion route (e.g., external carotid artery) for continuous administration of a contrast agent is secured. Examples include any branch of the internal carotid artery, and preferably, the pterygopalatine artery (PPA).

[0015] In step (A) of the production method of the present invention, the puncture (or perforation) site is the Circle of Willis, preferably from the internal carotid artery within the Circle of Willis to the anterior cerebral artery, specifically, from the tip of the internal carotid artery to the origin of the anterior cerebral artery, more preferably around the origin of the anterior cerebral artery. The puncturing instrument is preferably withdrawn promptly from the Circle of Willis to the origin of the insertion route (preferably PPA) after puncture so as not to obstruct the blood flow in the internal carotid artery.

[0016] In step (B) of the production method of the present invention, at least during the period from the start of bleeding due to puncture in step (A) to natural hemostasis, a desired amount of contrast agent can be continuously administered into the Circle of Willis, and 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 this period, the method of continuous administration of the contrast agent is not particularly limited. For example, it can be performed using a syringe pump or the like.

[0017] As a method of administering the contrast agent in step (B) of the production method of the present invention, for example, intra - carotid artery administration using a catheter inserted into the external carotid artery can be mentioned. In this case, the tip of the catheter is preferably inserted and fixed so as not to protrude more into the internal carotid artery side than the external carotid artery bifurcation so as not to obstruct the blood flow in the internal carotid artery.

[0018] The contrast agent is not particularly limited as long as the hematoma distribution and hematoma volume in the subarachnoid space can be imaged by computed tomography (CT) described later. For example, iodine compounds (such as organic iodic acids such as iodo - carboxylic acid, iodoform, triiodophenol, tetraiodoethylene, iohexol, etc.) can be mentioned. Preferred contrast agents include iohexol (300 mg / ml) etc. which have been proven not to cause neuropathy and can be administered into the spinal canal of the human body. The concentration of the contrast agent is not particularly limited as long as the hematoma distribution and hematoma volume in the subarachnoid space can be visualized, and it can be appropriately selected within the concentration range usually used for image diagnosis of head CT. For example, 100 mg / ml - 300 mg / ml, preferably 200 mg / ml - 300 mg / ml can be mentioned.

[0019] The contrast agent can maintain a constant concentration of the contrast agent contained in the blood flowing through the puncture site from the start of bleeding due to puncture in at least step (A) until natural hemostasis, and can image the hematoma distribution and hematoma volume in the subarachnoid space by CT described later. Therefore, it is necessary to continuously administer a certain amount at a constant rate from before puncture until natural hemostasis is completed. For example, the administration of the contrast agent should be started before puncture, specifically, 1 to 5 minutes before puncture, so that the blood concentration of the contrast agent is already maintained at a constant level during the puncture of the circle of Willis in step (A). In addition, considering the period from after the puncture of the circle of Willis until natural hemostasis is completed, the administration of the contrast agent continues for, for example, 1 to 10 minutes after puncture, preferably about 3 minutes after puncture.

[0020] In step (b) of the production method of the present invention, the contrast agent is continuously administered (i.e., continuously infused) into the internal carotid artery for the period as described above. The dosage and administration rate of the contrast agent are not particularly limited. For example, as the contrast agent solution, an amount of 0.5 ml to 0.8 ml, preferably 0.6 ml to 0.7 ml, can be administered at a rate of 6 ml / h to 10 ml / h, preferably 7 ml / h to 9 ml / h.

[0021] The present invention also provides a SAH model animal by the EP method, which can visualize a hematoma (e.g., hematoma distribution, hematoma concern) by CT immediately and in a living state from immediately after the induction of SAH prepared by the above method. In the SAH model animal, the contrast agent is continuously administered through a route different from the puncture instrument without blocking the anterograde blood flow of the internal carotid artery. Therefore, from the moment of puncture until natural hemostasis, the blood concentration of the contrast agent in the internal carotid artery and the cerebral perfusion pressure are maintained constant. Thus, the severity of bleeding and the amount of contrast agent leaking into the subarachnoid space show a high correlation, and the severity of the model animal can be evaluated without performing a SAH grade evaluation on the excised brain afterwards.

[0022] 2. Method for visualizing hematoma distribution and hematoma volume in the subarachnoid space of the present invention The present invention is as follows: A method for visualizing the hematoma distribution and hematoma volume in the subarachnoid space of a subarachnoid hemorrhage model animal, comprising the following: (a) A step of puncturing the circle of Willis to induce hemorrhage into the subarachnoid space; (b) A step of continuously administering a contrast agent into the circle of Willis while maintaining the anterograde blood flow from the common carotid artery to the internal carotid artery, at least from the puncture until hemostasis; and (c) A step of imaging the head of the subarachnoid hemorrhage model animal by computed tomography (CT). A visualization method comprising the above steps is provided.

[0023] Regarding the method for visualizing the hematoma distribution and hematoma volume in the subarachnoid space of the present invention, steps (a) and (b) of the visualization method shall incorporate all the contents of "1. Method for creating a subarachnoid hemorrhage model animal of the present invention" described above.

[0024] In step (c) of the visualization method of the present invention, regarding the hematoma distribution and hematoma volume in the subarachnoid space of the head of the subarachnoid hemorrhage model animal, as long as they can be depicted to such an extent that an evaluation at least equivalent to the macroscopic subarachnoid hemorrhage severity assessment (SAH grading) using the conventional six-division method is possible, there are no particular limitations regarding the settings of computed tomography (CT), etc., and they may be set by methods known per se. Also, from the perspective of resolution, it is preferable to use micro-CT for computed tomography (CT). Based on the head CT images obtained in step (c), the above hematoma distribution and hematoma volume can be determined by methods known per se.

[0025] 3. Method for evaluating the severity of subarachnoid hemorrhage in a subarachnoid hemorrhage model animal of the present invention The present invention provides the following: A method for evaluating the severity of subarachnoid hemorrhage in a subarachnoid hemorrhage model animal, comprising the following: (a) A step of puncturing the circle of Willis to induce hemorrhage into the subarachnoid space; (b) A step of continuously administering a contrast agent into the circle of Willis while maintaining the anterograde blood flow from the common carotid artery to the internal carotid artery, at least from the puncture until hemostasis; (c) Imaging the head of the subarachnoid hemorrhage model animal by computed tomography (CT), and (d) Based on the imaging, evaluating the severity of subarachnoid hemorrhage in the subarachnoid hemorrhage model animal An evaluation method comprising: is provided.

[0026] Regarding the method for evaluating the severity of subarachnoid hemorrhage in the subarachnoid hemorrhage model animal of the present invention, steps (a) to (c) of the evaluation method shall incorporate all the contents of the above-mentioned "1. Method for creating a subarachnoid hemorrhage model animal of the present invention" and "2. Method for visualizing the hematoma distribution and hematoma volume in the subarachnoid space of the present invention".

[0027] In step (d) of the evaluation method of the present invention, as long as 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), the evaluation method is not particularly limited. As the evaluation method, for example, the severity of the CT image may be evaluated based on the self-known SAH grading system (Sugawara et al. 2008) classification, etc., or, for example, after independently setting the evaluation criteria for SAH regarding the hematoma distribution, hematoma volume, etc., the evaluation may be performed. For example, in the case of 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).

[0028]

Table 1

[0029] When performing the evaluation method of the present invention, when considering the possibility of bleeding at different time phases (for example, delayed re-rupture), for example, a neurological behavioral test at 24 hours after the onset of SAH can be used as a pre-experimental screening test, and cases that are significantly deviated from the neurological symptoms predicted from the visualized hematoma volume may be further screened, such as being excluded from the samples.

[0030] The evaluation method of the present invention can immediately evaluate the severity of SAH in a subarachnoid hemorrhage model animal. Therefore, for example, in the sample sorting of model animals for an intervention experiment, the results of this evaluation method can be used to equalize the severity among samples. Thus, the present invention also provides a population of subarachnoid hemorrhage model animals with uniform severity using the said evaluation method. This population is useful as a parent population of SAH model animals for an intervention experiment.

Example

[0031] Micro-CT system All imaging in the example was performed using Cosmo Scan GX (Rigaku Corporation). Scanning was performed promptly after the induction of SAH. Micro-CT data was acquired at an X-ray tube voltage of 50 kVp and 160 μA (for CT acquisition). For high-resolution CT acquisition, the nominal resolution was 90 μm and the exposure time was 4 minutes.

[0032] Experimental animals As experimental animals, 16-week-old male Sprague-Dawley (SD) rats (Japan SLC, Inc.) (body weight 295 - 340 g) were used. The experimental animals were always anesthetized during imaging by CT to control the behavior and escape of the animals. All of the above animal experiments have been approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Occupational and Environmental Health. In this experiment, anesthesia was usually applied to animals inhaling isoflurane through an intubated tube. Continuous infusion of a contrast agent (iohexol 300 mg / ml) was applied by inserting a 24-gauge catheter into the ipsilateral external carotid artery and connecting it to a mechanical syringe pump.

[0033] Creation of an EP method SAH model and administration of contrast agent into the 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, the microtube and tungsten wire required for puncture were inserted through the pterygopalatine artery (PPA) rather than through the external carotid artery as in the conventional method. This enabled the catheter required for contrast agent administration to be inserted through the external carotid artery (Fig. 1). By combining arterial puncture with continuous infusion into the internal carotid artery, extravasation of the contrast agent was induced, allowing indirect visualization of the distribution of SAH.

[0034] Briefly, it was created as follows (see also Figure 2). (1) General anesthesia was induced with 3% isoflurane, and a 16G catheter was inserted. Anesthesia was maintained with inhalation isoflurane at 1-3%. Forced ventilation was initiated using a ventilator. (2) A midline neck incision was made to expose the left common carotid artery (CCA), internal carotid artery (ICA), and external carotid artery (ECA) from the carotid triangle, and the ECA was secured by dissecting it away from the surrounding tissue. (3) The pterygopalatine artery (PPA) was secured at the distal part of the left ICA, an 8-0 silk suture was placed at the bifurcation with the ICA, and the CCA and ICA beyond the PPA bifurcation were temporarily blocked with a vascular clip, after which the distal side of the PPA was ligated and severed. (4) A polytetrafluoroethylene (PTFE) tube (Braintree Scientific, SUBL-120, ID: 0.006 inch; OD: 0.012 inch) and a tungsten wire (Scientific Instruments Services, Inc., catalog number W91, diameter: 0.076 mm; length: 47 mm) were inserted into the PPA stump, and the bifurcation was ligated with 8-0 silk suture to secure the tube to the insertion site. (5) The left ICA and CCA were released from the occlusion, and antegrade blood flow was resumed (the occlusion time was approximately 1 to 2 minutes). Subsequently, the ECA bifurcation was temporarily occluded with a vascular clip. (6) A 24G catheter was inserted into the left ECA and ligated with two 8-0 silk sutures. The blockage was then released, and continuous infusion of iohexol (300 mg / ml) was initiated at 8 ml / h using a syringe pump. (7) The microtube was advanced distal to the ICA and inserted 18 mm from the PPA bifurcation, followed by the tungsten. (8) The tungsten wire was advanced so that it deviated 1.5 mm from the tip of the tube and inserted, 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 the antegrade blood flow. (9) Three minutes after the puncture, the continuous administration of contrast medium was terminated. To prevent bleeding, the ICA was again temporarily blocked with a vascular clip, the microtube and 24G catheter were removed from the PPA and ECA, respectively, and the stumps were ligated, after which the blockage was released. (10) Inhalation anesthesia with isoflurane and forced ventilation were continued, and micro-CT scans were performed within 30 minutes while the patient was attached to a ventilator. (11) Anesthesia was gradually terminated, and after spontaneous respiration stabilized, the ventilator was discontinued. The tube was extubated, and the wound was sutured closed to complete the surgery.

[0035] Image analysis of CT imaging The visualization capability of CT imaging was compared with the macroscopic findings of brain tissue immediately removed after imaging. The correlation between CT imaging findings and SAH grading was evaluated for hematoma thickness in six segments (the bil. ICA terminal, bil. IC-Pcom bifurcation, BA pons, and around the BA medullary portion) evaluated by SAH grading. Each segment was assigned a CT grade of 0 to 3 depending on the amount of subarachnoid thrombus within the segment, as shown in Table 1 below.

[0036] [Table 2]

[0037] Pathological evaluation After visually evaluating the SAH grading, pathological evaluation was performed using the brain fixed with 10% neutral buffered formalin solution. The brain was embedded in paraffin, sectioned, and evaluated with H&E. Regarding the parts where macroscopic observation from the bottom of the brain was difficult to evaluate, especially the distribution of subarachnoid hemorrhage and the presence or absence of intracerebral hemorrhage formation, including those within the cerebral ventricle and the interhemispheric fissure, the correlation with CT findings was evaluated.

[0038] Data analysis Simple regression analysis and Spearman's correlation coefficient by rank test were performed to evaluate the correlation between the SAH grade and the CT grade, and between the CT grades of each researcher. The data are presented as mean ± standard error of the mean (SEM). The statistical differences between various groups were evaluated by one-way analysis of variance using Holm-Sidak post hoc analysis. For comparison between two groups, an unpaired t test was used. A P value < 0.05 was considered statistically significant. A value of r > 0.4 was considered a significant correlation, and r > 0.7 was considered a strong correlation. Statistical analysis was performed using StatView version 5.0 for Windows.

[0039] Result 1 (Visualization of SAH immediately after induction by micro-CT (immediacy)) Comparison between the head CT image taken immediately after induction of SAH and the photograph of the excised brain is shown (Figure 3). By continuous administration of the transarterial contrast agent, successful visualization of the hematoma distribution of SAH by the EP method immediately after induction was achieved. The success of SAH induction could be confirmed immediately, and the hematoma volume and puncture site could also be inferred from the hematoma distribution (Figure 4).

[0040] Result 2 (Evaluation of the bleeding scale using micro-CT: comparison with SAH grading (detectability)) Visualization of the hematoma distribution on axial and sagittal CT imaging (Table 1 above) enabled severity classification comparable to that of the conventional 6-division method for SAH grading (Table 2). Comparison of the grading by micro-CT and the SAH grading of the excised brain is shown (Figs. 3 and 4). As understood from the results, a correlation was shown (P = 0.0109, r = 0.657).

[0041] Result 3 (Distribution of SAH and coexistence of intracerebral hemorrhage (diagnostic ability)) On micro-CT, it was observed that the distribution of the contrast agent did not remain in the basal cisterns but was widely distributed in the interhemispheric fissure, the cerebral ventricle, and the posterior cranial fossa. When diagnosing the hematoma distribution in the subarachnoid space on tissue sections of the excised brain, agreement with the imaging findings was confirmed (Fig. 5A). In addition, retention of the contrast agent was observed in the brain parenchyma, and multiple cases (n = 3) were found to be diagnosable as intracerebral hemorrhage. In cases diagnosed as intracerebral hemorrhage on CT images, it was confirmed to be intracerebral hemorrhage also by pathological diagnosis (Fig. 5B). There were cases where SAH was widely distributed in the cerebral ventricle, the interhemispheric fissure, and the posterior cranial fossa, which were not evaluated in the conventional SAH grading, not only in cases with coexistence of intracerebral hemorrhage, and it was suggested that there was a large variation in the hematoma volume among cases.

[0042] Points to note An important point in the above examples is to prevent obstruction of the antegrade blood flow in the ICA in order to keep the contrast agent concentration in the blood and the cerebral perfusion pressure at the puncture site constant, and the tip of the catheter inserted into the ECA needs to be inserted and fixed to such an extent that it does not protrude to the ICA side from the ECA branch. Also, at the time of puncture, it is necessary to quickly insert the microtube and the tungsten wire and pull them back until the tip reaches the origin of the PPA immediately after puncture.

[0043] Conventional SAH grading and new severity evaluation by micro-CT The imaging capabilities of the method described in the above example were verified by comparing it with SAH grading, and a correlation between imaging findings and macroscopic findings was confirmed. Therefore, by using this method, which can faithfully reproduce the severity of SAH, to immediately evaluate the success and severity of induction when creating an SAH model, it is possible to achieve uniformity between samples before intervention experiments. By replacing the currently widely used macroscopic SAH grading with CT imaging, retrospective severity assessment is no longer necessary and long-term prognosis observation becomes possible, which is a groundbreaking development that is expected to contribute to the future development of SAH research.

[0044] Quantitative and qualitative diagnostic capabilities of CT imaging (compared with pathological findings) In the above example, when an SAH model was actually created and visualized, it was found that not only the severity but also the distribution of hematomas within the subarachnoid space varied from case to case, and that in some cases small intracerebral hematomas, which had previously been difficult to evaluate, could also occur. In cases where intracerebral hematoma has formed, local focal symptoms such as paralysis will naturally appear, and the functional prognosis will inevitably be poor, so it is desirable to exclude them from the experimental sample.Until now, there have been cases where cerebral blood flow has been confirmed with a laser Doppler and intracranial pressure has been monitored to indirectly determine whether SAH induction has been successful and to consider whether to perform a second puncture.However, with this method, it is difficult to distinguish between cases where only ICH has occurred after puncturing the brain parenchyma and cases where the cerebral blood vessels have not been punctured, and it has become clear that there are cases where the conventional method is problematic. In this example, a comparison with pathological findings showed that this method was capable of diagnosing very small intracerebral hemorrhages and visualizing small subarachnoid hemorrhages, and was also useful for visualizing hematomas distributed in the ventricles and interhemispheric fissures, which could not previously be evaluated by observing the base of the brain alone. This will enable detailed and immediate confirmation of the overall state of bleeding, which is expected to dramatically advance future SAH research into the relationship between bleeding patterns and treatment outcomes. It may also help establish a puncture method that is less likely to cause intracerebral hematomas and more likely to induce SAH of uniform severity.

[0045] Study on the effects of contrast media The iohexol (300 mg / ml) used in this example is also used clinically in cerebrospinal imaging, so there is no possibility that it will affect neurological function. However, just in case, we investigated the effects of leakage of the contrast agent into the spinal cavity on brain damage and neurological symptoms by intracisternal injection of the contrast agent, and found no effects on nerves or other organs (Figure 5). [Industrial Applicability]

[0046] The production method of the present invention is useful because it allows the production of a subarachnoid hemorrhage animal model suitable for visualizing SAH distribution (hematoma distribution and hematoma volume in the subarachnoid space) and evaluating the severity of SAH. Furthermore, the visualization method of the present invention is useful because it allows for immediate and detailed visualization of SAH hematoma distribution and hematoma volume, and the results obtained by this visualization method correlate with macroscopic SAH grading, providing excellent visualization capabilities and eliminating the need to sacrifice animals for visualization. Furthermore, the evaluation method of the present invention is useful because it allows for the evaluation of SAH severity before intervention experiments, thereby avoiding bias.

Claims

1. A method for creating a subarachnoid hemorrhage model non-human mammal, comprising the following: (A) a step of puncturing the circle of Willis using a puncturing instrument inserted from the pterygopalatine artery to induce hemorrhage into the subarachnoid space, and (B) a step of continuously administering a contrast agent into the internal carotid artery through the external carotid artery while maintaining the anterograde blood flow from the common carotid artery to the internal carotid artery, at least from the puncture until hemostasis, thereby enabling visualization of the hematoma by computed tomography immediately after the induction of the hemorrhage. A method for creating the model non-human mammal.

2. The method for creation according to claim 1, wherein the puncture site of the circle of Willis is the anterior cerebral artery from the internal carotid artery.

3. The method for creation according to claim 1 or 2, wherein the continuous administration of the contrast agent is performed from before the puncture until 3 minutes after the puncture.

4. A method for visualizing the hematoma distribution and the amount of hematoma in the subarachnoid space in a subarachnoid hemorrhage model non-human mammal, comprising the following: (a) a step of puncturing the circle of Willis using a puncturing instrument inserted from the pterygopalatine artery to induce hemorrhage into the subarachnoid space, (b) a step of continuously administering a contrast agent into the internal carotid artery through the external carotid artery while maintaining the anterograde blood flow from the common carotid artery to the internal carotid artery, at least from the puncture until hemostasis, and (c) a step of imaging the head of the subarachnoid hemorrhage model non-human mammal by computed tomography (CT) A visualization method.

5. The visualization method according to claim 4, wherein the computed tomography (CT) is micro-CT.

6. A method for evaluating the severity of subarachnoid hemorrhage in a subarachnoid hemorrhage model non-human mammal, comprising the following: (a) a step of puncturing the circle of Willis using a puncturing instrument inserted from the pterygopalatine artery to induce hemorrhage into the subarachnoid space, (b) a step of continuously administering a contrast agent into the internal carotid artery through the external carotid artery while maintaining the anterograde blood flow from the common carotid artery to the internal carotid artery, at least from the puncture until hemostasis, (c) a step of imaging the head of the subarachnoid hemorrhage model non-human mammal by computed tomography (CT), and (d) a step of evaluating the severity of subarachnoid hemorrhage in the subarachnoid hemorrhage model animal based on the imaging An evaluation method.

7. The evaluation method according to claim 6, wherein the computed tomography (CT) is micro-CT.

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