Construction and evaluation method of post-stroke constipation animal model

Abstract

The application relates to the technical field of animal model construction and evaluation, in particular to a construction and evaluation method of a post-stroke constipation animal model, which comprises the following steps: selecting healthy animals, adaptively feeding the animals, performing a transient middle cerebral artery occlusion operation on the animals by adopting a thread plug method, reperfusing the animals after ischemia, and constructing a stroke model; after the construction is successful, daily gavage administration of loperamide hydrochloride is carried out on the animals, and the administration is continuously performed until the model construction is completed; the model constructed by the application has stable phenotypes and high repeatability.

Description

Technical Field

[0001] This invention relates to the field of animal model construction and evaluation technology, specifically to a method for constructing and evaluating an animal model of constipation after stroke. Background Technology

[0002] Stroke is a highly prevalent and disabling cerebrovascular disease in my country. While improved treatment techniques have significantly increased patient survival rates, managing various complications remains crucial for improving prognosis. Post-stroke constipation is a common complication, with an incidence rate ranging from 30% to 60%. It refers to secondary functional bowel disorders that occur during the acute and recovery phases of a stroke, characterized by reduced bowel movement frequency, difficulty defecating, hard stools, and prolonged intervals between bowel movements.

[0003] Constipation not only severely impacts patients' quality of life and neurological function recovery, but the straining and breath-holding caused by difficulty in defecation can also lead to increased intracranial pressure, significantly increasing the risk of recurrent stroke. Furthermore, prolonged accumulation of feces in the intestines allows some bacteria to metabolize and circulate through the intestines, potentially further exacerbating damage to the central nervous system.

[0004] Currently, research on post-stroke constipation largely focuses on clinical efficacy observations. However, basic experimental research lacks stable animal models that can mimic post-stroke constipation, hindering the exploration of its related mechanisms and key molecular targets. Previous studies have used stroke-only models (such as the MCAO model) to induce constipation, but this method exhibits significant individual variability and instability; in some rats, defecation function recovers spontaneously over time, resulting in an unstable constipation phenotype. Furthermore, constipation models induced solely by loperamide only simulate intestinal transit disorders and fail to reflect the neurological damage background following stroke, thus differing from the exact pathogenic factors of post-stroke constipation. Therefore, there is an urgent need to establish a phenotypically stable and highly reproducible animal model of post-stroke constipation. Summary of the Invention

[0005] The technical problem that this invention aims to solve is to provide a method for constructing and evaluating an animal model of constipation after stroke, which has stable phenotype and high reproducibility.

[0006] This invention provides a method for constructing an animal model of constipation after stroke, comprising the following steps: Healthy animals were selected and acclimatized before a transient middle cerebral artery occlusion surgery was performed using the suture occlusion method. After ischemia, reperfusion was performed to construct a stroke model. After successful model construction, loperamide hydrochloride was administered by gavage daily until the model construction was completed.

[0007] The preferred procedure for transient middle cerebral artery occlusion is as follows: after anesthetizing the animal, the left common carotid artery, internal carotid artery and external carotid artery are separated, the proximal end of the common carotid artery and the external carotid artery are ligated, the internal carotid artery is clamped with an arterial clamp, a suture is inserted into the middle cerebral artery through the incision in the common carotid artery, and the suture is removed after 2 hours to achieve reperfusion.

[0008] Preferably, the insertion depth of the plug is 16-20mm.

[0009] Preferably, after reperfusion, once the animal is fully awake, the Zea-Longa scoring method is used to assess the animal's neurological deficits in order to determine whether the modeling was successful.

[0010] Preferably, after successful construction, wait 24 hours before administering loperamide hydrochloride by gavage.

[0011] Preferably, the dosage of loperamide hydrochloride is 3 mg / kg / day.

[0012] This invention provides an evaluation method for an animal model of post-stroke constipation constructed using the aforementioned method, which evaluates the model by detecting the following indicators: a) Neurological deficit score b) Infarct volume; c) Constipation-related indicators, including 24-hour stool particle count, 3-hour stool water content, and small intestinal propulsion rate; d) Histopathological morphology of the colon; e) Serum levels of substance P and motilin.

[0013] Preferably, the neurological deficit score is assessed using the Zea-Longa scoring method; the scoring criteria are as follows: 0 points indicate no neurological deficit, 1 point indicates inability to fully extend the forepaws when the tail is lifted, 2 points indicate rotation or circling to one side when walking, 3 points indicate tilting to one side when walking or standing, and 4 points indicate inability to walk spontaneously, impaired consciousness, or coma.

[0014] Preferably, the method for detecting the small intestinal propulsion rate is as follows: the animal is given an oral gavage of activated charcoal suspension, and after a period of time, the rat is sacrificed. The distance from the pylorus to the ink front and the total length of the small intestine from the pylorus to the ileocecal junction are measured, and the small intestinal propulsion rate is calculated according to the following formula: Small intestinal propulsion rate = (ink propulsion length / total small intestinal length) × 100%.

[0015] The beneficial effects of this invention are that the model exhibits stable phenotype and high reproducibility. This invention combines a transient middle cerebral artery occlusion (tMCAO) model with loperamide hydrochloride induction. Utilizing the stable and quantifiable inhibitory effect of loperamide on intestinal peristalsis, it effectively compensates for the shortcomings of the simple MCAO model, which suffers from unstable and insignificant constipation phenotypes due to large individual differences. Experimental data (such as the coefficient of variation of fecal particle number and small intestinal propulsion rate) confirm that this method successfully constructs a phenotypically consistent, stable, and highly reproducible animal model of post-stroke constipation.

[0016] This invention employs a tMCAO model with reperfusion after 2 hours of ischemia, accurately simulating the typical scenario of blood flow reconstruction in stroke patients after thrombolysis or thrombectomy. Simultaneously, the administration of loperamide 24 hours post-surgery, during the acute phase, effectively simulates the acute constipation caused by neurological dysfunction and bed rest in the early post-stroke period, making the model more clinically relevant.

[0017] This invention provides a multi-dimensional model evaluation method from macroscopic to microscopic levels. This system not only covers core stroke indicators such as neurological deficit scores and infarct volume, but also includes constipation behavioral indicators such as fecal characteristics and intestinal transit function, and further extends to molecular biological levels such as colonic histopathology and serum gastrointestinal hormones (SP, MTL). This systematic evaluation method can comprehensively and objectively verify the reliability of the model, providing a solid foundation for subsequent mechanism research and drug screening. Attached Figure Description

[0018] Figure 1 A comparison chart of Zea-Longa neurological deficit scores in rats from different groups.

[0019] Figure 2 Images of TTC-stained brain tissue from rats in each group.

[0020] Figure 3 A graph comparing the percentage of cerebral infarction volume in each group of rats.

[0021] Figure 4 HE-stained images of colon tissue from rats in each group.

[0022] Figure 5 The graph shows the results of serum SP and MTL levels in rats of each group detected by ELISA. Detailed Implementation

[0023] The present invention will be further described in detail below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0024] Example 1 A method for constructing an animal model of constipation after stroke, comprising the following steps: 1. Materials and Methods 1.1 Materials Experimental animals: SPF-grade male SD rats, weighing (200±10) g.

[0025] Main reagents: thread plug, isoflurane, loperamide hydrochloride, activated charcoal, gum arabic, TTC staining solution (2,3,5-triphenyltetrazolium chloride), ELISA kit, etc.

[0026] 1.2 Methods 1.2.1 Animal grouping and modeling Thirty rats were acclimatized for one week and then randomly divided into three groups: a sham group (n=9), an MCAO group (n=9), and an MCAO+Lop group (n=9). In the Sham group, the corresponding site was only incised and sutured without inserting a suture embolus. The MCAO group was a middle cerebral artery occlusion model group, and the MCAO+Lop group was a middle cerebral artery occlusion combined with loperamide.

[0027] MCAO model establishment: The rat was fasted for 12 hours preoperatively but allowed free water. It was weighed, and after anesthesia, the rat was placed in a supine position with its limbs fixed to a maintenance anesthesia board with tape. After hair removal and disinfection with iodine, the skin was prepared. A 0.5cm incision was made lateral to the left side of the thyroid cartilage. Using forceps, the muscles and fascia were bluntly dissected layer by layer until the space formed by the carotid triangle was observed. The common carotid artery and its accompanying vagus nerve were located below this space. The vagus nerve was gently separated from the common carotid artery. The internal and external carotid arteries were located superiorly along the common carotid artery. The proximal end of the common carotid artery was ligated, and a slipknot was tied at the distal end. The external carotid artery was ligated near its bifurcation, and the internal carotid artery was clamped. A small incision was made near the proximal end of the common carotid artery, a suture plug was inserted, the slipknot was tightened, and the vascular clamp was released, allowing the suture plug to enter the internal carotid artery from the common carotid artery. After entering the skull, the suture plug blocked the middle cerebral artery. The insertion depth was approximately (18±2mm). Insertion was stopped when slight resistance was felt, and the skin was sutured. Two hours later, after anesthetizing the rats, the suture plug was gently removed. When the suture plug head felt resistance, that is, when the suture plug head was withdrawn to the bifurcation point between the inner and outer neck, the removal of the suture plug was stopped, the excess suture plug was cut off, and blood flow was restored.

[0028] After the rats were fully awakened from anesthesia and the model was established for 4 hours, the Zea-Longa scoring method was used to preliminarily assess the rats' neurological deficits. 0 points: no neurological deficit; 1 point: inability to fully extend the right forearm when tail is lifted; 2 points: turning or circling to the right when walking; 3 points: leaning to the right when walking or standing; 4 points: inability to walk spontaneously, impaired consciousness, or coma. Rats scoring 1-3 points were included in the study; rats with a score of 0 (no deficit) and those with a score of 4 (inability to walk spontaneously or coma) were excluded from the experiment.

[0029] Loperamide induces constipation: The MCAO+Lop group received loperamide 3 mg / kg daily via gavage 24 hours after modeling, while the sham surgery group and MCAO group received the same dose and frequency of normal saline via gavage. The neck incision was observed before and after each gavage, and any redness, swelling, effusion, suture loosening, or dehiscence was recorded.

[0030] Example 2 An evaluation method for an animal model of constipation after stroke, comprising the following steps: I. Detection Indicators and Methods 1. General testing: After modeling, the rats were observed daily for activity, mental state, fur hygiene, diet, and weight changes.

[0031] 2. Detection of the extent of cerebral infarction: The day after model establishment, three rats from each group were randomly selected for TTC staining to verify the successful establishment of the MCAO model. After anesthesia, rats were quickly euthanized by decapitation, and the intact brain tissue was removed from the skull and frozen at -20°C for 20 minutes. The brain was then sliced ​​into 5-6 2mm thick sections along the coronal plane and rapidly placed in 2% TTC staining solution. Incubation was performed at 37°C in the dark for 30 minutes, with the sections being flipped once every 15 minutes to ensure uniform staining. After staining, the sections were fixed in 4% paraformaldehyde. After fixation, images were taken, and ImageJ software was used to analyze the infarct area. The infarct ratio was calculated as infarct area / total volume × 100%.

[0032] 3. Constipation index test: 24-hour fecal particle count: Rats were housed individually for 7 days after modeling, during which time they had free access to food and water. Feces were collected over 24 hours, and the number of fecal particles was counted.

[0033] 3-hour fecal moisture content: During the 7-day modeling period, the wet weight of feces in each group was recorded at the same time for 3 hours. The feces were then placed in a drying oven and dried completely for 24 hours before being weighed dry. The fecal moisture content was then calculated. Fecal moisture content = (wet weight - dry weight) / wet weight × 100%.

[0034] Small intestinal propulsion rate: Rats were fasted for 12 hours. One hour after the last administration of loperamide, each rat was administered 2 ml of 10% activated charcoal suspension by gavage. After 30 minutes, the rats were anesthetized and sacrificed. Intestinal tissue was harvested to measure the distance from the pylorus to the ink front and the total length of the small intestine from the pylorus to the ileocecal junction. Activated charcoal suspension was administered to each rat at 5-minute intervals to ensure that the charcoal suspension remained in the stomach for 30 minutes for each rat when intestinal tissue was harvested. Excessive traction on the intestinal segment should be avoided during intestinal dissection and measurement. Small intestinal propulsion rate % = (Ink propulsion length / Total small intestinal length) × 100%.

[0035] 4. HE staining to observe colon tissue morphology Rats were anesthetized and euthanized. Colonic tissue approximately 5 cm in diameter was quickly removed, and a portion was fixed in 4% paraformaldehyde solution for later use. The paraformaldehyde-fixed colonic tissue was then embedded in paraffin, sectioned, and subjected to dewaxing, gradient ethanol hydration, hematoxylin staining, differentiation with differentiation solution, blue reversion with inverted blue solution, eosin staining, xylene clearing, and mounting with neutral resin. Morphological changes in the colonic tissue were then observed under a microscope.

[0036] 5. ELISA detection of serum substance P and motilin levels The collected supernatant was diluted to prepare a standard working solution. Blank wells, standard wells, and sample wells were prepared. After adding samples, the plates were sealed with sealing film and incubated at 37°C for 30 min. The plates were then prepared with solution, washed, enzyme added, and incubated and washed again. A chromogenic reagent was added and developed in the dark for 10 min. After adding stop solution, the OD value was measured at 450 nm using a microplate reader. A standard curve was plotted, and the serum substance P (SP) and motilin (MTL) levels were calculated based on the standard curve.

[0037] II. Statistical Analysis SPSS 29.0 statistical software was used for analysis, and Prism 10 software was used for plotting. Quantitative data conforming to a normal distribution are expressed as x±s. When comparing multiple groups, if the variances are homogeneous, one-way ANOVA was used, and pairwise comparisons between groups were performed using the LSD-t test. If the variances are unequal, Dunnett's T3 test was used. A p<0.05 was considered statistically significant.

[0038] result 1. General condition of rats During the study, rats in the Sham group were in good spirits, active, and had smooth, clean fur. Their food and water intake gradually returned to normal after the sham surgery. Compared with the Sham group, rats in the MCAO group and the MCAO+Lop group were in poorer spirits, less active, had dull fur, and reduced food and water intake. After modeling, the body weight of both the MCAO group and the MCAO+Lop group showed a decreasing trend compared with the Sham group (Table 1), indicating that MCAO surgery can lead to weight loss, and the difference was statistically significant (P<0.05). There was no statistically significant difference in body weight between the MCAO group and the MCAO+Lop group.

[0039] Table 1 Comparison of rat body weight among groups (x±s, n=6, g)

[0040] 2. Rats' neurological deficit score After the model was established and the rats in each group were fully awake, their neurological deficit scores were assessed using the Zea-Langa scale. Higher scores indicated more severe neurological damage. Compared with the Sham group, the neurological deficit scores of rats in both the MCAO and MCAO+Lop groups were significantly higher (p<0.01), while there was no significant difference between the MCAO and MCAO+Lop groups. Figure 1 ).

[0041] 3. Detection of the extent of cerebral infarction After MCAO modeling, three rats from each group were randomly selected for TTC staining. After TTC staining, the brain tissue of the sham-operated group rats appeared uniformly red, with no infarct foci. Compared with the sham-operated group, the cerebral infarction volume increased in both the MCAO group and the MCAO+Lop group (p<0.01). There was no statistically significant difference between the MCAO and MCAO+Lop groups. Figure 2 , Figure 3 ).

[0042] 4. Constipation index test results Fecal particle count and fecal water content are important indicators for evaluating gastrointestinal function in constipation. 24-hour fecal particle count reflects overall colonic transit function and defecation frequency; 3-hour fecal water content assesses the colon's ability to reabsorb water; and small intestinal propulsion rate reflects small intestinal peristalsis speed. In all three indicators, the MCAO group showed significantly lower values ​​compared to the Sham group (P<0.05). These results suggest that stroke itself can induce intestinal motility disorders and constipation tendency through autonomic nervous system dysfunction. However, the large intra-group variability indicates that the constipation phenotype induced by MCAO alone is not very stable. In contrast, the MCAO+Lop group showed significantly lower values ​​for all three indicators compared to the sham-operated group and the MCAO group. Compared to the MCAO group, the MCAO+Lop group showed significantly smaller intra-group variability in 24-hour fecal particle count, 3-hour fecal water content, and small intestinal propulsion rate, with a smaller intra-group standard deviation, indicating a more consistent constipation phenotype. This suggests that combined modeling can obtain a more stable and reproducible constipation phenotype (see Table 2).

[0043] Table 2 Results of constipation indicators in each group

[0044] 5. Colonic tissue morphology In the Sham group, the colonic mucosa structure was intact, the glands were well-arranged, and no obvious edema or inflammatory cell infiltration was observed in the submucosa. In the MCAO group, mild epithelial damage, mild glandular disorder, partial atrophy, and scattered inflammatory cell infiltration and mild edema were observed in the submucosa. The MCAO+Lop group, however, showed mucosal epithelial damage, significant glandular atrophy and disordered arrangement, significant inflammatory cell infiltration, and submucosal edema (see [link to relevant documentation]). Figure 4 ).

[0045] 6. Serum SP and MTL levels Compared with the Sham group, the serum SP and MLT levels in the MCAO+Lop group were significantly lower (P<0.01). Compared with the Sham group, there was no significant difference in serum SP levels in the MCAO group, but the decrease in serum MLT levels was statistically significant (P<0.05). Compared with the MCAO group, the SP and MLT levels in the MCAO+Lop group were further reduced (see...). Figure 5 ).

[0046] This invention employs a 2-hour ischemia-reperfusion model to simulate the typical clinical scenario of blood flow reconstruction after stroke thrombolysis and thrombectomy. Compared to the permanent occlusion model, the reperfusion model more closely reflects the post-clinical condition of most patients. Loperamide, as a peripheral μ-opioid receptor agonist, can inhibit intestinal peristalsis and increase water absorption. This invention utilizes the stable and quantifiable effects of loperamide to transform the originally highly individualized and phenotypically unstable intestinal inhibitory effect after stroke into a more stable and repeatable post-stroke constipation phenotype.

[0047] Clinical data shows that constipation after stroke is not a chronic sequela but can occur in the acute phase, which is related to autonomic dysfunction, bed rest, and dehydration after stroke. Animal experiments have also confirmed that the pathophysiological process of intestinal dysfunction after stroke begins in the early stages after the stroke. Therefore, intervention within 24 hours after surgery effectively simulates the pathological state of acute constipation in clinical practice. In addition, the interference of anesthesia and acute surgical stress on intestinal peristalsis has been eliminated within 24 hours after surgery, so gavage was chosen in the acute phase 24 hours after MCAO. Post-stroke constipation most often presents as a complex constipation dominated by chronic transit constipation, and loperamide inhibits the release of enteric neurotransmitters and slows down intestinal peristalsis, which is a classic modeling method for chronic transit constipation. Its stable model phenotype compensates for the deficiency of unstable constipation phenotype in the simple MCAO model, increasing the controllability and reproducibility of post-stroke constipation.

[0048] The experimental results showed that, compared with the sham-operated group, both the MCAO group and the MCAO+Lop group had lower levels of three indicators: 24-hour fecal particle count, 3-hour fecal water content, and small intestinal propulsion rate. Further comparison revealed that the MCAO+Lop group showed significantly lower levels of all three indicators compared to the MCAO group, indicating that combined gavage administration of loperamide could further aggravate the constipation phenotype. Although the MCAO group exhibited constipation, the phenotype was unstable, with a large standard deviation and significant intra-group variability. In particular, the standard deviation of 24-hour fecal particle count was ±7.48 (coefficient of variation CV = 51.56%), suggesting that relying solely on autonomic dysfunction after stroke cannot guarantee stable constipation in every rat. In contrast, the MCAO+Lop group had a smaller standard deviation and reduced variability, indicating that combined loperamide not only further aggravated the severity of constipation but, more importantly, reduced intra-group individual variability and improved the reproducibility of the model operation.

[0049] In summary, while the simple MCAO model can simulate post-stroke neurological damage, its constipation phenotype is unstable and exhibits significant individual variability, making it unreliable as a model for studying post-stroke constipation. Individual differences may also affect research results during the screening of related mechanisms and drugs. However, this invention, using a 2-hour ischemia-reperfusion MCAO model combined with intragastric administration of loperamide within 24 hours, establishes a stable post-stroke constipation model. Utilizing the stable and quantifiable intestinal peristalsis inhibition effect of loperamide, the significantly individualized autonomic neurogenic intestinal inhibition effect after stroke is transformed into a stable and reproducible constipation phenotype, resulting in good consistency in constipation function indicators and colonic pathological changes. Therefore, this model can serve as a stable, controllable, and reproducible animal model for studying post-stroke constipation.

[0050] This invention successfully established a stable, controllable, and reproducible rat model of post-stroke constipation using a combination of tMCAO and loperamide gavage. While the MCAO model alone can simulate post-stroke neurological damage, the induced constipation phenotype is unstable and exhibits significant individual variability. This invention utilizes loperamide as a peripheral μ-opioid receptor agonist to stably inhibit intestinal peristalsis, transforming the unstable intestinal inhibitory effect after stroke into a stable constipation phenotype. Experimental results show that this model exhibits typical post-stroke constipation characteristics at the behavioral, histopathological, and molecular biological levels, providing an ideal animal model for related mechanism research and drug screening.

[0051] Those skilled in the art should understand that the discussion of any of the above embodiments is merely exemplary and is not intended to imply that the scope of protection of this application is limited to these examples; within the framework of this application, the technical features of the above embodiments or different embodiments can also be combined, the steps can be implemented in any order, and there are many other variations of different aspects of one or more embodiments of this application as described above, which are not provided in detail for the sake of brevity.

[0052] One or more embodiments in this application are intended to cover all such substitutions, modifications, and variations that fall within the broad scope of this application. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of one or more embodiments in this application should be included within the protection scope of this application.

Claims

1. A method for constructing an animal model of constipation after stroke, characterized in that, Includes the following steps: Healthy animals were selected and acclimatized before a transient middle cerebral artery occlusion surgery was performed using the suture occlusion method. After ischemia, reperfusion was performed to construct a stroke model. After successful model construction, loperamide hydrochloride was administered by gavage daily until the model construction was completed.

2. The construction method as described in claim 1, characterized in that, The specific procedure for transient middle cerebral artery occlusion surgery is as follows: After anesthetizing the animal, the left common carotid artery, internal carotid artery and external carotid artery are separated, the proximal end of the common carotid artery and the external carotid artery are ligated, the internal carotid artery is clamped with an arterial clamp, a suture is inserted into the middle cerebral artery through the incision in the common carotid artery, and the suture is removed 2 hours later to achieve reperfusion.

3. The construction method as described in claim 2, characterized in that, The insertion depth of the plug is 16-20mm.

4. The construction method as described in claim 1, characterized in that, After reperfusion, once the animal is fully awake, the Zea-Longa scoring method is used to assess the animal's neurological deficits in order to determine whether the modeling was successful.

5. The construction method as described in claim 1, characterized in that, After successful construction, wait 24 hours, and then administer loperamide hydrochloride by gavage.

6. The construction method as described in claim 1, characterized in that, The dosage of loperamide hydrochloride is 3 mg / kg / day.

7. An evaluation method for an animal model of post-stroke constipation constructed according to any one of claims 1-6, characterized in that, The evaluation is conducted by measuring the following indicators: a) Neurological deficit score b) Infarct volume; c) Constipation-related indicators, including 24-hour stool particle count, 3-hour stool water content, and small intestinal propulsion rate; d) Histopathological morphology of the colon; e) Serum levels of substance P and motilin.

8. The method of claim 7, characterized in that, The neurological deficit score was assessed using the Zea-Longa scoring method. The scoring criteria were as follows: 0 points indicated no neurological deficit, 1 point indicated inability to fully extend the forepaws when the tail was lifted, 2 points indicated turning to one side or circling to one side when walking, 3 points indicated falling to one side when walking or standing, and 4 points indicated inability to walk spontaneously, impaired consciousness, or coma.

9. The evaluation method according to claim 7, characterized in that, The method for detecting the small intestinal propulsion rate is as follows: the animal is given an oral gavage of activated charcoal suspension, and after a period of time, the rat is sacrificed. The distance from the pylorus to the ink front and the total length of the small intestine from the pylorus to the ileocecal junction are measured. The small intestinal propulsion rate is calculated according to the following formula: Small intestinal propulsion rate = (ink propulsion length / total length of small intestine) × 100%.

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