Cat magnesium ammonium phosphate stone model and construction method and application thereof

Abstract

The invention relates to a cat magnesium ammonium phosphate calculus model and a construction method and application thereof, and belongs to the technical field of medical animal models. The construction method of the cat magnesium ammonium phosphate calculus model comprises the following steps: inserting a sterile catheter into the bladder of a modeling animal through the urethra; the proteus mirabilis bacterial liquid is injected into the bladder of the modeling animal through the catheter, the catheter is pulled out, and when it is observed that magnesium ammonium phosphate crystals and / or magnesium ammonium phosphate stones exist in the bladder and / or urine of the modeling animal, the cat magnesium ammonium phosphate stone model is obtained. The modeling method aims at safely and effectively simulating the key pathological process of cat struvite urolithiasis induced by upstream urinary tract infection (proteus mirabilis infection), and the defects that due to operator factors, the individual severity degree difference is large, other complications are difficult to control, repeatability is poor and the like can be overcome.

Description

Technical Field

[0001] This invention relates to the field of medical animal model technology, and in particular to a cat magnesium ammonium phosphate stone model, its construction method and application. Background Technology

[0002] Urolithiasis (magnesium ammonium phosphate stones) in cats is a serious urinary tract disease that can cause acute symptoms such as difficulty urinating, frequent urination, hematuria, and urethral obstruction. It can also lead to bladder damage, kidney dysfunction, and even uremia. Certain pathogens, such as Proteus mirabilis (…), can also cause this condition. Proteus mirabilis Magnesium ammonium phosphate (MgA) can break down urea in urine to produce ammonia, significantly increasing urine pH and promoting the crystallization of MgA. These crystals can aggregate to form stones, irritating the urethral mucosa, causing severe inflammation, and even urinary tract obstruction. More seriously, if the urethral obstruction is not relieved in time, it can rapidly lead to acute post-renal renal failure, hyperkalemia, and uremic shock, which is particularly fatal to male cats. In addition, recurrent MgA stones and accompanying urinary tract infections (UTIs) can lead to chronic cystitis, urethral stricture, and permanent kidney damage, greatly threatening the cat's health and quality of life.

[0003] Clinically, key factors leading to the formation of magnesium ammonium phosphate stones include: urine alkalization (high pH), excessively high concentrations of magnesium and ammonium ions in the urine, and infection with specific urease-positive bacteria. Among these, *Proteus mirabilis* (…) Proteus mirabilis () is a representative urease-positive pathogen.

[0004] Based on this, common methods for constructing feline urolithiasis models include: ① feeding a special diet high in phosphorus and magnesium that can lead to urine alkalization (e.g., a high-plant protein diet) to simultaneously increase the concentration of magnesium and phosphorus ions in the urine and maintain an alkaline environment, thereby promoting the formation of magnesium ammonium phosphate crystals; ② surgically implanting sterile or sterile foreign objects (such as zinc plates, stones, etc.) into the bladder to act as the core for the precipitation and attachment of magnesium ammonium phosphate crystals, accelerating stone formation. However, the above two methods have the following drawbacks: Method ① has a long modeling cycle, requires high control over the diet formula, is greatly affected by individual differences, and is prone to causing other metabolic problems; Method ② involves invasive procedures that cause significant trauma to the animals, have a certain operational threshold, and are prone to postoperative complications.

[0005] Therefore, there is an urgent need to develop a more convenient, faster, and more repeatable method for constructing a cat magnesium ammonium phosphate stone model. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a cat magnesium ammonium phosphate stone model with high repeatability, consistency and standardized operation, as well as its construction method and application.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a method for constructing a cat-like magnesium ammonium phosphate stone model, comprising the following steps: S1. Insert a sterile urinary catheter into the bladder of the model animal via the urethra; S2. Inject Proteus mirabilis bacterial solution into the bladder of the model animal through a urinary catheter, remove the urinary catheter, and obtain the cat magnesium ammonium phosphate stone model when magnesium ammonium phosphate crystals and / or magnesium ammonium phosphate stones are observed in the bladder and / or urine of the model animal.

[0008] Proteus mirabilis can produce urease, which decomposes urea in the bladder to produce ammonia, leading to urine alkalization and promoting the precipitation and aggregation of magnesium ammonium phosphate crystals to form stones. This invention involves the artificial introduction of Proteus mirabilis (… Proteus mirabilis The study induced the formation of struvite crystals and stones in animal models, accurately simulating the complete pathophysiological process of ascending urinary tract infection leading to struvite formation in cats, including bacterial colonization, urease-mediated urinary alkalization, crystal precipitation and stone formation, thus simulating the real state of spontaneous struvite urolithiasis in cats and constructing a cat magnesium ammonium phosphate stone model.

[0009] This invention, by injecting Proteus mirabilis bacterial solution into the bladder of model animals, allows for precise control of the amount of Proteus mirabilis solution, resulting in reproducibility, consistency, and standardization. Compared to commonly used methods such as diet-induced modeling and foreign body implantation modeling, the modeling method of this invention aims to safely and effectively simulate the key pathological processes of feline avian urolithiasis induced by ascending urinary tract infection (Proteus mirabilis infection), avoiding the drawbacks of large individual variability in severity due to operator factors, difficulty in controlling the occurrence of other complications, and poor reproducibility.

[0010] In a preferred embodiment of the construction method described in this invention, in step S2, the concentration of the *Proteus mirabilis* bacterial solution is 10. 8 ~10 10 CFU / mL.

[0011] In a preferred embodiment of the construction method described in this invention, in step S2, the concentration of the *Proteus mirabilis* bacterial solution is (5 × 10⁻⁶). 8 )~(5×10 9 CFU / mL.

[0012] In a preferred embodiment of the construction method described in this invention, in step S2, the concentration of the *Proteus mirabilis* bacterial solution is 1 × 10⁻⁶. 9 CFU / mL.

[0013] In a preferred embodiment of the construction method of the present invention, in step S2, the injection volume of the Proteus mirabilis bacterial solution is 0.5-2 mL.

[0014] In a preferred embodiment of the construction method of the present invention, in step S2, the injection volume of the Proteus mirabilis bacterial solution is 1 mL.

[0015] In a preferred embodiment of the construction method of the present invention, in step S2, the *Proteus mirabilis* in the bacterial culture is positive and / or strongly positive for urease detection. A positive and / or strongly positive urease detection is achieved by inoculating *Proteus mirabilis* into a culture medium and culturing it; if the culture turns pink, it is considered positive; or by detecting *Proteus mirabilis* according to the instructions of the urease detection kit.

[0016] As a preferred embodiment of the construction method of the present invention, in step S2, the Proteus mirabilis bacterial solution is mainly prepared by the following method: Proteus mirabilis is inoculated into a culture medium and cultured.

[0017] In a preferred embodiment of the construction method of the present invention, in step S2, the culture medium includes, but is not limited to, nutrient agar medium and / or LB medium.

[0018] As a preferred embodiment of the construction method of the present invention, in step S2, the culture conditions are culturing at 35-37℃ and 180-220 rpm for 18-24 h.

[0019] In a preferred embodiment of the construction method of the present invention, in step S2, the Proteus mirabilis bacterial solution is injected once a day and six times a week.

[0020] Secondly, the present invention provides a cat magnesium ammonium phosphate stone model, which is mainly prepared by the above-mentioned construction method.

[0021] Thirdly, this invention provides the application of the aforementioned feline magnesium ammonium phosphate (MgMP) stone model in feline urolithiasis research for non-diagnostic or non-therapeutic purposes. The feline MgMP stone model of this invention can be used to construct different types of feline MgMP stone models according to experimental needs, which helps in studying the hemodynamic and inflammatory cytokine pathophysiological changes in different types of feline urolithiasis, and aids in screening therapeutic drugs for feline urolithiasis and developing new treatment regimens.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) This invention, by injecting Proteus mirabilis bacterial solution into the bladder of model animals, can precisely control the amount of Proteus mirabilis bacterial solution, making it reproducible, consistent, and standardized. Compared with commonly used methods of diet-induced modeling and foreign body implantation modeling, the modeling method of this invention aims to safely and effectively simulate the key pathological process of feline avian urolithiasis induced by ascending urinary tract infection (Proteus mirabilis infection), avoiding the disadvantages of large individual differences in severity due to operator factors, difficulty in controlling the occurrence of other complications, and poor reproducibility.

[0023] (2) The present invention, through direct intravesical instillation of high concentrations of live bacteria, ensures the effective establishment of Proteus mirabilis infection focus, and can induce struvite formation in a short period of time (several days to three weeks). The choice of urinary catheter instillation technique is less invasive than bladder puncture, simpler to operate, and more in line with routine veterinary clinical operation standards, reducing the risk of complications (such as intraperitoneal hemorrhage, bladder wall injury), and improving the reproducibility of model construction and animal welfare. At the same time, it directly simulates the key process of struvite formation caused by ascending urinary tract infection (bacterial colonization of the bladder, urease action, local urine alkalization, crystal precipitation and stone formation), and can accurately reflect the core pathogenesis of this type of stone. Attached Figure Description

[0024] Figure 1 The results of bladder ultrasound examination of the experimental cats before and after modeling in Example 1 of this invention are shown in Example 1. Figure 2 The results of microscopic examination of urine from experimental cats before and after modeling in Example 1 of the present invention are shown in Example 1. Figure 3 The results of blood biochemical index detection in experimental cats before and after modeling in Example 1 of the present invention; Figure 4 The results of routine urine tests on experimental cats before and after modeling are shown in Example 1 of the present invention. Detailed Implementation

[0025] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0026] Unless otherwise specified, all materials and reagents used in the following examples, comparative examples, and effect examples are commercially available.

[0027] The Proteus mirabilis used in the following examples can be obtained commercially, as long as it is confirmed that the urease test of this Proteus mirabilis is strongly positive.

[0028] The modeling animals used in the following examples and comparative examples were experimental cats (aged 1 - 5 years) with no history of urinary system diseases. They were adaptively raised under the conditions of a standard experimental animal house, fed a standard full - price maintenance cat food, and had free access to water. Before the experiment, a systematic physical examination was conducted on all selected cats, including taking urine for routine urine tests, detecting indicators such as pH value and urine specific gravity; microscopic examination of urine sediment and bladder ultrasound examination were carried out to confirm no obvious crystals or stones. The experimental animal use license has been obtained (License number: SYXK (Gan) 2023 - 0011). The breeding environment was single - cage breeding according to the breeding requirements of GB14925 and DB13 / T2411—2016. All experimental designs have been reviewed and approved by the Experimental Animal Ethics Committee of Gannan Institute of Innovation and Translational Medicine, and all experimental operations strictly follow the requirements of the "Administrative Measures for Experimental Animal Ethics Review" and relevant regulations.

[0029] In all the following efficacy examples, all data were expressed as mean ± standard deviation (SD) of the results, and paired t - test was used for pre - and post - group comparison within the group.

[0030] Example 1 This example provides a magnesium ammonium phosphate stone model for cats and its construction method. The construction method includes the following steps: (1) After 7 days of adaptive breeding in a constant temperature and humidity environment, the external genitalia / prepuce area of the experimental cats was strictly cleaned and disinfected. Under aseptic operation, a sterile urinary catheter was gently inserted into the bladder through the urethra; (2) Proteus mirabilis was inoculated into NB medium and cultured at 37 °C and 180 rpm for 24 h to obtain a Proteus mirabilis bacterial solution. The concentration of the bacterial solution was adjusted to 1×10 9 CFU / mL with NB medium for standby; (3) Take 1 mL of the 1×10 9 CFU / mL Proteus mirabilis bacterial solution obtained in step (2) and inject it into the bladder of the experimental cat through the urethra via the urinary catheter, and then remove the urinary catheter. Inject it once a day and six times a week. After 12 times of bacterial challenge, take urine for microscopic examination of urine sediment. If a large amount of magnesium ammonium phosphate crystals are observed, the modeling is considered successful; if only sporadic crystals are seen or no crystals are seen, continue to inject Proteus mirabilis according to the above frequency (once a day and six times a week). When a large amount of magnesium ammonium phosphate crystals (at least more than 3 or 2 clusters of magnesium ammonium phosphate crystals) are visible in the microscopic examination of the animal's urine sediment, or the B - ultrasound examination shows that stones have formed in the bladder, the bacterial challenge can be stopped. Animals that meet any of the above conditions are considered to have successfully constructed a magnesium ammonium phosphate stone model for cats.

[0031] Example 2 This embodiment provides a cat-like magnesium ammonium phosphate stone model and its construction method. The construction method is similar to that of Embodiment 1, except that in step (3), 1×10 9 The injection volume of CFU / mL Proteus mirabilis bacterial suspension was 2 mL, with all other parameters remaining unchanged.

[0032] Example 3 This embodiment provides a cat magnesium ammonium phosphate stone model and its construction method. The construction method is similar to that in Embodiment 1, except that the concentration of Proteus mirabilis in step (2) is adjusted to 1×10⁻⁶. 10 CFU / mL, with other parameters and conditions remaining unchanged.

[0033] Example 4 This embodiment provides a cat magnesium ammonium phosphate stone model and its construction method. The construction method is similar to that in Embodiment 1, except that the concentration of Proteus mirabilis in step (2) is adjusted to 1×10⁻⁶. 8 CFU / mL, with other parameters and conditions remaining unchanged.

[0034] Example of effect Urine and blood samples were collected before animal modeling in Example 1 and on day 21 after modeling, and bladder ultrasound imaging was performed. The collected urine samples underwent routine urinalysis and microscopic examination of urine sediment. The collected blood samples were analyzed for complete blood count and blood biochemical indicators, including creatinine (CRE), blood urea nitrogen (BUN), serum phosphorus (P), and magnesium ions. The results are shown in [the table below]. Figure 1-4 See Table 1-3.

[0035] Table 1. Partial analysis results of complete blood count (CBC) before and 21 days after modeling (x̅±SD) Table 2. Results of blood biochemical index analysis before and 21 days after modeling (x̅±SD) Table 3. Urine routine analysis results before modeling and 21 days after modeling (x̅±SD) like Figure 1 As shown, preoperative bladder ultrasound examination of all experimental cats revealed smooth bladder walls and anechoic areas within the bladder cavity, with no abnormal hyperechoic masses or crystalline deposits detected. In Example 1, after infection induction, single or multiple round hyperechoic stones with significant acoustic shadowing were observed in the experimental cats.

[0036] like Figure 2As shown, no crystalline components were detected in the urine sediment of all experimental cats before modeling by microscopic examination (centrifugation sedimentation method). After infection induction, typical crystals were observed in the urine sediment of the experimental cats in Example 1, mainly consisting of a small amount of non-aggregated magnesium ammonium phosphate crystals (characteristic coffin lid-like or prismatic structure). This dynamic change process directly reflects the change in the urine stone-forming environment caused by urease-positive bacterial infection, providing key microscopic evidence for the effectiveness of the model.

[0037] As shown in Table 1, 26 blood routine indicators of the experimental animals were measured before and 21 days after modeling. The total white blood cell count on 21 days after modeling (13.19 ± 2.81 K / μL) remained stable compared to before modeling (13.13 ± 4.17 K / μL), with no significant change. However, the total neutrophil count on 21 days after modeling (8.11 ± 2.25 K / μL) was significantly higher than before modeling (5.97 ± 6.19 K / μL) (an increase of 35.8%). A more significant change was observed in the lymphocyte count, which decreased dramatically on 21 days after modeling (3.5 ± 0.88 K / μL) compared to before modeling (6.12 ± 3.15 K / μL) (a decrease of 42.8%). The above results indicate that a significant inflammatory response was successfully induced in the experimental cats 21 days after the model was established, characterized by an increase in neutrophils and a significant decrease in lymphocytes, suggesting an alteration in the body's immune status.

[0038] like Figure 3 As shown in Table 2, there were no significant differences in creatinine and serum phosphorus levels (P>0.05), while urea nitrogen and magnesium ion levels showed significant changes: the urea nitrogen level before modeling was 1.89 ± 0.53 mmol / L (below the normal range), while it significantly increased to 8.12 ± 0.92 mmol / L 21 days after modeling; the magnesium ion level before modeling was 1.24 ± 0.11 mmol / L, while it decreased to 0.96 ± 0.05 mmol / L 21 days after modeling, and the differences were statistically significant (P<0.05).

[0039] like Figure 4 As shown in Table 3, urine specific gravity remained stable; urine pH increased slightly (pre-modeling: 6.5±0.71, 21 days after modeling: 6.83±0.29), and occult blood increased sharply from 0 ± 0 Cell / μL before modeling to 136.67 ± 109.7 Cell / μL on 21 days after modeling, with statistically significant differences (P<0.05).

[0040] In summary, the urine of the experimental cats constructed using the method of this invention contained stones and / or crystals, and showed kidney or urinary system damage, characterized by disruption of the glomerular / tubular basement membrane integrity, leading to leakage of red blood cells into the urine. Combined with the previously observed significant increase in blood urea nitrogen (329.6% increase), this further indicates that the model animals not only exhibited magnesium ammonium phosphate stones but also symptoms of acute kidney injury (AKI), demonstrating the successful construction of the feline magnesium ammonium phosphate stone model of this invention. Furthermore, the construction method of this invention has the advantages of being simple, easy to operate, and highly reproducible, enabling faster construction of the feline magnesium ammonium phosphate stone model and improving the success rate of model construction.

[0041] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for constructing a cat-like magnesium ammonium phosphate stone model, characterized in that, Includes the following steps: S1. Insert a sterile urinary catheter into the bladder of the model animal via the urethra; S2. Inject Proteus mirabilis bacterial solution into the bladder of the model animal through a urinary catheter, remove the urinary catheter, and obtain the cat magnesium ammonium phosphate stone model when magnesium ammonium phosphate crystals and / or magnesium ammonium phosphate stones are observed in the bladder and / or urine of the model animal.

2. The construction method as described in claim 1, characterized in that, In step S2, the concentration of the Proteus mirabilis bacterial solution is 10. 8 ~10 10 CFU / mL.

3. The construction method as described in claim 1, characterized in that, In step S2, the injection volume of the Proteus mirabilis bacterial solution is 0.5-2 mL.

4. The construction method as described in claim 1, characterized in that, In step S2, the *Proteus mirabilis* in the bacterial culture is positive and / or strongly positive for urease detection.

5. The construction method as described in claim 1, characterized in that, In step S2, the Proteus mirabilis bacterial solution is mainly prepared by the following method: Proteus mirabilis is inoculated into a culture medium and cultured.

6. The construction method as described in claim 5, characterized in that, In step S2, the culture conditions are 35-37℃ and 180-220 rpm for 18-24 h.

7. The construction method as described in claim 1, characterized in that, In step S2, the Proteus mirabilis bacterial solution is injected once a day, six times a week.

8. A cat magnesium ammonium phosphate stone model, characterized in that, It is mainly prepared by the construction method described in claims 1-7.

9. The feline magnesium ammonium phosphate stone model as described in claim 8, used in feline urolithiasis research for non-diagnostic or non-therapeutic purposes.

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