Introduction

Animal models of urethral injury are of significant clinical value in guiding the evaluation of treatment efficacy for urethral injuries1. Male rats, due to their larger size, visible external genitalia, and strong reproductive and growth capabilities, are often the preferred animals for urethral injury models2. However, the random and concealed nature of animal excretory behaviors poses challenges in determining the endpoints of experimental animal models, potentially increasing the number of animals used in experiments, which contradicts the principles of animal welfare. Given the scarcity of methodological research on the establishment of endpoints in urethral injury animal models, this study focuses on exploring the methodology for model establishment, imaging evaluation methods at endpoints, and their effectiveness. This research was conducted as an additional study within a project on urethral stricture, without exceeding the original experimental design’s bodily trauma to the rats.

Materials and methods

General information

Twenty-four male Sprague-Dawley (SD) rats, clean grade, were purchased from the Experimental Animal Center of Fujian Medical University, the animal qualification certificate number: SCXK (Min) 2016-0002, with individual net weight ranging from 350 to 400 g. They were housed in a closed environment with artificially simulated alternating 12-hour light-dark cycles, and had ad libitum access to food and water.

Experimental reagents

(1). TGF-β1 cytokine, sourced from Sigma-Aldrich Corporation, USA; (2).Preparation of MSC-Exo solution: The MSC-Exo solution was extracted from human umbilical mesenchymal stem cells using differential centrifugation, aproximately the final concentration is 0.5 µg/µl .Umbilical mesenchymal stem cells (UMSC) were purchased from Salial Company (Guangzhou, China; http://www.saliai.com) and maintained in a medium containing 10% fetal bovine serum (FBS; Thermo Fisher Scientific Life Sciences) at 37 °C and 5% CO2.

Rat model preparation (the grouping process is shown in Fig. 1)

Six rats were randomly selected as the control group, and the remaining rats (n = 18) form the surgery group. After anesthesia with intraperitoneal pentobarbital sodium (50 mg/kg), all rats (n = 24) are intubated. The control group (n = 6) receives 100ul of saline solution injected into the urethral wall, while the remaining rats (n = 18) receive TGF-β solution (1 mg TGF-β1 per 100 ml saline solution) 100ul injected into the urethral wall. Five minutes after injection, use a surgical blade to make transverse incisions on the mid-ventral side of the penis in the surgery group rats, perpendicular to the longitudinal axis of the penis, until the catheter is visible. Make four consecutive cuts at even intervals, ensuring smooth blade movement and avoiding intersection between the incisions. Then, use 5 − 0 absorbable sutures to stitch the skin of the penis, remove the catheter. Twenty-four hours later, the control group rats receive a second injection of 100ul saline solution into the urethral wall. One group of rats were randomly selected from the surgery group (n = 6) to simply receive 100ul of TGF-β1 solution (labeled as TGF group), and the remaining surgery group rats (n = 12) receive 100ul of TGF-β1 solution plus 30ug mesenchymal stem cell exosomes (MSC-Ex) (labeled as MSC group). One month later, all groups of rats underwent ultrasound examination blindly, and then they were immediately euthanized by intraperitoneal injection of 100 mg/kg of 2% pentobarbital sodium. The animals’ vital signs were reassessed after a 10-minute interval following the administration. Penile and urethral tissues are collected for pathological histological examination.

Fig. 1
figure 1

Grouping flowchart.

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Histology

Penile tissues from the incision and injection sites were carefully harvested and fixed with 10% neutral buffered formalin (NBF) for further histological examination. Following standard operating procedures, these samples were then subjected to Masson’s trichrome staining and Hematoxylin and Eosin (HE) staining3. In conducting histopathological analysis, we used the Olympus (Model: BX51) microscope along with a Nikon (Model: Digital Sight 10) camera.

Instruments and methods

The GE E10 ultrasound diagnostic apparatus with a probe model 11 L-D and a frequency of 4.5–12.0 MHz was used. After intraperitoneal anesthesia with water-soluble pentobarbital sodium (50 mg/kg), the rat was placed in a supine position, and its hind legs were held by an assistant to expose the external genitalia. The rat was then immersed in a water basin up to the level of its waist ( Fig. 2). The high-frequency probe covered with a sterilized sheath (to prevent cross-contamination) was inserted into the water basin, and the ultrasound beam was directed parallel to and overlapping with the long axis of the rat’s penis to obtain a clear image of the entire urethral penile segment. The images were interpreted by a radiologist with 10 years of experience in ultrasound imaging, who was blinded to the grouping information of the rats. The images were evaluated based on criteria such as "Continuity of central line of urethral hyperechoic bands; Width of urethral hyperechoic bands; Echo intensity of urethral hyperechoic bands; Echo intensity distribution uniformity of urethral hyperechoic bands” and corresponding scores were assigned as detailed in Table 1.

Fig. 2
figure 2

The ultrasound examination method in rats is illustrated.

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Table 1 Ultrasound manifestations of urethra in rats in surgical group.
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Statistical methods

Statistical analysis was performed using SPSS 22.0 software. The respective measurement indices of urethral echogenicity were normally distributed metric data, expressed as mean ± standard deviation \((\overline{x} \pm s)\). The independent samples t-test was employed for comparisons between two groups, while one-way analysis of variance (ANOVA) followed by the LSD post hoc test was used for comparisons among three or more groups. Additionally, receiver operating characteristic (ROC) curves were constructed for rat scores, and the area under the curve (AUC) was calculated to determine the cutoff value. A significance level of P < 0.05 was considered statistically significant.

Results

Ultrasonographic echogenicity of urethra in different groups

All rats survived healthy until the completion of ultrasonographic image acquisition. In the normal group, there were 6 rats whose urethral long axis exhibited a straight and strong echogenic stripe, with the strongest echo at the central line and weakened but still noticeable echoes at the edges, which were stronger than the surrounding corpus cavernosum echoes (Fig. 3). The overall stripe appeared as a “three-line pattern,” and on the short axis, it presented as a circular central echo with the strongest echo forming a target-shaped high echo area (Fig. 4). The mean width \((\overline{x} \pm s)\) of the long axis stripe (based on the outer edge) was 0.89 ± 0.11 mm. All surgical group rats showed a narrowing of the strong echogenic stripe width in the middle segment of the penile urethra (Fig. 5), with a mean length of narrowing of 0.22 ± 0.06 mm, ranging from 0.14 to 0.30 mm. The mean width \((\overline{x} \pm s)\) of the strong echogenic stripe in the narrowed segment of the urethra (recorded at the narrowest point) was 0.40 ± 0.10 mm, ranging from 0.20 to 0.50 mm; among them: (1) The mean width \((\overline{x} \pm s)\) of the strong echogenic stripe in the urethra for the MSC group was 0.47 ± 0.05 mm, with a 95% confidence interval ranging from 0.41 mm to 0.52 mm; (2) The mean width \((\overline{x} \pm s)\) of the strong echogenic stripe in the urethra for the TGF group was 0.30 ± 0.06 mm, with a 95% confidence interval ranging from 0.23 mm to 0.37 mm.

Fig. 3
figure 3

Ultrasound image of the urethra of a normal rat, the solid arrow points to the strongest echo line at the center of the urethra, and the hollow arrow points to the area of weakened echoes at the urethral edge. (CS = Cavernous body, UL = urethral lumen).

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Fig. 4
figure 4

Ultrasound images of the urethra of a normal rat are illustrated, with a cross-section on the left and a long-axis section on the right (CS = Cavernous body, UL = urethral lumen).

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Fig. 5
figure 5

The surgical group (MSC group) exhibited a noticeable constriction in the width of the hyperechoic band located at the midpoint of the penile urethra, indicative of stenosis (red arrow). (CS = Cavernous body, UL = urethral lumen) (The ultrasound image was obtained one month after surgery.)

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The urethral stripe echogenicity decreased in all surgical groups (Fig. 6), with some stripes becoming blurred and discontinuous (Fig. 7), detailed changes are shown in Table 1. All measurement data were normally distributed and expressed as continuous variables. Comparisons between two groups were performed using the independent samples t-test, while comparisons among three or more groups were conducted using one-way analysis of variance (ANOVA) followed by the LSD post hoc test. Statistical analysis revealed significant differences in the urethral hyperechoic band width between the normal group and both the MSC and TGF groups, as well as between the MSC and TGF groups (P < 0.01).

Fig. 6
figure 6

The echo intensity of the urethra strips was reduced in the surgical group(MSC group) (red arrow) (CS = Cavernous body, UL = urethral lumen) (The ultrasound image was obtained one month after surgery.)

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Fig. 7
figure 7

The urethra strip in the surgical group (TGF group) was blurred with poor continuity (red arrow) (CS = Cavernous body, UL = urethral lumen) (The ultrasound image was obtained one month after surgery.)

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Scoring of changes in rat urethral echogenicity

Different scores were assigned to changes in the continuity of the central line, width, echo intensity, and echo intensity distribution uniformity of the urethral hyperechoic bands. Changes in the continuity of the central line include basic continuity and interruption. Changes in the width of the urethral hyperechoic bands include stability and stenosis. Changes in the echo intensity of the urethral hyperechoic bands include basic consistency, weakening of the central line echo with blurred edge lines, and weakening of the central line echo with disappearance of the edge line. The echo intensity distribution uniformity of the urethral hyperechoic bands includes basic uniformity and nonuniformity. Specific scoring criteria are detailed in Table 1, with a maximum possible score of 9 points. The cumulative score range for all rats is summarized in Table 2. All the control group rats’ urethral ultrasounds did not show any of the changes described above, so they were not included in the statistical tables for display.

Table 2 Distribution table of score assignment for rats in the surgical group.
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Surgical group rat urethral tissue pathology and ultrasound scoring comparison

All rats in the surgical group exhibited more pronounced urethral stenosis compared to the control group, with the TGF group showing more severe urethral stenosis than the MSC group. Histopathological examination revealed abundant dense collagen fibers at the urethral incision site in the TGF group, with disordered arrangement of newly formed collagen fibers. In contrast, the arrangement of collagen fibers at the incision site in the MSC group was also disordered but with fewer overall quantities, and there were significantly more blood sinuses within the surrounding corpus cavernosum tissue (Figs. 8, 9 and 10).

Fig. 8
figure 8

(A) shows urethral histopathology in normal rats; (B) shows a large number of fibroblasts are proliferative in the surgical group; (C) showed a large number of blood sinuses appeared in the surgical group (HE staining, the images were taken at a magnification of 10x, with the scale bar representing 100 μm). (This pathological image was obtained one month after surgery.)

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Fig. 9
figure 9

Pathological images of urethra of rats in the surgical group (HE Staining, The image was taken at a magnification of 10x, with the scale bar representing 100 μm); TGF group (A), MSC group (B), The MSC group, compared to the TGF group, exhibited similarly disorganized collagen fibers but in a lesser overall quantity. (This pathological image was obtained one month after surgery.)

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Fig. 10
figure 10

Pathological images of rat urethras in various groups (Masson’s trichrome staining, The image was taken at a magnification of 40x, with the scale bar representing 25 μm); Control group (A), TGF group (B), MSC group (C). The surgical group shows similarly disorganized collagen fibers under HE staining, but the overall quantity is less in the MSC group compared to the TGF group. (This pathological image was obtained one month after surgery.)

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Statistical analysis revealed that the ultrasound scores in the TGF group were significantly higher than those in the MSC group. In the TGF group, all 6 rats had ultrasound scores of 8 or above, with a mean score of (8.67 ± 0.52), while in the MSC group, only 2 out of 12 rats scored 8, and the remaining 10 rats scored below 8, with a mean score of (6.83 ± 0.94). The score difference between the two groups was statistically significant (P < 0.01). Further analysis using receiver operating characteristic (ROC) curve (Fig. 11) showed an area under the curve (AUC) of 0.972, indicating that at a cutoff value of 7.5, the sensitivity and specificity for distinguishing between the TGF and MSC groups were 100% and 83.3%, respectively. These results suggest that the TGF group had significantly higher ultrasound scores for urethra assessment compared to the MSC group, and the ROC curve analysis supports the effectiveness of this scoring system in differentiating between the two groups of rats.

Fig. 11
figure 11

ROC score of rats (Based on the ultrasound evaluation scores of TGF group and MSC group).

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Discussion

Urethral stricture, a prevalent condition in the realm of urology, presents a formidable challenge to practitioners within this medical field4. Its pathological progression is characterized by an overabundance of fibrosis in the urethral mucosa and the scarring of the surrounding spongiosum tissue5. In the contemporary clinical management of urethral stricture, invasive methodologies reign supreme6, including but not limited to, urethral dilation, urethrotomy, and a myriad of urethroplasty techniques. Among these procedures, urethroplasty emerges7as the vanguard surgical intervention in clinical practice, attributed to its refined surgical methodologies and commendable longitudinal outcomes. However, it still carries risks of complications such as a high recurrence rate 8.9and erectile dysfunction10.

Hence, many scholars are devoted to exploring more ideal treatment modalities or adjunctive therapeutic approaches. The establishment of an animal model for urethral stricture holds significant importance for both the fundamental understanding and clinical research of this condition. In this study, we induced urethral stricture in rats by injuring their urethra and investigated the methodology of model establishment, along with the efficacy of imaging evaluation methods for assessing the endpoint of model establishment.

Currently, there is no unified standard for establishing a rat urethral stricture model, particularly one that mimics the formation of urethral stricture resulting from abnormal wound healing. In actual practice, regardless of the establishment method, the formation of urethral stricture is primarily a pathological healing process characterized by fibrosis at the cellular and tissue levels, which is associated with inflammatory reactions, abnormal or excessive activation of fibroblasts11.

Studies have found that both human and rat urethras contain a large number of TGF-β1 receptors12. After the injection of TGF-β1 into urethral tissues, the expression of type I and III collagens significantly increases. Therefore, most experiments induce urethral fibrosis using TGF-β1 to construct rat urethral stricture models2.

Mesenchymal stem cells (MSCs) are a type of multipotent stem cells derived from the mesoderm with the potential for multidirectional differentiation13. In addition to their differentiation ability, MSCs can promote tissue repair by regulating cellular responses in structure and immune cells, thereby creating a regenerative and anti-inflammatory environment. It is well known that inflammatory mediators can attract MSCs and alter their secretions, thereby promoting immune responses14and improving the therapeutic progress of inflammatory diseases, such as urethral stricture.

Many studies have shown that during cell therapy, MSCs primarily enhance wound healing efficiency through paracrine mechanisms15,16, and in the repair of urethral stricture, they may improve repair rates through the secretion of extracellular vesicles17. Extracellular vesicles derived from MSCs typically possess many properties associated with MSCs, including tissue healing, attenuation of inflammatory reactions, and reduction of fibrosis18,19,20. Meanwhile, extracellular vesicles derived from MSCs may inhibit the differentiation of myofibroblasts during the wound healing process21.

On the other hand, TGF-β1, as an inflammatory factor, also has the effect of inducing MSCs to inhibit inflammation22. The histopathological results of this study showed that compared to the TGF group alone, the severity of urethral stricture in the MSC group rats was milder, indicating that MSCs play an important role in the repair of urethral stricture.

The excretion behavior of rats is secretive and highly random, making it impossible to assess the endpoint of model establishment or stratify the severity of strictures based on excretion behavior changes. Moreover, relying on pathological tissue assessment to determine the endpoint of urethral stricture model establishment would require an increase in the number of experimental rats, which contradicts the optimization principle of the “3R” principle of animal welfare23.

Ultrasound, as a technique with good repeatability, convenience of use, and clear and reliable imaging, naturally possesses advantages as a method for determining model endpoints. The high-frequency ultrasound probe used in this study is widely used in clinical practice, eliminating the need to purchase expensive small animal ultrasound equipment and thus saving experimental costs.

The immersion method used in this study effectively removes air from the rat’s fur and increases the distance between the high-frequency ultrasound probe and the scanning site, avoiding near-field interference of the ultrasound beam and further enhancing image clarity. Additionally, the immersion method avoids the process of shaving the rats, reducing physical and emotional harm to the experimental animals, improving their survival rate, and reducing the number of animals used in experiments, thereby adhering to the “3R” principle of animal welfare.

Under high-frequency ultrasound, the normal rat urethra appears as a “three-line pattern” with a central strong echo and slightly weaker edges on the longitudinal axis. The central line represents a thin continuous band formed by the compression of the urethral lumen, while the slightly weaker bands at the edges represent the smooth muscle tissue of the urethral wall. On the short axis, the rat urethra appears as a target-shaped high echo area.

Due to surgical injury affecting the urethral wall, the urethral strong echo bands in the surgical group rats showed continuous changes. After postoperative scar repair, the proliferation of collagen fibers replaced the original smooth muscle tissue, leading to urethral lumen and surrounding tissue contraction, resulting in uneven distribution of echo intensity and changes in band width on ultrasound images. There was a statistically significant difference in urethral echo band width between the control group and the surgical group measured by ultrasound. The repair degree of urethral injury in the MSC group rats was higher than that in the TGF group, as reflected in the histopathological examination in this study.

High-frequency ultrasound, due to its high spatial resolution, can correlate changes in echo intensity and form with changes in tissue structure, thus distinguishing between urethral changes in the MSC group and the TGF group. There was a significant statistical difference in the width of the urethral strong echo bands between the two groups of rats, and the corresponding evaluation scores also revealed differences in urethral echo band form changes between the two groups.

The results of this study confirm that high-frequency ultrasound combined with the immersion method has a good imaging evaluation effect in establishing rat urethral stricture models and has good judgment effects on the endpoint of model establishment and the degree of stricture among different groups. However, the sample size in this study was small, which may have biased the determination of scoring thresholds between groups.