Abstract
Urodynamic studies in rats and mice are broadly used to examine the
pathomechnisms of several diseases and to identify and test the therapeutic
targets. The first study conducted a meta-analysis aimed to highlight the
effects of the anesthetics on the lower urinary tract function in order to select
the protocols that allow smooth anesthesia and recovery for repeated
measurements while preserving the bladder and urethral function under study.
The data included a comparison of urodynamic studies with various types of
anesthesia, and the relevant urodynamics performed in awake animals.
Urethane was recommended as an anesthetic for the investigation of the lower urinary tract function, but it was appropriate for acute urodynamic studies only. The major advantages of urethane were its stability and ability to preserve the micturition reflex. However, its toxicity and carcinogenicity limited its use for recovery procedures.
The meta-analysis evaluated available alternative anesthetic protocols including propofol, isoflurane, combinations of ketamine-xylazine, ketamine-medetomidine, and/or fentanyl-fluanisone-midazolam. Different effects have been shown among these drugs on the urinary bladder, the urethral sphincter, as well as on their neuroregulation. The lowest incidence of adverse effects was observed with the use of a combination of ketamine and xylazine.
In vivo laboratory experiments were performed to establish and validate a rat model of urethral sphincter injury and to develop a method for leak point pressure (LPP) measurements that can be performed repeatedly in the same animal.
Twenty-four Sprague-Dawley female rats were used in the second study. All animals were operated microscopically with bladder and epidural catheter implantation. Five days after catheterization, cystometry was performed using continuous saline infusion, under the effect of anesthesia with isoflurane, ketamine-xylazine (KX) or fentanyl-fluanisone-midazolam (FFM). After three micturition cycles, intrathecal bupivacaine was injected to suppress the bladder reflex contractions. LPP measurements were performed using the vertical tilt technique. After the initial LPP measurement, partial resection (~30%) of the striated urethral sphincter was performed. The effect was evaluated 6 weeks after surgery, by repeating the LPP measurement in the same animals.
Results from the validation study demonstrated 10 out of 19 animals that showed full micturition cycles under isoflurane, and 9 animals under KX anesthesia. No significant difference in micturition pressures (Mean ± SEM; 30.1 ± 2.3 vs. 26.8 ± 1.6 mmHg) and LPP (31.0 ± 2.4 vs. 28.0 ± 0.9 mmHg) was observed between isoflurane and KX groups, respectively.
Reflex micturition was suppressed in FFM group. Spinal anesthesia using bupivacaine led to overflow incontinence in all cases. Sphincter injury caused fibrotic changes and a significant increase in LPP (26.4 ± 2.3 before vs. 46.9 ± 4.6 mmHg after injury, p < 0.05).
It was concluded that KX anesthesia preserves bladder contractions. Intrathecal bupivacaine eliminates micturition reflex, allowing for repeated LPP measurements in the same animal. Resection of the striated sphincter resulted in increased LPP 6 weeks post injury. The site of urethral sphincter resection healed with local fibrosis of the striated muscle.
Cell therapy has become clinically relevant for treatment of small muscle damages e.g., sphincter injury. Yet, early cell death of transplanted myogenic cells has shown to be an obstacle in successful cell therapy via injection of myogenic stem cells. Cell protective scaffolds have been suggested as a strategy to enhance survival of the transplanted cells. Thus, the third pilot study examined survival and integration of the transplanted myoblasts in combination with different materials such as fibrin, alginate, or buffer. In addition, the concern of cell labeling was addressed. The analyses of tissue compatibility of the scaffolds were extracted.
It was found that the injected material in the intact muscle tissue tends to be distributed along the perimysial connective tissue with a minor contact to the muscle fascicles. When cell-enriched scaffold materials were implanted in the surgical lesion of the thigh muscle, both fibrin and alginate embedding in hydrogels and incorporation in myoblast appeared to protect the cells.
After fourteen days, significant indications were found for integration of labelled cells. Regarding tissue response, no foreign body reaction was detected in the fibrin and alginate implants. This pilot study points to further experiments to be performed with hydrogels and delivery techniques
The histological and immunohistochemical studies showed greater numbers of surviving MDSCs, increased muscle/collagen ratio as well as increased microvessel density at the injection sites of the matrix. Fibrinalginate may potentially enhance the action of transplanted MDSCs to restore the histology and function of the external urethral sphincter in a SUI rat model. Injection of MDSCs with fibrin-alginate may offer a promising cell therapy for treating stress urinary incontinence.
Urethane was recommended as an anesthetic for the investigation of the lower urinary tract function, but it was appropriate for acute urodynamic studies only. The major advantages of urethane were its stability and ability to preserve the micturition reflex. However, its toxicity and carcinogenicity limited its use for recovery procedures.
The meta-analysis evaluated available alternative anesthetic protocols including propofol, isoflurane, combinations of ketamine-xylazine, ketamine-medetomidine, and/or fentanyl-fluanisone-midazolam. Different effects have been shown among these drugs on the urinary bladder, the urethral sphincter, as well as on their neuroregulation. The lowest incidence of adverse effects was observed with the use of a combination of ketamine and xylazine.
In vivo laboratory experiments were performed to establish and validate a rat model of urethral sphincter injury and to develop a method for leak point pressure (LPP) measurements that can be performed repeatedly in the same animal.
Twenty-four Sprague-Dawley female rats were used in the second study. All animals were operated microscopically with bladder and epidural catheter implantation. Five days after catheterization, cystometry was performed using continuous saline infusion, under the effect of anesthesia with isoflurane, ketamine-xylazine (KX) or fentanyl-fluanisone-midazolam (FFM). After three micturition cycles, intrathecal bupivacaine was injected to suppress the bladder reflex contractions. LPP measurements were performed using the vertical tilt technique. After the initial LPP measurement, partial resection (~30%) of the striated urethral sphincter was performed. The effect was evaluated 6 weeks after surgery, by repeating the LPP measurement in the same animals.
Results from the validation study demonstrated 10 out of 19 animals that showed full micturition cycles under isoflurane, and 9 animals under KX anesthesia. No significant difference in micturition pressures (Mean ± SEM; 30.1 ± 2.3 vs. 26.8 ± 1.6 mmHg) and LPP (31.0 ± 2.4 vs. 28.0 ± 0.9 mmHg) was observed between isoflurane and KX groups, respectively.
Reflex micturition was suppressed in FFM group. Spinal anesthesia using bupivacaine led to overflow incontinence in all cases. Sphincter injury caused fibrotic changes and a significant increase in LPP (26.4 ± 2.3 before vs. 46.9 ± 4.6 mmHg after injury, p < 0.05).
It was concluded that KX anesthesia preserves bladder contractions. Intrathecal bupivacaine eliminates micturition reflex, allowing for repeated LPP measurements in the same animal. Resection of the striated sphincter resulted in increased LPP 6 weeks post injury. The site of urethral sphincter resection healed with local fibrosis of the striated muscle.
Cell therapy has become clinically relevant for treatment of small muscle damages e.g., sphincter injury. Yet, early cell death of transplanted myogenic cells has shown to be an obstacle in successful cell therapy via injection of myogenic stem cells. Cell protective scaffolds have been suggested as a strategy to enhance survival of the transplanted cells. Thus, the third pilot study examined survival and integration of the transplanted myoblasts in combination with different materials such as fibrin, alginate, or buffer. In addition, the concern of cell labeling was addressed. The analyses of tissue compatibility of the scaffolds were extracted.
It was found that the injected material in the intact muscle tissue tends to be distributed along the perimysial connective tissue with a minor contact to the muscle fascicles. When cell-enriched scaffold materials were implanted in the surgical lesion of the thigh muscle, both fibrin and alginate embedding in hydrogels and incorporation in myoblast appeared to protect the cells.
After fourteen days, significant indications were found for integration of labelled cells. Regarding tissue response, no foreign body reaction was detected in the fibrin and alginate implants. This pilot study points to further experiments to be performed with hydrogels and delivery techniques
The histological and immunohistochemical studies showed greater numbers of surviving MDSCs, increased muscle/collagen ratio as well as increased microvessel density at the injection sites of the matrix. Fibrinalginate may potentially enhance the action of transplanted MDSCs to restore the histology and function of the external urethral sphincter in a SUI rat model. Injection of MDSCs with fibrin-alginate may offer a promising cell therapy for treating stress urinary incontinence.
| Original language | English |
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| Awarding Institution |
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| Date of defence | 31. Jan 2022 |
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| DOIs | |
| Publication status | Published - 31. Jan 2022 |
| Externally published | Yes |