The use of halofuginone in limiting urethral stricture formation and recurrence: an experimental study in rabbits

PURPOSE: We developed a reproducible animal model for the induction of urethral stricture in the rabbit and evaluated the role of halofuginone in limiting stricture formation. MATERIALS AND METHODS: A total of 20 New Zealand male rabbits were used in the first phase of the experiment. Bulbar urethral stricture was induced by electrocoagulation. The animals were then randomly assigned to 2 groups of 10 each, which received a diet containing halofuginone or a normal diet. In the second phase electrocoagulation induced stricture was treated with visual internal urethrotomy in 45 rabbits. These rabbits were randomly assigned to 2 groups, namely a halofuginone and a control group. RESULTS: In the first phase stricture developed in 2 study rabbits (20%) vs 10 controls (100%). In the second phase 37 rabbits were evaluable (8 died). Recurrent stricture was observed in 5 of the 18 study rabbits (27%) vs 14 of the 19 controls (73%). CONCLUSIONS: Halofuginone is effective in limiting the occurrence of de novo urethral stricture and recurrent stricture after visual internal urethrotomy. This antifibrotic molecule may become an important therapy to treat urethral stricture and/or recurrence following endoscopic manipulation of stricture in humans.     

The effect of halofuginone, a specific inhibitor of collagen type 1 synthesis, in the prevention of pancreatic fibrosis in an experimental model of severe hyperstimulation and obstruction pancreatitis

BACKGROUND: The aim of this paper is to assess the effects of halofuginone, a specific inhibitor of synthesis of collagen Type 1, on fibrogenetic process in an experimental model of early pancreatic fibrosis. METHODS: Thirty rats were divided into three equal groups: group 1, sham laparotomy; group 2, severe hyperstimulation and obstruction pancreatitis (SHOP) with no treatment; group 3, SHOP with halofuginone treatment group. SHOP model was induced by complete pancreatic duct obstruction and daily cerulein hyperstimulation (50 microg/kg, intraperitoneally). Halofuginone was administered daily from the operative day (5 mg/kg, intraperitoneally). All of the animals were sacrificed, and blood and pancreatic tissue samples were obtained for biochemical and histopathological examination on the 5th postoperative day. RESULTS: No mortality was observed in any group. Serum amylase, lipase, hyaluronic acid, and nitric oxide levels were significantly higher in groups 2 and 3 compared with group 1 (P < 0.05), but were significantly lower in group 3 compared with group 2 (P < 0.05). No significant differences were observed regarding serum malondialdehyde and glutathione levels between groups 1 and 3. Tissue hydroxyproline levels were found to be significantly higher in groups 2 and 3 compared with group 1 (P < 0.001), but were significantly lower in group 3 compared with group 2 (P < 0.001). Although tissue hydroxyproline levels were significantly higher in the halofuginone treatment group compared with the control group, histopathological evaluation did not reveal a significant difference between these groups regarding collagen deposition. When group 3 was compared with group 2, halofuginone significantly reduced inflammation and acinar atrophy in the pancreas as well (P < 0.05). CONCLUSION: Halofuginone was found to be effective in reducing SHOP-related inflammation, acinar atrophy, and fibrosis in the pancreas.     

The effect of halofuginone, a specific inhibitor of collagen type 1 synthesis, in the prevention of esophageal strictures related to caustic injury

BACKGROUND: To assess the effects of halofuginone, a specific inhibitor of synthesis of collagen type 1, which is the major constituent of fibrosis, on esophageal stricture formation due to caustic ingestion. METHODS: Sixty rats were divided into four equal groups: control group; sham laparotomy group; caustic injury without treatment group; caustic injury with halofuginone treatment group. Caustic injuries were done by 50% sodium hydroxide. Halofuginone was administered by the first postoperative day. All animals were sacrificed on day 21; and the results were evaluated by hydroxyproline levels, stenosis index, lumen diameter, histopathological evaluation, wall thickness, and animal weights. RESULTS: Mortality differences were significant comparing group 3 with group 1 and 2 (P = 0.006) and group 4 (P = 0.03). According to hydroxyproline levels, the differences are significantly higher (P <0.001) comparing group 3 with group 1, 2, and 4. The P value was considered significant in all other parameters (P <0.001) for all the groups but group 1 versus group 2 (P >0.05). CONCLUSIONS: Halofuginone, a specific inhibitor of collagen type 1 synthesis, significantly reduced esophageal stricture occurrence.     

Topical treatment of cutaneous chronic graft versus host disease with halofuginone: a novel inhibitor of collagen type I synthesis

BACKGROUND: In chronic graft-versus-host disease (cGvHD), skin fibrosis, contractures, and an increase in collagen content form the hallmark. We report a successful treatment of a cGvHD patient by topical application of halofuginone, an inhibitor of collagen alpha1(I) gene expression. METHODS: Halofuginone-containing ointment was applied daily on the left side of the neck and shoulder of a cGvHD patient. Collagen alpha1(I) gene expression and collagen content in skin biopsy specimens were evaluated by in situ hybridization and sirius red staining, respectively. RESULTS: After 3 and 6 months, a marked reduction in skin collagen synthesis was observed, accompanied with increase neck rotation on the treated side. After cessation of treatment, the sclerosis, skin tightness, and collagen alpha1(I) gene expression returned to baseline level. No adverse effects were observed, and no plasma levels of halofuginone could be detected. CONCLUSIONS: Halofuginone may provide a promising novel and safe therapy for cGvHD patients.     

The effect of hyaluronan on adult and fetal fibroblast proliferation and collagen synthesis: an in vitro study

European Journal of Plastic Surgery -     

The effect of botulinum neurotoxin type A on capsule formation around silicone implants: the in vivo and in vitro study

This study confirms that botulinum neurotoxin type A (BoNT‐A) decreases capsular contracture and elucidates a possible mechanism. Silicone blocks were implanted subcutaneously in 20 mice. The experimental groups received BoNT‐A (1, 2·5 or 5 U) instilled into the subcutaneous pocket. After 30 days, periprosthetic capsules were harvested and evaluated. The effect of BoNT‐A on the differentiation of human dermal fibroblasts to myofibroblasts in culture was examined by Western blot analysis. Changes in transforming growth factor‐beta1 (TGF‐β1) expression in cultured fibroblasts were determined by enzyme‐linked immunosorbent assay (ELISA). In in vivo study, the thickness of capsules (P < 0·05) and the number of alpha‐smooth muscle actin (α‐SMA)⁺ cells in capsules (P < 0·05) were significantly decreased in the experimental groups. TGF‐β1 was significantly underexpressed in the experimental groups (P < 0·05). In in vitro study, BoNT‐A did not significantly affect fibroblast viability. Western blot analysis showed that α‐SMA protein levels were significantly decreased in the experimental groups (P < 0·05). Based on ELISA, the amount of TGF‐β1 was significantly decreased in the experimental groups (P < 0·05), especially cells treated with a high dose of BoNT‐A (P < 0·001). This study confirms that BoNT‐A prevents capsular formation around silicone implants, possibly by blocking TGF‐β1 signalling and interrupting the differentiation of fibroblasts to myofibroblasts.     

The effects of a selective noradrenaline reuptake inhibitor on the urethra: an in vitro and in vivo study

OBJECTIVE: To identify the effects of a selective noradrenaline reuptake inhibitor (venlafaxine) on urethral perfusion pressure (UPP) in rabbits and rats, and thus assess its therapeutic potential for treating stress urinary incontinence. MATERIALS AND METHODS: Strips of bladder and proximal urethra were prepared from female New Zealand White rabbits. Each strip was electrically stimulated and the contractile responses of controls strips compared with those after pretreatment with venlafaxine (100 micromol/L). In separate experiments using 80 adult female Sprague-Dawley rats (250-300 g), changes in intravesical pressure and UPP after the intra-arterial and intra-urethral administration of phenylephrine, phentolamine, fluoxetine and venlafaxine were monitored using double-lumen catheters. RESULTS: Pretreatment with venlafaxine significantly decreased the contraction of bladder strips (P=0.01) and significantly increased the contraction of urethral strips (P=0.008). In vivo, phenylephrine administered by both routes significantly increased UPP (P=0.02); phentolamine (arterial) significantly decreased UPP (P=0.001); fluoxetine (arterial) had no effect on UPP, and venlafaxine (both routes) significantly increased UPP (both P<0.001). The intravesical pressure was not changed significantly in any animal. CONCLUSIONS: Venlafaxine effectively increased UPP both in vitro and in vivo; these results imply that venlafaxine may be useful for treating stress urinary incontinence, by increasing the UPP.     

The preventive effect of halofuginone on posterior glottic stenosis in a rabbit model.

OBJECTIVE: The aim of this study was to investigate the preventive effect of halofuginone on posterior glottic stenosis (PGS) in an animal model. STUDY DESIGN: A randomized, controlled animal study. SUBJECTS AND METHODS: Sixteen male New Zealand White rabbits were used for this study. After the mucosa of posterior glottis was removed for producing PGS, the study group (eight rabbits) was given intraperitoneal halofuginone at 0.1 mg/kg/day for 4 weeks and saline was injected into peritoneum in the control group. At 4 weeks after injury, postsurgical changes of posterior glottis were evaluated by gross and histologic examination. RESULTS: PGS was induced by the mucosal stripping of the posterior glottis. The halofuginone-treated group showed less scarring and granulation tissue formation. Also, the degree of synechia was significantly less than that of control group. Histologic analysis showed the decreased fibrosis in the halofuginone-treated group. CONCLUSION: This study suggests that halofuginone can be helpful in preventing PGS after laryngeal injury.     

The distribution of neuronal and inducible nitric oxide synthase in urethral stricture formation

PURPOSE: The distribution of neuronal (n) and inducible (i) nitric oxide synthase (NOS) may have a role in the maintenance of normal urethral spongiosum and during the development of spongiofibrosis in urethral stricture disease. MATERIALS AND METHODS: Eight normal and 33 strictured human bulbar urethras were studied by histological and immunohistochemical techniques for the neuronal markers S-100, nNOS and iNOS. The smooth muscle-to-collagen ratio was calculated by morphometric analysis of Masson's trichrome sections. Immunohistochemical staining patterns of the neuronal markers in normal urethral tissue was compared to that in urethral stricture tissue with spongiofibrosis. RESULTS: The smooth muscle-to-collagen ratio was significantly lower in the strictured urethra compared to that in the control group (p = 0.001). In the strictured bulbar urethra nNOS immunoreactivity was decreased compared to that in normal urethral tissue. The severity of spongiofibrosis corresponded to the loss of nNOS immunoreactivity. iNOS immunoreactivity was found in strictured urethral epithelium and spongiosal tissue, whereas the control group was nonimmunoreactive to iNOS. CONCLUSIONS: Urethral stricture formation is a fibrotic process associated with significant changes in NOS metabolism. Abnormal collagen synthesis following urethral trauma may be stimulated by inappropriate iNOS activity. A functional nerve supply to the urethral spongiosum seems to be crucial in the maintenance of the unique ultrastructure of the urethral spongiosum.     

Biodegradable urethral stents seeded with autologous urethral epithelial cells in the treatment of post‐traumatic urethral stricture: a feasibility study in a rabbit model

OBJECTIVE

To evaluate the adhesion and growth of rabbit urethral epithelial cells (UECs) on a biodegradable unbraided mesh urethral stent, and to assess the feasibility and effect of the cell‐seeded urethral stent for treating post‐traumatic urethral stricture (PTUS) in a rabbit model.

MATERIALS AND METHODS

Rabbit UECs were collected by biopsy from adult rabbit urethra and seeded onto the outer layer of a mesh biodegradable urethral stent. The growth of UECs in cell‐scaffolds was assessed by scanning electron microscopy, immunohistochemical and fluorescence staining. In all, 32 male New Zealand rabbits were used, with either PTUS or uninjured, as a control group. Cell‐seeded stents were implanted into the rabbits strictured urethra. The histological and immunohistochemical findings were assessed after death at 1, 2, 8, 12 and 24 weeks, respectively. The reconstruction and function were evaluated by urethroscopy and retrograde urethrography.

RESULTS

The cultured UECs adhered to the stent and grew well. Immunohistochemistry showed that the cells were stained positively for cytokeratin. At 4 weeks, vs 2 weeks, the thickness of the papillary projections of the epithelium decreased and inflammatory cell infiltration diminished. At 24 weeks the injured urethra was completely covered by integrated regeneration of three to five layers of urothelium. There was no evidence of voiding difficulty, stricture recurrence or other complications.

CONCLUSIONS

The unbraided mesh biodegradable urethral stent with autologous UECs seemed to be feasible for treating PTUS in the rabbit urethra, and provides a hopeful avenue for clinical application allowing reconstruction of PTUS.     

Laser induced collagen remodeling: a comparative study in vivo on mouse model

BACKGROUND AND OBJECTIVE: Many lasers have claimed the clinical efficacy on skin rejuvenation. In this study, the mechanisms of laser induced collagen remodeling were explored systematically on a Kunming (KM) mouse model in vivo by comparing the different non-ablative laser effects using four different laser treatment modalities. MATERIALS AND METHODS: The dorsal skin of KM mice was exposed by depilation before the laser treatments. Four laser treatment modalities were used: the 595-nm pulsed dye laser (PDL) (10 ms), 1,320-nm neodymium-yttrium-aluminum garnet (Nd:YAG) laser (0.35 ms), 1,064-nm Nd:YAG laser with Q-switched (5 ns), and long-pulsed (0.3 ms) mode. Each modality exposed one side of the mouse dorsal skin leaving the other side as the contralateral control. Then skin histology, fibroblast number, and the genesis of collagen type I and III were studied by comparing the treatment site and control site at 1 hour, 1 day, 1 week, 3 weeks, 4 weeks, and 8 weeks after laser treatment. Hydroxyproline content of the skin tissue was measured 4 weeks and 8 weeks after laser exposure. RESULTS: All laser treatments led to marked improvements in dermal layer thickness and collagen fiber density, and the increase in fibroblast number and hydroxyproline content compared with their own controls. Collagen synthesis and remodeling induced by the Q-switched 1,064-nm laser was most effective 4 weeks after treatment, while there was no significant difference among the other three modalities. Among the new collagen genesis after the different laser treatments, collagen type III increased sharply after the Q-switched 1,064-nm laser treatment whereas more collagen type I was elicited by the other laser treatment modalities. CONCLUSIONS: The efficacy of photo-mechanical effects in promoting more effectively the synthesis of collagen type III, whereas the photo-thermal effect favored more the formation of collagen type I.     

Effects of green tea on urinary stone formation: an in vivo and in vitro study

PURPOSE: We evaluated whether epigallocatechin gallate (EGCG), a main constituent of green tea polyphenols, could protect against cellular toxicity by oxalate and whether green tea supplementation attenuates the development of nephrolithiasis in an animal model. MATERIALS AND METHODS: Cells of the NRK-52E line were incubated with different concentrations of oxalate with and without EGCG, and toxicity and malondialdehyde assays were done to investigate the cytotoxic effect of oxalate and the anti-oxalate effect of EGCG.. In a second series of experiments, male Sprague-Dawley rats were divided into three groups. Group 1 animals (controls) were fed regular chow and drank water ad libitum; group 2 animals were fed chow containing 3% sodium oxalate with the administration of gentamicin (40 mg/kg) and drank water ad libitum; group 3 animals were fed the same diet as group 2 with gentamicin administration and drank only green tea. Rats were killed 4 weeks later after a 24-hour urine collection, and the kidneys were removed for morphologic examination. RESULTS: As oxalate concentrations increased, the number of surviving cells decreased, and the formation of free radicals increased. The administration of EGCG inhibited free-radical production induced by oxalate. Green tea supplementation decreased the excretion of urinary oxalate and the activities of urinary gammaglutamyltranspeptidase and N-acetylglucosaminidase. The number of crystals within kidneys in group 3 was significantly lower than in group 2. CONCLUSIONS: Green tea has an inhibitory effect on urinary stone formation, and the antioxidative action of EGCG is considered to be involved.     

The effect of pregnancy on hiatal dimensions and urethral mobility: an observational study

Introduction and hypothesis

Childbirth is an established risk factor of pelvic floor dysfunction. The role of pregnancy is, however, not fully understood. This study was designed to evaluate the potential effect of pregnancy on pelvic floor function. The hypothesis was: Pregnancy has no effect on urethral mobility and levator hiatal dimensions.

Methods

This was a reanalysis of the translabial 3D/4D ultrasound volume data of 688 nulliparous pregnant women seen in the late 3rd trimester and again 4?months postpartum and that of 74 nulliparous, nonpregnant volunteers in previously reported studies. Hiatal dimensions and urethral mobility were determined as the outcome parameters. Multivariate regression analysis was performed after adjusting for age and BMI between the pregnant and nonpregnant cohorts.

Results

Comparison of 3rd trimester data of the pregnant cohort with that of the nonpregnant nulliparae revealed a 27?% and 41?% increase in hiatal area at rest and on Valsalva and an increase in segmental urethral mobility by 64?% to 91?% in late pregnancy. About 70?% of this difference in hiatal dimensions, but virtually identical differences in urethral mobility, were observed when comparing nonpregnant controls with women 4?months after prelabour or 1st stage caesarean section.

Conclusion

Both hiatal dimensions and urethral mobility were markedly higher in women in late pregnancy and at 4?months after prelabour/1st stage caesarean section compared to nulliparous controls. The hormonal and mechanical changes of pregnancy may have an irreversible effect on the pelvic floor.     

The effect of local anesthetics and amitriptyline on peroxidation in vivo in an inflammatory rat model: preliminary reports

We studied the inhibition of peroxidation by local anesthetics in an inflammatory animal model. Inflammatory lipid peroxidation was assessed by the thiobarbituric assay in plasma from rats injected or not injected with carrageenan (Carra) and killed 1, 2, 4, 6, 12, and 24 h thereafter. Thiobarbituric acid reactive substances (TBARS) values in inflammatory animals were maximal 6 h after Carra administration. This result, in accordance with the evolution of paw edema width during time, supports that TBARS reflect the intensity of inflammation. Local anesthetics (bupivacaine, lidocaine, ropivacaine, or bupivacaine-loaded microspheres) or amitriptyline were injected in clinically relevant concentrations as a sciatic nerve block or intraperitoneally in inflamed animals. Ropivacaine did not exhibit any protective effect on Carra-induced lipid peroxidation in rats. With all the other drugs administered as a sciatic nerve block, the maximal TBARS increase was not observed at 6 h. Our conclusion is that bupivacaine (plain or encapsulated), lidocaine, and amitriptyline in clinically relevant concentrations administered via the sciatic nerve showed antioxidant properties toward lipid peroxidation induced by Carra inflammation. Intraperitoneal injection of those drugs gave the same effect as nerve block; this result suggests that their mechanism of action is not strictly limited to the nerve. IMPLICATIONS. We investigated the antioxidant effects of local anesthetics and amitriptyline in an inflammatory rat model. Amitriptyline exhibits antioxidant properties per se, whereas lidocaine and bupivacaine (plain or encapsulated) seem to inhibit the peroxidation process. This may have future application in limiting toxic oxygen metabolite production during the inflammatory process.     

Biomimetic mineralization of charged collagen matrices: in vitro and in vivo study

Polyanionic collagen matrix prepared by hydrolysis side chain amides of asparagine and glutamine was mineralized in vivo, without inflammatory response, biodegradation, or resorption, with calcium phosphate deposited in close resemblance to the D-periodicity of collagen fibrils assembly. In vitro results with the same material produced mineralized collagen fibers with a similar morphology and chemical characteristics, suggesting that amide hydrolysis may have introduced into this matrix, signs for the controlled mineralization of collagen fiber. TEM indicated that amide hydrolysis occurred near the OVERLAP and GAP zones, as suggested by the significant reduction in inter-band distances in these regions. The lack of an inflammatory response associated to the similar mineralization pattern observed in vivo and in vitro suggests not only the biomimetic behavior of polyanionic collagen matrix, but also its potential uses as scaffold for bone tissue reconstruction. Based on these results, a model for the in vitro mineralization was also proposed.     

Effects of collagen nerve guide on neuroma formation and neuropathic pain in a rat model

BACKGROUND: Posttraumatic neuroma formation is a major cause of neuropathic pain that can occur after elective surgery, amputation, or trauma. This study examined the use of biosynthetic collagen nerve guides to prevent the development of posttraumatic neuromas. METHODS: Collagen nerve guides were applied after neurectomy in a rat sciatic nerve model in an effort to stimulate linear neuronal outgrowth and reduce random axon sprouting. Animals were monitored for evidence of neuropathic pain--autotomy scores were recorded for 8 weeks posttransection--after which proximal stumps were excised and processed for histologic analyses. RESULTS: Moderate to severe autotomy was observed in 88% (7 of 8) of the control (neurectomy) animals. In contrast, 13% (1 of 8) of animals receiving collagen nerve guides developed autotomy, which was significantly less than controls (P < .01). Qualitative analyses of neurofilament and Schwann cell-labeled nerve sections showed a significant enhancement in Schwann cell migration away from the proximal stump and advanced linear axonal regrowth in the collagen nerve guide-treated animals. CONCLUSIONS: Collagen nerve guides alter the regrowth of transected nerves and reduce the severity of symptoms associated with neuropathic pain.     

Effects of phosphodiesterase type 5 inhibitor on the contractility of prostate tissues and urethral pressure responses in a rat model of benign prostate hyperplasia

AIM: This study was performed to investigate the effect of DA-8159, a selective phosphodiesterase 5 (PDE5) inhibitor, on benign prostatic hyperplasia (BPH) both in vitro and in vivo. METHODS: We assessed the influence of DA-8159 on the contractility of rat prostate tissues in an organ-bath experiment. In addition, in order to investigate whether chronic administration of DA-8159 prevents the increase of electrostimulation-induced intraurethral pressure (IUP) responses associated with BPH, BPH was induced by steroid hormones (testosterone plus 17beta-estradiol) and DA-8159 (5, 20 mg/kg) was concomitantly administered once a day for 8 weeks. After that the electrostimulation-induced IUP responses were measured. Finally, we investigated the acute treatment effect of DA-8159 on IUP responses in an established BPH model after a single intravenous injection of DA-8159 (0.3, 1 mg/kg). RESULTS: DA-8159 concentration-dependently reduced the contraction of the isolated prostate strips with an IC50 value of 70 microM. In chronic treatment study, while the BPH control rats showed a significantly increased IUP both at the baseline and by electrostimulation, the chronic DA-8159 treatment significantly attenuated the increase in IUP responses in a dose- and frequency-dependent manner. In the acute treatment study, a single intravenous injection of DA-8159 also prevented the increase in urethral pressure in a dose-dependent manner. CONCLUSIONS: These results suggest that DA-8159 may be beneficial on lowering the urethral pressure associated with BPH via dilatation of the prostate, but a further evaluation of the efficacy on humans needs to be performed.