Dr Shi-Chung Chang, Department of Urology, Tzu-Chi College of Medicine and Hospital, No 701, Section 3, Chung-Yang Road, Hualien, Taiwan.E-mail: firstname.lastname@example.org
Objective To assess the urinary and reproductive function of rats after inducing protoporphyrin IX with 5-aminolaevulinic acid (ALA) and subsequent intraurethral photodynamic therapy (PDT).
Materials and methods Twelve female Wistar rats were given ALA orally or intravesically (four each), followed by intraurethral PDT through a 10-mm cylindrical fibre at 100 mW for 500 s (argon laser, 632 nm). Urinary frequency, the number of gestations and histological changes were then evaluated and compared with a group of eight control rats.
Results There was only a slight increase in urinary frequency during the first 2 weeks after PDT in the group given oral ALA, while the same degree of urinary frequency was evident for up to 4 weeks in those given intravesical ALA. Despite the occurrence of urinary incontinence from undefined causes in two rats, reproductive function remained unchanged. There was no histological evidence of damage to the reproductive system adjacent to the bladder.
Conclusions PDT of the urethra with ALA is relatively safe and carries few risks of inducing permanent urological complications.
Photodynamic therapy (PDT) is a technique that produces nonthermal selective photochemical tissue destruction through the combined action of a photosensitizer, light of the appropriate wavelength and the presence of tissue oxygen [1,2]. In recent years, this technique has been used successfully in the treatment of a variety of malignancies [2–6]. Photofrin® (QLT Phototherapeutics Plc, Vancouver, Canada) is the most popular photosensitizer developed to date and is the only agent approved officially for clinical use in some countries . Despite its promising effectiveness, prolonged skin photosensitivity and occasional light- associated bladder dysfunction have been encountered [7,8].
The use of 5-aminolaevulinic acid (ALA), an endogenous photosensitizer precursor, is an important advance in PDT because it is clinically efficient and can be eliminated from the body within 24–48 h after administration. ALA is an intermediary in the biosynthetic pathway for haem in living cells. The introduction of exogenous ALA in large quantities results in the accumulation of protoporphyrin IX (PpIX), the active compound responsible for photodynamic tissue effects . This agent has been extensively tested for a variety of cancer treatments in clinical conditions , in addition to in vivo animal studies [11–13]. Our previous study showed that PpIX accumulated preferentially in the urothelium after bladder instillation  and caused less lamina propria and muscularis damage than the equivalent dose of ALA given orally . Preliminary clinical experience with intravesical ALA for bladder cancer has shown favourable therapeutic responses . The present study assessed urinary and reproductive function in female rats after intraurethral PDT with ALA-induced PpIX. The results of this study may be used to further clarify the safety of PDT for future clinical trials.
Materials and methods
Twenty female Wistar rats (160–200 g body weight) were used for the experiments; a group of mature male rats were mated with the 20 female rats in the second part of the study. Before administering ALA and intraurethral PDT, the rats underwent inhalation anaesthesia with halothane/O2 (volume ratio 1:2). A supplementary intramuscular injection with 0.1 mL Hypnorm® (fentanyl and fluanisone, Jansen Pharmaceuticals Ltd., Sweden) was administered to rats to ensure bladder retention of ALA for at least 2 h before voiding.
As in the previous study , a 10% ALA solution was used (200 mg/kg body weight; ALA-HCl, 98% pure powder, Sigma Chemical Co., St Louis, MO, USA) for both oral and bladder instillation, and a 5% solution (100 mg/kg body weight) only for oral sensitization. These solutions were prepared immediately before administration, by titration with saturated sodium bicarbonate to a final pH of 5.5.
For oral administration, ALA solutions at 100 and 200 mg/kg body weight were given through a bulb-tip gavage needle into the stomach under inhalation anaesthesia. As rats are incapable of vomiting or regurgitation, all animals ingested the full delivered dose of ALA and resumed normal activity within 5 min of terminating inhalation anaesthesia.
For bladder instillation, ALA was delivered as a 10% solution at 200 mg/kg (0.2 mL/100 g body weight) through an 18.5 G PTFE cannula placed transurethrally under inhalation anaesthesia. To ensure that the ALA was retained in the bladder, a supplementary intramuscular injection of Hypnorm® (0.1 mL) was given to the anaesthetized rats to ensure complete sedation for 2 h.
The control groups comprised four rats each in groups A and B; group A received neither ALA nor light and group B had only light but no ALA. The 12 experimental animals were divided equally into groups C, D and E; group C received intravesical ALA (200 mg/kg), group D oral ALA (100 mg/kg) and group E oral ALA (200 mg/kg). They all received doses of red light (632 nm) at 50 J (100 mW for 500 s) from an argon laser (Innova-70) and pumped-dye laser (model 599, both from Coherent Systems, Santa Clara, CA, USA) through a 10-mm-tipped 1.2 mm cylindrical diffuser fibre (Medlight SA, Lausanne, Switzerland). The intensity of light emission through the tip of the diffuser fibre is shown in Fig. 1.
The optimal interval between drug delivery and illumination for intravesical ALA was 5 h (2 h of ALA retention under full anaesthesia plus a 3-h interval after retention) , and 3 h for oral ALA . A small episiotomy (1–2 mm) was made over the vestibule to expose the urethral meatus and to facilitate insertion of the diffusing fibre through the urethra. As the rat urethra at this body weight was 9–10 mm long, a mark was made on the laser fibre at 12 mm from the diffusing tip so that it traversed the length of the urethra and protruded 2–3 mm into the bladder cavity. The power output from the diffuser fibre was calibrated before and after each treatment. The calculated radiant exposure of the urethra at the surface of the diffusing fibre was:
while the irradiance was:
for the bladder, assuming a bladder surface of 2 cm2 on distension. After PDT, the animals were kept alive for 3 months for further observation of their urinary frequency and fertility.
Before PDT, the 20 rats (eight control and 12 experimental) were kept separately (one in each cage) for 2 weeks to record their daily voiding patterns. The pattern was assessed by counting the number of urine spots on water-absorbent paper placed over the waste-collection tray laid 3–4 cm beneath the floor of the cage. This design allowed the urinary frequency to be estimated daily. Because all the rats had a small episiotomy at the time of intraurethral PDT, the initial 3-week phase of observation was not started until 1 week after PDT to minimize discrepancies in voiding patterns caused by wound pain. A second observation period was started again 4 weeks later (56 days after PDT) and lasted for 2 weeks.
Immediately after completing the assessment of urinary frequency the rats were evaluated for fertility. The 20 rats were placed in their original groups (four per cage). A mature male Wistar rat (300 g body weight) was placed into each cage to attempt mating. A semen plug present in the vagina and vestibule in the subsequent 24 h was considered evidence of insemination; the male rats were then removed from the cages. However, if there was no evidence of insemination in a particular female rat, a second mating was tried 2 days later, with the same male alone with the female rat for another 48 h. If the same male failed to inseminate the female during the second session, a different male, with a confirmed ability to inseminate, was used in a third mating attempt with the same female. Two weeks after insemination an experienced animal technician estimated the number of gestations by palpating the rats over their abdomen. On the 18th day of pregnancy, the rats were killed by CO2 narcosis and the number of gestations counted.
The bladder, urethra and reproductive system were removed en bloc and preserved in 10% formalin for histological examination. Sections from various levels of the urethra and reproductive system were taken longitudinally or transversely, and stained with haematoxylin and eosin, Masson’s trichrome or Van Gieson stains, and assessed using light microscopy.
Signs of distress, such as unsteady gait and shivering, were observed immediately after PDT, but the behaviour returned to normal within 24 h. Bloody spotting, but not haematuria, occurred in most rats within 6 h of PDT, possibly resulting from bleeding in the vestibule; the bleeding resolved by the end of the first day. Haematuria was found in three rats, causing pink urinary spotting on the paper for 2 or 3 days. No rats died during or after PDT.
Two patterns of urinary spotting were identified on the paper; urine stains of > 4 cm in diameter were considered ‘large’ spots, whereas spot patterns 1–4 cm in diameter were considered ‘small’. Urine spots of < 1 cm in diameter were not uncommon, but were regarded as the terminal phase of voiding rather than a separate episode of urination. There was no fixed voiding pattern in the five groups of rats before PDT, but the total number of all large spots plus half the number of small spots was relatively constant in all rats. To facilitate comparisons of voiding frequency, this urinary frequency index (UFI) was used as a reference. Another voiding pattern, occasionally seen in a few rats in the early stages of the initial phase of assessment, was a diffuse spreading of small urine spots (usually < 1 cm in diameter) and no large stains. This probably represented an increased urinary frequency secondary to bladder or urethral irritation, or a voiding pattern resumed at a later stage.
The UFI before and after PDT is shown in Table 1. All rats in groups C–E had a greater UFI 2 weeks after PDT than before treatment. The differences were not significant except for group C, which had a significantly greater UFI for those 4 weeks after PDT ( Table 1). Groups D and E had considerably lower UFIs in the second phase than in first stage of the initial phase of observation ( Table 1).
Table 1. The daily urinary frequency index before and after intraurethral PDT.
Mean (d) UFI in group
Number of observations
Measurements obtained by daily recording of four rats in each group over a period of 7 or 14 days. *P < 0.05 and †P < 0.001, significant difference from before PDT; ‡P < 0.01, significant difference with second phase.
Three rats (two in group C and one in group D) were suspected of having urinary incontinence in the first 2 weeks after PDT. Their pattern of urinary spotting comprised large stains of urine (usually 4–7) spread throughout the paper. Another indication of incontinence was the absence of small urinary spots ( 4 cm in diameter) routinely found in the spotting patterns of normal rats. Of these three rats, one in group C resumed a normal voiding pattern 15 days after treatment but the remaining two rats (one each in group C and D) continued to be incontinent until death.
Of the rats in the second part of the study, all but one (in group A) became pregnant. The mean (range) number of gestations per rat at the termination of pregnancy (18 days) was 8.4 (2–11). The difference between the mean ( sd) number in the control and experimental groups was insignificant, at 8.3 (2.3) vs 8.6 (2.5) (P > 0.2). The rat from group A that failed to become pregnant after repeated attempts was finally deemed infertile and further attempts discontinued. However, its infertility had no effect on the significance of the differences. One rat in group E had only two gestations but showed a normal voiding pattern and UFI.
The vagina and urethra from the control rats (group A) showed no evidence of tissue destruction or inflammatory cell infiltration. The experimental groups had nests of round cell infiltration in the vaginal wall ( Fig. 2a). The uterus and ovaries were normal histologically. The urethral specimen from group B showed only sparsely distributed inflammatory cell infiltration in the lamina propria ( Fig. 2b). Rats in groups C–E had a slight increase in round- and plasma-cell infiltration, indicating the presence of chronic inflammation ( Fig. 2c). In groups C–E, despite the presence of chronic urethral inflammation, the vagina had no signs of fibroblast infiltration or muscle degeneration. Stepwise sectioning of the urethra from one of the two rats with urinary incontinence showed suspicious but undefined evidence of fibrosis in the smooth muscle bundle, indicating possible muscle damage with tissue repair ( Fig. 2d). The collagen content in the suburethral lamina propria had increased substantially in some animals after PDT ( Fig. 2e).
PDT is becoming a promising option in the management of a variety of cancers [2,6,17] but it has not yet been widely accepted by the medical profession. The major urological adverse effects associated with PDT are voiding irritation, bladder shrinkage with reflux and obstructive uropathy, and prolonged skin photosensitivity [7,8,18]. To reduce urological complications, experience shows that the appropriate selection of photosensitizers with correspondingly refined illuminating techniques is important. Following these guidelines, there has been greater success in animal models [13,19] and in human studies [16,20,21]. Other options, e.g. the administration of iron chelators to retard the conversion of PpIX to haem  or the use of electromotive devices to facilitate the accumulation of PpIX in the tumour and urothelial layers of the bladder , may also further reduce the likelihood of light-induced bladder side-effects. Although the clinical effectiveness and safety of ALA-based PDT for bladder cancer treatment has been documented , there is no published report on changes in urinary and reproductive function after ALA-based PDT of the urethra and bladder. Thus the present study assessed urinary continence and reproductive function in female rats undergoing transurethral PDT of the urethra and bladder.
Human urinary continence is controlled by two principal elements; a stable bladder and an effective outlet resistance mechanism. The stability of the bladder is a function of the detrusor muscle , but the outlet resistance is dominated by both intrinsic and extrinsic mechanisms . Intrinsic mechanisms, involving the interaction of the bladder neck, urethral mucosa and internal urethral sphincter, account for 40% of total continence control ; pelvic support accounts for the remainder . The mechanisms maintaining urinary continence in rodents are as yet unclear. In the present study, both bladder and intrinsic mechanisms seemed to be involved in the pathogenesis of urinary frequency and incontinence. The superficial destruction of the urothelium with subsequent regeneration led to a complete recovery of urinary frequency within 4 weeks after PDT. Apart from the urothelial factor, deeper damage to the urethral musculature was a likely cause for urinary incontinence, as the urethra received a relatively higher irradiance (104 J/cm2) than the bladder (13 J/cm2). Two of the present 16 rats had urinary incontinence after urethral PDT; although the type of incontinence in these two rats remains undefined, it might be partly attributable to smooth muscle damage to the sphincter from urethral PDT (total incontinence) rather than arising from stress incontinence occurring after increased abdominal pressure [27,28]. Other possible causes of urinary incontinence include persistent urinary tract infection and excessive urethral manipulation during PDT. The absence of definitive evidence of smooth muscle damage, and the relatively mild inflammatory cell infiltration of the urethra and underlying tissues, after ALA-based urethral PDT may imply either greater safety of this treatment or a relatively low efficiency of this agent for bladder or urethral PDT. Despite the occurrence of sparsely distributed chronic inflammatory cells in the urethra and the increase of collagen and elastic fibres in the suburethral lamina propria and smooth muscle bundles, there was no obvious evidence of tissue derangement related to PDT.
Stewart et al. used a rolling-paper device to determine the UFI, but the present method for assessing urinary frequency was simpler. However, the accuracy of this method in evaluating the real voiding frequency remains to be clarified. Moreover, the physiological implication of the confluence of large urine stains into a very extensive wet area, which in this study was interpreted as total incontinence, needs further investigation, as urinary overflow incontinence may be another possible factor accounting for the persistent leakage of urine. Because the UFI derived from daily urinary spots for rats of moderate size (160–200 g) over the 2 weeks before PDT was so consistent, this method was adopted for the present study. Despite the differences in the animal model, animal size and experimental design from that of Stewart et al., the increased voiding irritation seen in the first 2 weeks after PDT in the present study was comparable to that reported in theirs, which used Photofrin II (10 mg/kg) in the H3C mouse bladder [29,30]. They concluded that the UFI and the time interval to return to normal voiding was linearly related to the dose of light. Post et al. showed a substantial increase of urinary frequency (a factor of 5–7) 2 weeks after whole-bladder PDT with Photofrin, meso-tetrahydroxyphenylchlorin (mTHPC) and bacteriochlorin (BCA) at various drug and light doses. PDT based on these photosensitizers was probably so potent as to cause extensive bladder damage, and this resulted in severe urinary irritation for several weeks after PDT. In the present study, a 40% increase in the UFI using ALA and 26 J/cm2 for irradiating the bladder may imply suboptimal incident irradiance to the lower urinary tract, a greater resistance to light by the rat urothelium than in mice, greater safety or less efficacy of ALA than with Photofrin, mTHPC or BCA.
The UFI was slightly higher in rats in group C than in D and E in the early phase after PDT, but not significantly. Our previous study indicated that PDT with oral ALA (100 or 200 mg/kg) usually led to patchy necrosis of the urothelium, unlike the lesions induced by intravesical ALA, which tended to be more homogeneously distributed . The greater voiding irritation observed probably represents a more extensive urothelial destruction of the bladder and urethra induced by intravesical ALA. It may indirectly provide additional support for the observation that intravesical ALA is a more appropriate route for PDT of the lower urinary tract. Using other photosensitizers in an animal model, PDT induced urinary irritation that sometimes persisted for 30 weeks if the light dose was beyond a certain threshold .
The wavelength used for the present study was 632 nm and not that commonly used (635 nm, at the peak of the absorption spectrum of PpIX in the presence of serum protein) . As the initial part of this study was conducted in London, where the optimum wavelength suggested for ALA at the National Medical Laser Centre was then 632 nm, the study was completed using the original protocol at this wavelength. Stocker et al., in a chemically induced bladder tumour model, showed that tumour and urothelial necrosis was significantly greater at 635 nm. From the absorption spectrum of PpIX, it is likely that light at 635 nm causes more tissue damage than at 632 nm, although empirical data are needed to confirm the better effectiveness and adverse-effect profile of 635 nm over 632 nm.
Despite the urinary tract being close to reproductive organs, there was no detectable reduction in fertility in rats after PDT. The uterus and ovaries would be expected to accumulate some PpIX after oral or intravesical ALA, as in situ porphyrin synthesis contributes to PpIX accumulation in most internal organs . The absence of PDT damage in the ovaries and uterus may result from either insufficient PpIX accumulation in these reproductive organs or that light does not reach the uterus and ovaries through the urethra and bladder. Despite large differences in the genitourinary tracts of humans and rodents, the absence of histological and functional abnormalities of the reproductive system further supports the safety of urethral and bladder PDT for female patients. This was confirmed clinically in a preliminary report by Kreigmair et al., although they did not evaluate reproductive function in that study. In the clinical setting there is less chance of inducing light-associated tissue damage to the reproductive system in women because the human bladder and urethra are much thicker than in the rat and the ovaries lie deep in the peritoneal cavity, preventing light penetration. The possibility of damage to the uterus would probably be even less than that to the ovaries because of the protection provided by its dense smooth muscle layers. Nevertheless, caution is needed if more potent photosensitizers are to be used in the future, particularly as the absorption peak of the more potent photosensitizers requires longer wavelengths, which in turn could cause more tissue damage.
The clinical implication of the present study is that it is theoretically safe for female patients to receive urethral PDT. The effects on the bladder neck and urethra are unlikely to result in severe tissue damage, loss of urinary continence or reproductive function. It is not unusual to find the bladder neck wide open during whole-bladder PDT; this is a result of bladder distension created deliberately to unfold the bladder mucosa and so optimize effective treatment. Back-scattering of light into the urethra during whole-bladder PDT, although possible, is unlikely to damage the internal sphincter and cause dysfunction of urinary control, because the light escaping is far less than the therapeutic light dose needed to induce substantial tissue damage. Fertility is unlikely to be affected in ALA-based urethral PDT. The safety implications of bladder or urethral PDT with most newer photosensitizers remain poorly defined and need further study.
We gratefully acknowledge the research grant provided by the National Science Council, The Republic of China (NSC87–2314-B-320–013, NSC88–2314-B-320–013) and Tzu Chi College of Medicine (TCMRC-8609). We also thank Dr Hsu YH, consultant pathologist at Tzu Chi General Hospital, for his assistance in histological interpretation.
S.-C. Chang, MD, PhD, Consultant Urologist and Associate Professor.