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Abstract

Objectives

This study was undertaken to verify the possible modifications caused by hormonal deprivation in the extracellular matrix in the penises of neutered cats.

Methods

Twenty-seven penises from domestic shorthair cats were collected: 14 samples from intact cats and 13 from neutered cats. Sections were stained with Weigert’s resorcin-fuchsin, hematoxylin and eosin, and picrosirius red. Histomorphometric analysis was performed using light microscopy and image analysis software. The following parameters were analyzed: density of the elastic fibers and collagen fibers in the corpus spongiosum; density of the elastic fibers in the tunica albuginea of the corpus cavernosum and the tunica albuginea of the corpus spongiosum; luminal area of the urethra; area of the corpus spongiosum; area of the corpus cavernosum; and thickness of the urethral epithelium. The data were analyzed using the Shapiro–Wilk test to verify the normal distribution, and groups were compared using Student’s t-test; P <0.05 indicated statistically significant differences.

Results

Significant differences were observed between intact cats and neutered cats in the density of elastic fibers in the tunica albuginea of the corpus cavernosum (8.13% ± 1.38% vs 3.11% ± 0.66%), tunica albuginea of the corpus spongiosum (4.37% ± 1.08% vs 3.30% ± 1.01%) and corpus spongiosum (6.28% ± 3.03% vs 4.10% ± 2.19%), and density of collagen fibers in the corpus spongiosum (34.11% ± 10.86% vs 44.21% ± 12.72%).

Conclusions and relevance

The results show a significant decrease in the density of the elastic fibers and a significant increase of the density of the collagen fibers in the corpus spongiosum in neutered animals. This suggests that the compliance of the periurethral region is reduced, and these changes could be a predisposing factor for urethral obstructive disease.

Introduction

Feline lower urinary tract disease (FLUTD) is frequently observed in domestic cats, and this condition involves several clinical signs observed in cats. The main causes include feline interstitial cystitis or feline idiopathic cystitis, urolithiasis, bacterial infections, anatomical malformations, neoplasia, and behavioral and neurological disorders. Common clinical signs include pollakiuria, dysuria, stranguria, hematuria and periuria.¹ FLUTD is classified into two types: obstructive and non-obstructive. The obstructive form occurs more frequently in males than in females, and also more frequently in neutered males than in intact males.^2,3 Anatomically, male cats are more prone to urethral obstruction (UO) because of their progressively narrowed urethra, which starts at a diameter of 2.4 mm at the vesicourethral junction and ends at a diameter of 0.7 mm at the penile urethra. Studies have reported that 18–58% of cats diagnosed with FLUTD have UO.^2,4–7 This information substantiates the magnitude of this pathology in the veterinary clinical setting.

The relationship between castration and UO is not well established, and the effective role of androgen deprivation in the feline penis remains unclear. Willeberg suggested that the mechanism of UO is correlated not only with androgen deprivation, but also with other metabolic changes.⁸ Various characteristics, such as type of food consumed, body condition, micturition frequency and volume, activity level, presence of diseases, breed, stress and castration, are considered predisposing factors for UO.^9,10 Notably, middle-aged, obese, indoor-living and dry food-consuming cats with low activity level are frequently presented to veterinary care for FLUTD.¹⁰

In addition to its effects on the male reproductive system, testosterone apparently plays an essential role in body development. One month after castration, a 21% reduction in the area of the corpus cavernosum was detected in rats.¹¹ Androgen deprivation in male rabbits and rats seemed to induce modifications in penile structures, including endothelial morphologic changes, reduction in trabecular smooth muscle and an increase in the number of adipocytes adjacent to the tunica albuginea of the corpus cavernosum.¹²

A recent survey demonstrated that many veterinarians from the UK considered that cats aged between 12 and 16 weeks to be at a higher risk of FLUTD, regardless of the lack of scientific studies indicating an increased risk of FLUTD in kittens neutered at an early age.¹³ Two studies compared male cats neutered during the prepubertal and pubertal periods and did not report any significant difference in the incidence of FLUTD and potential UO after follow-up periods of 2 and 3 years, respectively.^14,15 A voiding cystourethrogram study comparing the effects of castration in prepubertal and pubertal cats with those in intact cats demonstrated that the urethral diameter of both prepubertal and pubertal neutered male cats did not significantly differ from than that of intact cats.¹⁶ Herron evaluated the effects of early castration on male urethra and periurethral tissue and reported that intact cats showed a thicker urethral epithelium than did neutered felines.¹⁷ In the same study, the author also identified a higher density of fibrocytes in the penile matrix of neutered cats.¹⁷

The objective of the study was to compare the penises of intact domestic cats and neutered cats, and to investigate whether hormonal deprivation caused by castration leads to changes in the penile histoarchitecture.

Materials and methods

This research project was approved by the Ethics Committee on Animal Use of the Universidade Federal Fluminense (approval number 246). Twenty-seven penises collected from domestic shorthair cats were donated to the sector of Animal Anatomy, Department of Biology, Department of Pathology, Municipal Institute of Veterinary Medicine Jorge Vaitsman, for necropsy. Of these penises, 14 were from intact male cats and 13 from neutered cats. In both the intact and neutered groups, five animals were between 1 and 10 years of age, but the age of the remaining animals was unknown. For the animals of unknown age, the exclusion criteria included animals that did not have all fully developed teeth and those that had iris atrophy or lenticular sclerosis.¹⁸ Thus, the intact and neutered groups were comprised of young and mature adult animals and excluded very young and old animals. No animal showed any signs of pathology in the urogenital system. During necropsy, the penises were collected via a deep dissection and were then placed in containers with 10% buffered formalin. Thereafter, all samples were cleaved at the middle third and then submitted to histological processing. The material was processed using routine histological methods and was paraffin-embedded and sectioned at 5 μm. The samples were stained with hematoxylin and eosin, picrosirius red and Weigert’s resorcin-fuchsin. Histological slides stained with hematoxylin and eosin were used to verify the tissue integrity for all animals.

For evaluating the thickness of the urethral epithelium, images were obtained at × 400 magnification using a digital camera (Olympus DP-72) coupled with a light microscope (Olympus BX-51). The images were converted to digital signals and transferred to a computer. ImageJ software (NIH, Bethesda, MD, USA) was used to outline the basal and luminal limits of the epithelium, thus providing data on the epithelial area. For the epithelial thickness, five random lines were drawn perpendicularly between the basal layer and the luminal limit of the urethral epithelium. The arithmetic mean of the five measurements was used as the mean epithelial thickness (Figure 1a).

Figure 1

Screenshot illustrating how the measurements were performed using ImageJ. (a) Thickness of the urethral epithelium; five lines were drawn at the epithelium, and their mean was obtained. Urethra is indicated by the asterisk. Results are shown in the window at the right side of the image. (b) Luminal area of the urethra. The urethra (asterisk) is highlighted in yellow. Results are shown in the window at the right side of the image. (c) Density of elastic fibers in the corpus spongiosum. Each different color of the image was marked with different symbols (‘X’, ‘O’ and ‘+’), then the software tool (color segmentation) calculated the percentage occupied by each color in the whole image. Results are shown in the window at the right side of the image. (d) Density of the collagen fibers in the corpus spongiosum. As for part (c), each different color of the image was marked with different symbols (‘X’, ‘O’ and ‘+’) and then the software too calculated the percentage occupied by each color in the whole image. Results are shown in the window at the right side of the image

The urethral luminal area was measured in picrosirius red stained sections at × 200 magnification, using a tablet (Tablet Trust wireless TB-2100), which consisted of a pad and pen. This device allowed for a more precise selection of the area of interest for the study. Thus, the entire apical border of the urethral epithelium was outlined, indicating the luminal area of the urethra. After selection, ImageJ software was used to measure the selected area in μm² (Figure 1b).

The areas of the corpus cavernosum and corpus spongiosum were obtained from sections stained with picrosirius red, using ImageJ software and Tablet Trust. Both the corpus cavernosum and corpus spongiosum were outlined at × 100 magnification. After this procedure, ImageJ automatically provided a numerical analysis of each area in μm².

The percentage of elastic fibers in the corpus spongiosum, tunica albuginea of the corpus cavernosum and tunica albuginea of the corpus spongiosum was assessed using sections stained with Weigert’s resorcin-fuchsin, which provided a satisfactory contrast between the elastic fibers and the background. Photomicrographs of four different regions were taken at × 400 magnification, using CellSens software (Standard 1.8.1). The images were analyzed using ImageJ software, with the color segmentation resource via a specific plug-in. All color tones that appeared close to the staining of the elastic fibers were selected, and, at the end of the analysis, the corresponding percentages were obtained (Figure 1c).

The percentage of the collagen fibers of the corpus spongiosum was assessed in sections stained with picrosirius red and observed under polarized light at × 200 magnification. Four different regions were photographed using CellSens software (Standard 1.8.1). The images were analyzed using ImageJ software, with the color segmentation resource via a specific plug-in. All color tones of the birefringence fibers, as well as the black background related to the lacunar spaces, were selected, and at the end of analysis, the corresponding percentages were obtained (Figure 1d).

The data were analyzed using the Shapiro–Wilk test to verify the normal distribution, and the groups were compared using the Student’s t-test; P <0.05 indicated statistically significant differences.

Results

The density of the elastic fibers in the corpus spongiosum was lower in the neutered animals (4.10%) than in the intact animals (6.28%; P = 0.0434; Figure 2). In addition, statistical differences were observed between the neutered and intact cats in the density of the elastic fibers in the tunica albuginea of the corpus cavernosum (P <0.0001) and in the tunica albuginea of the corpus spongiosum (P = 0.0143; Table 1).

Figure 2

Photomicrograph of the corpus spongiosum of the penis of an intact cat (a) and a neutered cat (b), stained with Weigert’s resorcin-fuchsin; fewer elastic fibers are seen in the corpus spongiosum of the neutered cat, and elastic fibers in the wall of an arteriole (arrow) and urethra (asterisk) are stained. Magnification = × 400. Photomicrograph of the entire corpus spongiosum of the penis of an intact cat (c,e) and a neutered cat (d,f), stained with picrosirius red; a higher density of collagen fibers is seen in the penis of the neutered cat. (e,f) Same sections as shown in (c) and (d) observed under polarized light. Magnification = × 200. Scale bar = 50 µm

Table 1

Statistical analysis of the analyzed parameters in the penises of intact and neutered cats

Variable	Groups		P value
Variable	Intact	Neutered	P value
ECs (%)	6.28 ± 3.03	4.10 ± 2.19	0.0434
ETa (%)	8.13 ± 1.38	3.11 ± 0.66	<0.0001
ETaCs (%)	4.37 ± 1.08	3.30 ± 1.01	0.0143
CCs (%)	34.11 ± 10.86	44.21 ± 12.72	0.0355
TU (μm)	21.86 ± 5.84	21.96 ± 4.23	0.9573
LAU (μm²)	46,402 ± 15,683	58,127 ± 20,819	0.1092
ACc (μm²)	4,305,884 ± 1,274,133	3,382,835 ± 1,145,710	0.0596
ACs (μm²)	918,386 ± 585,680	1,276,190 ± 413,348	0.0807

Data are presented as mean ± SD

ECs = elastic fibers in the corpus spongiosum; ETa = elastic fibers in the tunica albuginea of the corpus cavernosum; ETaCs = elastic fibers in the tunica albuginea of the corpus spongiosum; CCs = collagen fibers in the corpus spongiosum; TU = thickness of the urethral epithelium; LAU = luminal area of the urethra; ACc = area of the corpus cavernosum; ACs = area of the corpus spongiosum

The thickness of the urethral epithelium and the luminal area of the urethra did not significantly differ between the neutered and intact animals. Similarly, the area of the corpus spongiosum and the corpus cavernosum did not significantly differ between the neutered and intact cats (Table 1).

Although no changes were noted in the birefringence pattern of the collagen fibers, more collagen fibers were present in the corpus spongiosum of the neutered animals than in the intact animals (Figure 2).

Discussion

UO is a very important pathology, and between 1980 and 1999 its proportional morbidity rate in cats was 1.5% among veterinary teaching hospitals in the USA.¹⁹ Therefore, a global understanding of the pathology associated with UO will help in the development of preventive and therapeutic measures. Orchiectomy is frequently associated with a predisposition for UO in cats,¹⁰ although no well-established theory to justify this correlation exists.

In the present study several significant differences in the penile histoarchitecture, such as in the density of elastic fibers in the corpus spongiosum and the tunica albuginea of the corpus cavernosum and corpus spongiosum and the density of the collagen fibers in the corpus spongiosum, were observed between intact and neutered animals. The increased density of collagen fibers corroborates the findings of Herron,¹⁷ who reported an increase in fibrocyte density in the penile matrix of neutered cats. The reduction of elastic fiber density and the augmentation of collagen fibers without a reduction in the corpus spongiosum area can alter the ratio between collagen and elastic fibers, and can make the corpus spongiosum of neutered cats firmer than that of in intact cats. This difference may reduce the compliance of the corpus spongiosum that surrounds the urethra. Thus, the structural changes caused by castration could facilitate plug or calculi retention in the urethral lumen. Although an association between castration and a higher incidence of UO has been reported, an explanation for this finding has not yet been provided.^2,10,15 Our findings regarding the alteration of the penile structure, especially the corpus spongiosum, in neutered cats may explain the increased frequency of UO in neutered cats than in intact cats.

UO more commonly occurs in male cats than in female cats because of the narrower urethra.¹ Although UO is also more frequent in neutered male cats, previous studies have demonstrated no difference in the urethral diameter between intact and neutered male cats.^8,17 In the present study, evaluation of the urethral lumen area showed no significant differences between intact and neutered cats.

Although Herron reported a thicker urethral epithelium in neutered cats than in intact cats,¹⁷ no differences in the urethral epithelium thickness between intact and neutered cats were observed in the present study. However, we evaluated the middle-third of the penis, whereas Herron assessed three distinct penile areas.¹⁷

Two studies evaluating castration in prepubertal and postpubertal periods revealed that the incidence of FLUTD in both groups was similar.^14,15 Despite the presence of reports regarding predisposing factors, such as urolithiasis, urethral spasm, strictures and neoplasia, to FLUTD and UO, none of these could be correlated to castration.¹ The histological changes promoted by castration, which were demonstrated in this study, especially in the corpus spongiosum of neutered cats, could cause reduced compliance in the periurethral region and may contribute to the retention of plugs and calculi in the urethra, which could justify the high incidence of UO in neutered male cats.

Although the present study revealed the important structural changes that occur in the penises of neutered cats, some limitations should be taken into account while interpreting the findings. Future studies should also be performed to confirm the correlation of these findings with FLUTD and UO. Most of the animals used in this study came from shelters, so it was not possible to determine their precise age. To minimize possible bias, exclusion criteria were adopted on the basis of dentition and ocular changes to eliminate very young and old animals from the sample. Nonetheless, a group of animals of varying ages were evaluated, so the mean age of the two groups could not be assessed. This may have resulted in a bias caused by the differences in ages between the two groups. However, testosterone deficiency in humans and animal models has been associated with penile fibrosis, with elastic fibers being replaced by collagen fibers.^20,21 This finding was consistent with the results of the present study. Another limitation is that although the animals did not present any signs of FLUTD at the time of necropsy, their clinical history was unavailable, so it was not possible to state that these cats were never affected by FLUTD. Furthermore, because of the lack of information regarding the history of these animals, the precise period of castration could not be identified. Therefore, it was also not feasible to determine the duration between castration and the structural analysis, which is relevant because different levels of penile structural alterations can occur depending on the duration of testosterone deficiency in the animals. Despite the limitations of the study, an important alteration of the corpus spongiosum was identified. This finding warrants further consideration and additional studies to confirm whether the testosterone deficiency caused by castration may be a predisposing factor for UO in FLUTD.

Conclusions

A significant decrease in the density of the elastic fibers and an increase in the density of the collagen fibers in the corpus spongiosum of neutered animals might reduce the compliance of the periurethral region, suggesting that these changes could be a predisposing factor for urethral obstructive disease.

Footnotes

Conflict of interest

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

This research received a specific grant from the Fundação de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ, Brazil)

Accepted: 22 December 2016

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Effects of castration on penile extracellular matrix morphology in domestic cats

Abstract

Objectives

Methods

Results

Conclusions and relevance

Introduction

Materials and methods

Results

Discussion

Conclusions

Footnotes

Conflict of interest

Funding

References