Phalloidin is a small molecule that can penetrate rapidly into the tightly packed cytoskeleton. This property and its highly specific affinity for actin make it an ideal probe for actin, and to this end, several fluorochrome-conjugated phalloidin derivatives have been employed to stain smooth muscle in various organs and tissues 9,13
. The staining procedure is applicable to both frozen and paraffin-embedded tissues 14
and can be completed in 30 min, in contrast to IF, which may require up to 24 h. As shown in the present study, phalloidin stain works synergistically with IF for various antigens including CD31, nNOS, and Col-IV. In contrast, double IF requires that two primary antibodies be generated from two different host species in order to incubate the antibodies together 15,16
. We have observed that double IF staining occasionally produced low-quality histology due to interference between the primary antibodies and/or between the secondary antibodies.
Phalloidin has been employed for assessing smooth muscle content in a cavernous nerve ablation rat model 17
. However, histological images in this study are not as sharp as what's shown in the present study. One possible explanation is that the earlier study used tetramethyl-rhodamine isothiocyanate-conjugated phalloidin, which has been shown to produce lesser quality images when compared to Alexa-conjugated phalloidin 8
. The exceptionally high optical resolution afforded by Alexa-conjugated phalloidin enabled us to recognize that the CSM was divided into a circular and a longitudinal compartment - characteristic structures of tubular organs such as intestines, urinary bladder, and urethra. Thus, the penile corpus cavernosum can possibly be considered as a multiunit tubular organ with each unit (sinusoid) consisting of a circular and a longitudinal smooth muscle. The circular CSM regulates the penile girth while the longitudinal CSM the length during tumescence and detumescence.
It is well known that each penile cavernous sinusoid is an equivalent of a blood vessel's lumen with both having a single layer of endothelial cells covering the luminal side of the smooth muscle layer. The endothelial cells can be easily identified by IF using either anti-RECA (highly specific for rat endothelial cells) or anti-CD31, as demonstrated in the present study and in many previously published studies 18,19
. However, in the present study we saw that certain smooth muscle bundles that otherwise resembled the longitudinal CSM contained centrally located RECA or CD31-positive cells. Based on this observation we propose that these are helicine arteries entering the cavernous sinusoids; while retaining the arterial endothelium on the luminal side, they also become covered by the cavernous endothelium on the abluminal side. Thus, it appears that the vascular and cavernous smooth muscle cells are “protected” from direct contact with the blood by the endothelium. This hypothesis is based on experiments in which de-endothelialization leads to platelet activation and aggregation 20
as well as SMC proliferation 21
Burnett et al 22
were the first to demonstrate the localization of nNOS-positive nerves in the penis. They used IHC stain to show that these nerves were mainly localized in the dorsal nerves and were also visible near the dorsal arteries and in the cavernous tissue. These findings are confirmed in the present study; additionally, our phalloidin stain enabled the visualization of the relationship between nNOS-positive fibers and smooth muscles in the dorsal arteries and the cavernous tissue. Specifically, these nerves were located outside of the arterial smooth muscle and therefore in the adventitia of the dorsal artery. Furthermore, their doted appearance in cross sectional view suggests an orientation parallel to the muscle fibers in the artery as well as in the cavernous tissue.
The cavernous ECM is mainly composed of collagen types I, III, and IV, with types I and IV being predominant 11
. Although many studies have examined collagen expression in the penis, few have looked into the specific localization of each collagen type. Five of these reports, all from the same group of researchers, used IHC to examine differences in Col-III expression between hypertensive and control animals 23,24
. To our knowledge, there has been no report that examined the localization of Col-IV in the penis. In blood vessels Col-IV is a major component of the endothelial basement membrane and of the basal lamina surrounding SMC; it also plays important roles in maintaining the contractile phenotype of SMC 12
. Thus, since the present study's main objective is to utilize the phalloidin stain to visualize the penile SMC and its surroundings, Col-IV was chosen to represent the cavernous ECM. Indeed, the results show that Col-IV was localized to the subendothelial area as well as in the surroundings of individual SMC. Very little Col-IV expression was found in the rest of the cavernous tissue, which is otherwise the main locale of collagen expression. That is, Col-IV is primarily associated with the endothelium and CSM while Col-I and Col-III in the remaining areas of the cavernous tissue.
We have previously published a rat ED model that simulates post-prostatectomy nerve injury 10
. The penile tissue samples of this study were used in the present study for the purpose of demonstrating the utilization of the phalloidin stain. The results show that the CSM and nNOS nerves were significantly reduced in rats with cavernous nerve injury while no statistical difference was found for the endothelium at 4 months after injury. In regard to Col-IV, while the absolute amount of Col-IV was lower, the relative amount to CSM was actually increased.
In addition to nerve injury, we have found that the phalloidin stain to be useful for the evaluation of other types of ED including diabetes-associated ED 25
and hyperlipidemia-associated ED 26