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Improved penile histology by phalloidin stain: circular and longitudinal cavernous smooth muscles, dual-endothelium arteries, and erectile dysfunction-associated changes - PubMed

Improved penile histology by phalloidin stain: circular and longitudinal cavernous smooth muscles, dual-endothelium arteries, and erectile dysfunction-associated changes

Guiting Lin et al. Urology. 2011 Oct.

Abstract

Objective: To investigate whether fluorochrome-conjugated phalloidin can delineate cavernous smooth muscle (CSM) cells and whether it can be combined with immunofluorescence (IF) staining to quantify erectile dysfunction (ED)-associated changes.

Methods: ED was induced by cavernous nerve crush in rats. Penile tissues of control and ED rats were stained with Alexa-488-conjugated phalloidin and/or with antibodies against rat endothelial cell antigen (RECA), CD31, neuronal nitric oxide synthase (nNOS), and collagen-IV (Col-IV).

Results: Phalloidin was able to delineate CSM as composed of a circular and a longitudinal compartment. When combined with IF stain for CD31 or RECA, it helped the identification of the helicine arteries as covered by endothelial cells on both sides of the smooth muscle layer. When combined with IF stain for nNOS, it helped the identification that nNOS-positive nerves were primarily localized within the dorsal nerves and in the adventitia of dorsal arteries. When combined with IF stain for Col-IV, it helped identify that Col-IV was localized around smooth muscles and beneath the endothelium. Phalloidin also facilitated the quantitative analysis of ED-related changes in the penis. In rats with cavernous nerve injury, RECA or Col-IV expression did not change significantly, but CSM and nNOS nerve contents decreased significantly.

Conclusion: Phalloidin stain improved penile histology, enabling the visualization of the circular and longitudinal compartments in the CSM. It also worked synergistically with IF stain, permitting the visualization of the dual endothelial covering in helicine arteries, and facilitating the quantification of ED-related histologic changes.

Copyright © 2011 Elsevier Inc. All rights reserved.

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Figures

Fig. 1
Fig. 1

Smooth Muscles in Rat Penis. Rat penis was stained with Alexa-488-conjugated phalloidin, which specifically detected smooth muscles (green stains) in dorsal arteries (DA), dorsal vein (DV), and cavernous tissue (CSM). Boxed areas in panels A and B are enlarged in panels B and C, respectively. These tissue sections were co-stained with DAPI for the visualization of cell nuclei (blue). The seemingly “missing nuclei” (phalloidin-stained cells without DAPI) is due to tissue sectioning at an angle or plane that missed the nuclei. Panels D and E are consecutive sections stained with phalloidin and anti-SMA, respectively. Inserts are images enlarged digitally from the boxed areas.

Fig. 2
Fig. 2

Endothelium in Rat Penis. Rat penis was stained with Alexa-488-conjugated phalloidin and with Alexa-594-conjugated anti-RECA (A-C) or anti-CD31 antibody (C). These stains provide visualization of smooth muscle in green and the endothelium in red. Boxed areas in panel A are enlarged in panels B and C, respectively. Arrows in Panels C and D point to the internal and external endothelia (red) of helicine arteries. Panel E shows the dorsal arteries and vein that were stained positive for RECA (red) in the luminal side but not the abluminal side of smooth muscle (green). All tissue sections were co-stained with DAPI for the visualization of cell nuclei (blue).

Fig. 3
Fig. 3

nNOS-Positive Nerves in Rat Penis. Rat penis was stained with Alexa-488-conjugated phalloidin and with Alexa-594-conjugated anti-nNOS. These stains provide visualization of smooth muscles in green and nNOS-positive nerves in red. (A) A representative dorsal nerve stained positive for nNOS but negative for smooth muscle. Blue stains are Schwann cell nuclei. (B) A representative dorsal artery with its smooth muscle stained positive by phalloidin. Red stains surrounding the smooth muscle are nNOS-positive nerves. Blue stains encircling the lumen are endothelial cell nuclei. (C) A representative CSM bundle with a few nNOS-positive nerves innervating at its periphery.

Fig. 4
Fig. 4

Collagen-IV in Rat Penis. Panels A and B are rat penis stained with Alexa-488-conjugated phalloidin and with Alexa-594-conjugated anti-Col-IV antibody. For enhanced clarity, Col-IV expression was shown in the absence of phalloidin stain in Panel A. For visualizing the relationship between Col-IV and SMC, phalloidin-stained image was added in Panel B. Panels C and E are rat penis stained with Alexa-488-conjugated Col-IV (green) and with Alexa-594-conjugated anti-RECA (red). Panels D and F are rat penis stained with Alexa-488-conjugated anti-RECA (green) and with Alexa-594-conjugated anti-Col-IV (red). Note that Panels E and F show a helicine artery in the center. All tissue sections were co-stained with DAPI for the visualization of cell nuclei (blue).

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