title: Improved penile histology by phalloidin stain: circular and longitudinal cavernous smooth muscles, dual-endothelium arteries, and erectile dysfunction-associated changes.
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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Urology, University of California, San Francisco, CA, USA.
authors.forename: Guiting
authors.initials: G
authors.lastname: Lin
authors.forename: Xuefeng
authors.initials: X
authors.lastname: Qiu
authors.forename: Thomas M
authors.initials: TM
authors.lastname: Fandel
authors.forename: Maarten
authors.initials: M
authors.lastname: Albersen
authors.forename: Zhong
authors.initials: Z
authors.lastname: Wang
authors.forename: Tom F
authors.initials: TF
authors.lastname: Lue
authors.forename: Ching-Shwun
authors.initials: CS
authors.lastname: Lin
mesh_terms: D000818:Animals; D004730:Endothelium, Vascular / Q000473:pathology*; D007172:Erectile Dysfunction / Q000473:pathology*; D005109:Extracellular Matrix / Q000473:pathology; D006801:Humans; D007091:Image Processing, Computer-Assisted; D008297:Male; D008856:Microscopy, Fluorescence / Q000379:methods; D009130:Muscle, Smooth / Q000473:pathology*; D009474:Neurons / Q000473:pathology; D010413:Penis / Q000473:pathology*; D010590:Phalloidine / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D010590:Phalloidine