In Vivo Detection of Apoptotic Cells in the Testis Using Fluorescence Labeled Annexin V in a Mouse Model of Testicular Torsion

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Abstract

Purpose

It is well established that experimental testicular torsion induces germ cell specific apoptosis. Annexin V (BD Pharmingen™) binds phosphatidylserine that becomes exposed on the cell membrane in apoptotic cells. In vivo detection of apoptotic cells with fluorescently labeled annexin V is an emerging technique that we evaluated for detecting apoptotic germ cells in a mouse model of testicular torsion.

Materials and Methods

Annexin V labeled with an indocyanine fluorophore (bisfunctional succinimidyl ester of cyanine 5.5) (Amersham, Little Chalfont, United Kingdom) was injected intravenously in mice 18 hours after the repair of unilateral 720-degree testicular torsion for 2 hours. Serial fluorescence images were obtained 21, 24, 28 and 42 hours after torsion repair. Relative fluorophore localization was visualized in vivo using an optical small animal imaging system mounted with a filter in near infrared light. Average fluorescence intensity in torsed and sham testes was quantified in images of testes in situ exposed through an abdominal incision and in ex vivo testes.

Results

A significant increase in fluorescence intensity was found in images of torsed vs sham operated testes. This was seen in ex vivo, exposed and in vivo testes (215%, 250% and 161%, respectively, p <0.05). Bisfunctional succinimidyl ester of cyanine 5.5 conjugated to dehydrogenase, a protein with a size similar to that of annexin V, was used to assess for capillary leakage. It was also more localized to the torsed testis relative to its contralateral sham control whether exposed or ex vivo (174% and 176%, respectively).

Conclusions

To our knowledge this study demonstrates for the first time the possibility of in vivo near infrared fluorescence imaging of apoptotic germ cells after testicular torsion in mice. It shows important confounding factors that must be considered as this new imaging technique is developed for detecting apoptotic cells in vivo in testes or in any other organ.

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