A method for identifying peripheral connections of perivascular nerves based on sensitive acetylcholinesterase staining via perfusion

To identify peripheral connections of perivascular nerves, which are usually hard to find and easily damaged, we developed a sensitive method for acetylcholinesterase (AChE) staining in rat. The procedure is based on primary staining via perfusion, which permits visualization of perivascular nerves before dissection and, after dissection, further staining via immersion for identification of peripheral connections of the earlier stained perivascular nerves. The AChE histochemistry is based on an intensification method originally described for sections. The procedure has been optimized for staining via vascular perfusion and can also be applied on whole-mount preparations via immersion. The results show intense nerve staining with minimal background. Although the procedure was initially developed for cerebrovascular nerves, the staining approach can also be applied in other regions, the main advantage being that perivascular neural structures can be identified without the damage that usually occurs during dissection.