AIMS: Muscarinic acetylcholine receptor agonist pilocarpine reduces intraocular 
pressure (IOP) of glaucoma mainly by stimulating ciliary muscle contraction and 
then increasing aqueous outflow. It is of our great interest to know whether 
pilocarpine has the additional properties of retinal neuroprotection independent 
of IOP lowering in vitro and in vivo models.
METHODS: In rat primary retinal cultures, cell viability was measured using an 
MTT assay and the trypan blue exclusion method, respectively. Retinal ganglion 
cells (RGCs) were identified by immunofluorescence and quantified by flow 
cytometry. For the in vivo study, the retinal damage after retinal 
ischemia/reperfusion injury in rats was evaluated by histopathological study 
using hematoxylin and eosin staining, transmission electron microscopy, and 
immunohistochemical study on cleaved caspase-3, caspase-3, and ChAT.
RESULTS: Pretreatment of pilocarpine attenuated glutamate-induced neurotoxicity 
of primary retinal neurons in a dose-dependent manner. Protection of pilocarpine 
in both retinal neurons and RGCs was largely abolished by the nonselective 
muscarinic receptor antagonist atropine and the M1-selective muscarinic receptor 
antagonist pirenzepine. After ischemia/reperfusion injury in retina, the inner 
retinal degeneration occurred including ganglion cell layer thinning and neuron 
lost, and the optic nerve underwent vacuolar changes. These degenerative changes 
were significantly lessened by topical application of 2% pilocarpine. In 
addition, the protective effect of pilocarpine on the ischemic rat retina was 
favorably reflected by downregulating the expression of activated apoptosis 
marker cleaved caspase-3 and caspase-3 and upregulating the expression of 
cholinergic cell marker ChAT.
CONCLUSIONS: Taken together, this highlights pilocarpine through the activation 
of muscarinic receptors appear to afford significant protection against retinal 
neurons damage and optic nerve degeneration at clinically relevant 
concentrations. These data also further support muscarinic receptors as 
potential therapeutic neuroprotective targets in glaucoma.