PACAP induces neurite outgrowth in cultured trigeminal ganglion cells and recovery of corneal sensitivity after flap surgery in rabbits.
OBJECTIVE
To evaluate the ability of pituitary adenylate cyclase-activating polypeptide (PACAP) to induce growth of neuronal processes in cultured trigeminal ganglion cells, and to accelerate neurite outgrowth and recovery of corneal sensitivity after creation of a corneal flap in a rabbit model of laser-assisted in situ keratomileusis (LASIK) surgery.


METHODS
Animal study.


METHODS
The cDNA of rabbit PACAP was sequenced, and the expression of PACAP receptors in the trigeminal ganglia from rabbits was quantified by quantitative real-time polymerase chain reaction. Trigeminal ganglion cells were isolated from rabbits and cultured for 48 hours with or without PACAP27 (bioactive N-terminal peptide from PACAP). Cells were stained with antibody against neurofilaments, and neurite outgrowth was quantified by cell counting. In the rabbit LASIK model, a corneal flap with a planned thickness of 130 microm and 8.5 mm diameter was created with a microkeratome. The rabbits then received eyedrops containing PACAP27 four times a day for eight weeks, and corneal sensitivity was measured. Neurite outgrowth was assessed by staining histologic sections of the flap area for cholinesterase.


RESULTS
The deduced amino acid sequence of PACAP in rabbit was identical to that of human. PACAP receptor, PAC1, was highly expressed in trigeminal ganglia from newborn and adult rabbits. PACAP27 at 1 microM induced growth of neuronal processes in cultured primary trigeminal ganglion cells. In the LASIK model, extensions of neuronal processes from amputated nerve trunks in cornea were observed after administration of eyedrops containing 1 or 10 microM PACAP27. The 10 microM PACAP27 treatment also greatly accelerated recovery of corneal sensitivity.


CONCLUSIONS
PACAP may be a candidate drug for ameliorating dry eye after LASIK surgery.
Fukiage
Chiho
C
Senju Laboratory of Ocular Sciences, Senju Pharmaceutical Co Ltd, Kobe, Hyogo, Japan.
Nakajima
Takeshi
T
Takayama
Yoshiko
Y
Minagawa
Yoko
Y
Shearer
Thomas R
TR
Azuma
Mitsuyoshi
M
D000818:Animals; D000831:Animals, Newborn; D001483:Base Sequence; D002469:Cell Separation; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003315:Cornea / Q000187:drug effects / Q000294:innervation / Q000502:physiology*; D003319:Corneal Stroma / Q000294:innervation / Q000601:surgery; D019084:Fluorescent Antibody Technique, Indirect; D006133:Growth Substances / Q000235:genetics / Q000494:pharmacology*; D020731:Keratomileusis, Laser In Situ; D008969:Molecular Sequence Data; D016501:Neurites / Q000502:physiology*; D009883:Ophthalmic Solutions; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide / Q000235:genetics / Q000494:pharmacology*; D011817:Rabbits; D051236:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide / Q000235:genetics / Q000378:metabolism; D020127:Recovery of Function / Q000502:physiology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013524:Surgical Flaps; D012668:Trigeminal Ganglion / Q000187:drug effects* / Q000378:metabolism
D006133:Growth Substances; D009883:Ophthalmic Solutions; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D051236:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide