Study On The Protective Effect Of Neuropeptide PACAP On Nerve Injury Of Eye

Objective (1) To evaluate the ability of pituitary adenylate cyclase activating polypeptide (PACAP) to accelerate neurite outgrowth and recovery of corneal sensitivity after creation of corneal flap in rabbit model surgery. The efficiencies of PACAP38 and PTD-PACAP38 in penetrating cornea were compared, which lay a foundation for the application of PACAP in bioremediation of corneal nerve. (2) To further develop the protective action on corneal nerve, PACAP38 was applied to cultured trigeminal ganglion cells. (3) MAX and PTD-MAX were used in rats treated with monosodium-glutamate (MSG) and retinas were processed for histological and functional examination. MAX and PTD-MAX were injected into the vitreous body of rats and their distribution status was studied. Our studies will provide theoretical basis for prevention and treatment of retinopathy of MAX and PTD-MAX.Methods (1) The corneal flap surgery was performed in the right eyes of 27 New Zealand white rabbits which divided into three groups.10μmol/L PACAP38 eye drops,5μmol/L PACAP38 eye drops or phosphate buffered saline (PBS) were topically administered in rabbits 4 times a day for 8 weeks respectively after operation. Corneal sensitivity was measured with Luneau ophthalmologie tester at 1-week interval through 8 weeks after operation. The growth of corneal nerve was evaluated with antibody staining against neurofilament (NF200). Rat eyes were treated with FITC-fluorescently labeled PACAP38 and PTD-PACAP38 (8mg/ml). The penetrating rate was compared by means of aqueous humor detection. (2) Trigeminal ganglion cells were isolated from 2-week-old New Zealand white rabbit and cultured in neurobasal medium.1μmol/L or 0.1μmol/L of PACAP38 or PBS were added into the medium respectively in 24 hours after culture for 1 week. Cultured cells were identified by immunofluorescence of NF200 stain. (3) There were MAX group, PTD-MAX group and PBS group.5μL MAX (100pM) was injected into the vitreous body of rats treated with MSG (4mM) on the following two days as MAX group. Experimental methods of PTD-MAX group and PBS group were the same as MAX group. After 2 weeks, retinas were processed for hematoxylin-eosin staining, transmission electron microscope (TEM) and flash electroretinogram (F-ERG) examination. Additionally, PTD-MAX with FITC, MAX with FITC or PBS was injected into the vitreous body of rats and the entire retina was carefully dissected from the eye with the photoreceptors facing upward. Flat mounts were examined by fluorescence microscopy, and the images were photographed.Results (1) After administration of PACAP38, corneal sensitivity was gradually enhanced with the increase of time following the surgery in all of the three groups, but the fastest restore of sensation was in 10μmol/L PACAP38 group. Significant differences were found in the corneal sensitivity value between 10μmol/L PACAP38 group and PBS group (P=0.000<0.01) or 5μmol/L PACAP38 group and PBS group (P=0.005<0.01). NF200 immunofluorescence stains revealed that neurites of PACAP38 group were the most among three groups. PTD-PACAP38 was superior to PACAP38 in penetrating cornea. The average efficiency of former was 27.22% while the latter was 8.10%. (2) More nerve neuronal processes and synapsis of trigeminal ganglion cells were exhibited in 1 week after culture in 1μmol/L PACAP38 group comparison with 0.1μmol/L of PACAP38 group or PBS group by the observations of invert microscope and NF200 Immunofluorescence stains. (3) The histological examination showed that PTD-MAX and MAX exhibited prominent capability of protecting cell forms, structures and thickness of IPL, ONL and INL in the retinal degeneration models induced by MSG in rats. TEM test showed that the retina in PBS group was degenerated seriously. The death and debris of cells in PBS group were the most in three groups. GCL layer was severely damaged but photoreceptor cell layer was seldom influenced in all three groups. The b and OPs-Wave amplitude of three groups had statistical differences (P<0.05). The FITC intensity results of retina flat mounts indicated that PTD-MAX was widespread in retina and more intense than MAX.Conclusion (1) PACAP38 can improve corneal sensitivity and reinnervate corneal nerve. PTD can enhance the capability of penetrating cornea of PACAP38. (2) PACAP38 can promote the proliferation and inhibit the death of rabbit trigeminal ganglion cells. Our studies in vivo and in vitro demonstrate that PACAP38 has neuroprotective effect for cornea, which suggest that neuropeptide PACAP has potential pharmaceutical value for nerve regeneration. (3) PTD-MAX precedes MAX in alleviating MSG-induced retinal toxicity. PTD increase distribution of MAX in retina. Our studies paved the way for the application of neuropeptide PACAP in retinopathy.