重组人白细胞介素-2玻璃体注射慢性高眼压大鼠神经节细胞层神经元的凋亡

BACKGROUND: The majority of studies addressing neuroprotection have focused on drugs, proteins and cytokines. A recent study reports that recombinant human interleukin-2 (rhIL-2) achieves good therapeutic effects, but the underlying mechanisms remain poorly understood.OBJECTIVE: To observe the neuroprotection of recombinant human interleukin-2 (rhIL-2) by intravitreous injection on the retinal ganglion cells (RGCs) following chronic elevated IOP model in Wistar rats. DESIGN, TIME AND SETTING: A randomized, controlled, animal experiment was performed at the Center Laboratory, Second Xiangya Hospital, Central South University, China, from July 2008 to March 2009.MATERIALS: LY294006 and AG490 were provided by Invitrogen, USA. Rabbit anti-AKT1 polyclonal antibody and rabbit anti-STAT3 polyclonal antibody were purchased from American Basic Gene Associate Bioscience, USA.METHODS: Chronic elevated IOP model of Wistar rats were established by cauterizing the limbal episcleral veins to block the reflux of aqueous humor by 532nm viridis-lite diode laser in right eyes. Intravitreous injection of 2µl PBS, rhIL-2 or rhIL-2 LY294002, AG490 mixed liquor for eyes were administered 1 week after chronic elevated IOP model established. Ocular tissue was immunostained to investigate ED-1 expressions. Rats were sacrificed after intravitreous injection 3 days, 14 days, 21 days to identify RGC, and the 3% fluorogold was used to the epithalamus 7 days before sacrifice. Western blot analysis Akt1 and STAT3 protein in the retina after intravitreous injection 3h,12h, 96h. MAIN OUTCOME MEASURES: ED-1 expression of activated microglia/macrophages was determined by immunofluorescence. The RGCs levels were detected using fluorogold staining. The efficacy of the intraocular rhIL-2 and rhIL-2, LY294002, AG490 mixed liquor injections in inhibiting PI3K/Akt、Jak/Stat3 signal transductions were confirmed by Western blot analysis.RESULTS: Numerous ED-1 positive macrophages were seen in the vitreous and along the inner retinal surface in rhIL-2 treatment group. The number of RGCs was much higher than that in control groups and PBS group (P<0.01). The numbers of RGCs in rhIL-2 and LY294002, AG490 mixed liquor group was as much lower than that in rhIL-2 group (P<0.01). Western blotting analysis showed very little Akt1 and STAT3 protein in the retina after 3h intravitreous injections of rhIL-2 in rats, started to increase rapidly at 12 reperfusion,96h decreased slightly. LY294002 and AG490 prevented the Akt1 and STAT3 protein expression in the retina.CONCLUTION: rhIL-2 has neuroprotective effection after intravitreous injection. It can decrease or avoid RGCs layer damage in chronic glaucoma model of rats. Intravitreal injection of rhIL-2 activates macrophages, which derived factors might enhance the RGCs survival. After intravitreal injection of rhIL-2 PI3K/Akt、Jak/Stat3 signal transductions may play an important role following a chronic elevated IOP model, and LY294002, AG490 can block this effect.

Neuronal changes in the retinal ganglion cell layer following recombinant human interleukin-2 intravitreal injection in a rat model of chronically elevated intraocular pressure

Intraperitoneal injection of recombinant human interleukin-2 (rhIL-2) inhibits neuronal apoptosis in the chronic ocular hypertension retinal ganglion cell layer. Intravitreous injection was performed on retinal ganglion cells in a Wistar rat model of chronically elevated intraocular pressure to observe the effects of LY294002 and AG490 on retinal ganglion cell survival, macrophage activation, and PI3K/Akt and JAK/STAT activation. The number of retinal ganglion cells in the rhIL-2 treatment group was much greater than in the normal control and phosphate-buffered saline groups. Western blot analysis revealed low Akt and STAT3 protein expression in the retina after 3-hour intravitreous injections of rhIL-2. However, protein expression was increased at 12 hours, but decreased again at 24 hours, with very low expression at 96 hours. LY294002 and AG490, which are inhibitors of the PI3K/Akt and JAK/STAT3 signal pathways, prevented upregulation of Akt and STAT3 protein expression in the retina, respectively. Intravitreous injection of rhIL-2 exhibited neuroprotective effects by decreasing retinal ganglion cell layer damage in a rat model of chronic glaucoma. These results suggest that intravitreal injection of rhIL-2 could induce the PI3K/Akt and JAK/STAT3 signaling pathways to protect retinal ganglion cells in chronically elevated intraocular pressure models.