Preclinical Study Of Intravitreal Injection Of Mouse Nerve Growth Factor For Retinitis Pigmentosa

Retinitis pigmentosa (RP) is a blinding disease characterized by progressive photoreceptors apoptosis. Studies demenstrated that the reduction of loss of several neurotrophic factors,such nerve growth(NGF), ciliary neurotrophic factor(CNTF), brain derived neurotrophic factor(BDNF), enhance the invasion and aggravation of RP. Thus one of the treatment strategy for RP is to make the neurotrophic factors of retina enough to the balance of retinal internal enviroment. Nerve growth factor(NGF) is one of the factors in neurotrophic factors family. The reduction or loss is correlated with the progress of RP. It indicated that NGF maybe one critical key factor implicated in RP. Eye local administration of exgenous NGF exerts a rescue effect on photoreceptors in the RP animal model. Furthermore, NGF not only protect photoreceptors itself, but also stimulate other biological mediators produced and released in the retina,such as brain-derived neurotrophic factor(BDNF), beta-fibroblast growth factor(b-FGF), transforming growth factor-beta(TGF), vascular endothelial factor (VEF)and neuropeptide-Y .The NGF ,a factor isolated from the mouse submandibular gland , has been the clinical i.m.medicine with CFA license,which gives the opportunity to the study of therapy of retinal degeneration. However, the limitation of NGF useness in eye diseases is troublesome because of the little concentration contained in eye through NGF system administration. Intravitreal injection is a good technique of direct intraocular administration and avoiding the blood-retinal barrier and blood-aque oculi barrier. But the concrete contents of intraocular tissues with NGF intravitreal injection are obscure. The affects of intravitreal injection of NGF have been confirmed in several secondary retinal degenerations, such as ischemic, retinal detachment. However, the study about the safty and efficacy of intravitreal injection of NGF remained unknown.RP is classified into typical RP and atypical RP in clinic. The common types in Asian are typical and Bietti's crystalline dystrophy(BCD), which is one of the atypical RP. Bietti's crystalline dystrophy(BCD) is a autosomal recessive hereditary disorder , characterized by numerous, tiny, yellow-white spots at the posterior pole of the retina and tapetoretinal degeneration with choroidal sclerosis. Much of the report about BCD remains to the gene analysis and case report. The natural course ,espectially the retinal functional defects of BCD is poorly understood. Therefore, it is important to understand the evaluative effects of the available visual functional examination, such as visual acuity, perimetry, electrophysiologic tests. Pupillary light reflex (PLR) is an important objective visual response. Doctor Liu has identified that relative papillary construction(RPC) may reflect the photoreceptors function of typical RP objectively and sensitively. However, the PLR features of BCD are indistinct.Not only typical RP but also BCD lack the suitable clinical treatment. It is proceeding at an optimistic pace on account of the effection of NGF on RP animal model and the clinical application of NGF. Our previous studies have comfirmed that intravitreal injection of NGF is safe to the end stage of typical RP. But we also find it is useless to a single administration of NGF. So it is important to identify the safety and effect of repetatus intravitreal injection of NGF on typical RP and BCD.We planned a research project about"Preclinical Study of Intravitreal Injection of Mouse Nerve Growth Factor for Retinitis Pigmentosa". This project was approved by medical ethic committee of Southwest Hospital, Third Military Medical University(license No KY2008001), and registered at the Registration center of Chinese clinical trails affiliated to World Health Organization (registration No ChiCTR-TNC-00000193). The contents and results of our research are described as follow:1. The results of animal experiments are as follow: 1) we investigated the ocular distribution and peak time of 125I-NGF after intravitreal injection and posterior juxtascleral injection in vivo using isotope tracer method and to analyze 125I-NGF penetration pathway and ability of both ocular delivery methods. The results showed The 125I-NGF contents through intravitreal injection were higher than the contents through posterior juxtascleral injection apparently. The gradients of 125I-NGF contents in intraocular tissue by intravitreal injection were vitreous body(471.75±4.42)>retina(118.32±18.74)>chorioid(79.13±8.82)>corpus ciliare choroideae(68.29±14.41)>aqua oculi(51.42±7.28)>sclerotic(36.64±4.16)>cornea(21.00±3.10). 2) we observed the safty of intravitreal injection of NGF in normal rabbits. The results showed no abnormal changes being found in their cornea, lens, vitreous body and retina after NGF intravitreal injection. And the each layer of retinal cells layout were regular by tissue morphological obserbvation on one month after treatment. 3) we evaluated the effect of repetatus intraveal injection of NGF on P30d RCS rats through the measurement the layer thickness of outer nuclear layer(ONL) and opoptotic index of photoreceptors in ONL. The results displayed that the thickness of ONL in the group of NGF treatment was thicker than the thickness in the group of normal saline and blank contral(P<0.05); the opoptotic index of photoreceptors in NGF treated group was more decrease than that in saline group and blank contral group(P<0.05).2. We observed that the safty of single intravitreal injection of NGF on twenty typical RP patients and 15 BCD patients. We found that the feeling of subject with BCD was better than that of typical RP patients at 1d, 7d, 1m after injection. The intraocular inflammatory reaction of typical RP was obvious after treatment, with 30% of exceeding 3 inflammatory score on 1d, 10% on 7d. And the inflammation disappeared on one month. Comparatively, the intraocular inflammatory reaction of BCD was sligt with 10% of exceeding 3 inflammatory score on 1d and disappeared inflammation on 7d. No abnormalitiea were observed in B scan, OCT, and fundus photograph of BCD and typical RP on 12m. The incidence of adverse effects was 20% of typical RP and 0 of BCD. We further performed the preclincal experiment about the security of repetatus intraveal injection of NGF on BCD through comparing the safty of NGF treatment with 3months interval and 1months interval. The results appeared both well subject feeling , slight intraocular inflammatory reaction and none of inflammatory superposition. The abnormalities were observed in OCT on long-time observation in two groups.3. We evaluated the disease progression of 15 patients with BCD through the fundus stages and visual function evaluation. Furthermore, we screened the potimal techniques for retinal disturbance of BCD. The results showed 1 patient in early stage of BCD, 9 in intermediate stage and 5 in advanced stage. The variable abnormality of fERG and sevious reduction , even extinguish responses of mERG correlated with the photoreceptors dysfunction. 85 degree visual field appeared an peripherial scotoma at early BCD, a midperipherial visual island at intermediate stage and blind visual field at end BCD. The results of chromatic PLR showed the RPC of blue and white light-induced PLR in all patients were lower than the normal subjects and decreased with the BCD stage progressive.4. We perform the preclinical evaluation of repetatus intraveal injection of NGF with 1month interval and 3 month interval on 15patients with BCD. The results appeared an improvement of subjects'visual acuity, visual field(VF), RPC under blue light and white light stimuli on 1month after intravitreal injection of NGF. And the improvements were maintained by 1month interval treatment rather than by 3month interval treatment. When the therapy stopping, the improvement of retinal function stoped and reversed to the pre-treatment.We concluded that:1. Intravitreal injection of 125I-NGF may gain higer contents in each ocular tissue than posterior juxtascleral injection, especially in retina. The intravitreal injection of NGF is safe to rabbits'ocular and effective to delay the retinal degeneration of RCS rats.2. The single intravitreal injection of NGF on BCD is safter than on typical RP. The repetatus intraveal injection of NGF on BCD with 1month interval and 3month interval are both safty.3. To BCD, the abnormality of fERG is more consistent with the aggravation of BCD than the changes of morphological expression . 85 degree perimetry may locate the lesion and remainence of visual function. mERG play an effective role of early diagnosis of BCD. Chromatic PLR testing is a potential technique in the objective assessment of photoreceptors dysfunction with BCD.4. Allotransplantation of fetal RPP cells is safe, the visual function of host can improved in 6 months follow-up, it will promotes us to carry out efficacy Study in the future.5. Intravitreal injection of NGF may improve the visual function of BCD. Furthermore, the improvement may be maintained through the repetatus intravitreal injection of NGF in 1 month interval.