Conformal geometry of the retinal nerve fiber layer

The nerve fiber layer of the human retina is made up of the retinal segments of ganglion cell axons. Its geometry can be described mathematically as a fibration of a 2D domain: a partition of a certain region into smooth curves. Here, we present a simple family of curves that closely models the observed geometry of the nerve fiber layer. For each retina, the pattern depends on 2 parameters, A and B: A computer program determines A and B for a given retina and the theory matches the retina with a standard deviation of ≈6–8°. These particular curves turn out to be the curves that would be generated if the growing ganglion cell axon tip moved down a gradient toward a source of diffusible neuroattractant at the disk and away from a weaker macular diffusible repellant. Thus, this model provides morphological evidence that diffusible substances provide positional information to the embryonic ganglion cell axons in finding their way to the optic nerve head.     

Vascular reactivity of optic nerve head and retinal blood vessels in glaucoma--a review

Glaucoma is characterized by loss of retinal nerve fibers, structural changes to the optic nerve, and an associated change in visual function. The major risk factor for glaucoma is an increase in intraocular pressure (IOP). However, it has been demonstrated that a subset of glaucoma patients exhibit optic neuropathy despite a normal range of IOP. It has been proposed that primary open angle glaucoma could be associated with structural abnormalities and/or functional dysregulation of the vasculature supplying the optic nerve and surrounding retinal tissue. Under normal conditions, blood flow is autoregulated, i.e., maintained at a relatively constant level, in the retina and ONH, irrespective of variation in ocular perfusion pressure. A number of factors released by the vascular endothelium, including endothelin-1 and nitric oxide, are suggested to play an important role in the regulation of local perfusion in the retina and ONH. Most work to-date has investigated homeostatic hemodynamic parameters in glaucoma, rather than the measurement of the hemodynamic response to a provocation. Future work should comprehensively assess blood flow in all the ocular vascular beds and blood vessels supplying the eye in response to standardized stimuli in order to better understand the pathophysiology of glaucomatous optic neuropathy.     

丹星通络汤对脑缺血再灌注大鼠神经生长因子表达的影响

目的 观察丹星通络汤( DXTLD)治疗脑缺血再灌注大鼠后脑组织神经生长因子(NGF)的变化,探讨其对脑缺血再灌注损伤的保护机制. 方法 SD大鼠40只随机分为5组(n=8):假手术组、缺血再灌注组(模型组)、尼莫地平组、DXTLD预处理小剂量组、DXTLD预处理大剂量组.线栓法建立大鼠大脑中动脉缺血再灌注模型.应用免疫组织化学染色、灰度分析等方法检测缺血2h再灌注24 h后脑组织海马区NGF的表达. 结果 各治疗组缺血侧海马区的NGF表达显著增加,与模型组比较差异有统计学意义(P＜0.05或P＜0.01).结论 DXTLD通过上调脑缺血再灌注损伤后脑组织内NGF的表达,起到保护脑的作用.     

Regulation of nerve growth factor in the heart: The role of the calcineurin-NFAT pathway

A heightened sympathetic tone accelerates the development of lethal arrhythmias after myocardial infarction (MI) and the progression of heart failure (HF). Cardiomyocytes control their local neural milieu by expression of nerve growth factor (NGF), which triggers sympathetic neural growth (sympathetic nerve sprouting: SNS). The molecular mechanisms that regulate NGF expression are largely unknown. During HF or MI the myocytes are exposed to increased mechanical load and adrenergic stimulation. Both stimuli induce myocyte hypertrophy. The angiotensin-II-calcineurin-NFAT (nuclear factor of activated t-cells) pathway is a well characterized signaling cascade in the pathogenesis of myocyte hypertrophy. The present study aims to investigate the molecular mechanisms by which mechanical stretch and/or alpha-1-adrenergic stimulation affect NGF expression in neonatal rat ventricular myocytes. Both stimuli resulted in a down-regulation of NGF gene and protein expression. Angiotensin-II type 1 receptor blockade with losartan blunted the stretch-induced NGF down-regulation. Specific calcineurin inhibition with cyclosporine A and FK506 or NFAT inhibition with 11R-VIVIT reversed the stretch or alpha-1-adrenergic induced decrease of NGF. Calcineurin over-expression increased NFAT-DNA binding activity and decreased NGF expression. The magnitude of NGF decrease was sufficient to reduce neurite outgrowth of cultured sympathetic neurons. In conclusion, mechanical stretch and alpha-1-adrenergic stimulation contribute to a decrease of cardiomyocyte NGF expression via the calcineurin-NFAT pathway. To evaluate if the calcineurin-NFAT is critically involved in the pathogenesis of SNS further in-vivo studies in models of HF and MI are required. Nevertheless, the calcineurin-NFAT pathway may provide promising starting points for new pharmacological strategies to prevent SNS in the heart.     

Studies on carrageenan-induced arthritis in adult rats: presence of nerve growth factor and role of sympathetic innervation

Summary Recent studies effected by our Institute indicate that various forms of human arthritis express both immunohistochemically and biologically active nerve growth factor (NGF) in the synovium. In the present study, we used a model of carrageenan-induced arthritis to further evaluate the effects of joint inflammation on NGF level. These studies showed that experimentally-induced arthritis in rats caused a significant increase in NGF in the perivascular area of the synovium. We also showed that injection into the synovium of purified NGF did not cause inflammation per se and that the destruction of peripheral sympathetic innervation significantly reduced both the inflammation and the level of NGF following carrageenan injection.     

大鼠心肌梗死后神经生长因子受体的表达

目的:观察大鼠心肌梗死(MI)后不同时间点(1 d、7 d、30 d)神经生长因子(NGF)受体的表达情况。方法:采用开胸结扎冠状动脉法建立大鼠MI模型,在术后1 d、7 d、30 d处死动物,取出心脏,假手术大鼠(只开胸不结扎)作为对照研究。分别用实时定量PCR方法及Western blot方法检测梗死边缘区心肌中高亲和性受体酪氨酸激酶A(Trk A)和低亲和力神经受体P75(P75NTR)mRNA及蛋白的表达。结果:与对照组相比,MI后1 d,7 d,30 d,P75NTR mRNA及蛋白表达都没有明显差异,Trk A mRNA及蛋白表达在MI后7 d及30 d较对照组明显下降(P0.05),而且呈动态下降趋势。结论:大鼠MI后心脏NGF受体Trk A和P75NTR的表达发生了变化,P75NTR表达程度在NGF介导的MI后交感神经增生中不起决定作用。     

A vitamin D3 derivative (CB1093) induces nerve growth factor and prevents neurotrophic deficits in streptozotocin-diabetic rats

Abstract Aims/hypothesis. Streptozotocin-diabetic rats show impaired neurotrophic support by deficient nerve growth factor (NGF) in muscle and skin. We, therefore, examined a novel agent (CB1093; 1(S),3(R)-Dihydroxy-20(R)-(1-ethoxy-5-ethyl-5-hydroxy-2-heptyn-1-yl)-9,10-seco-pregna-5(Z),7(E),10(19)-triene), which induces expression of endogenous nerve growth factor. Methods. We gave CB1093 orally followed by measurements of mechanical nociception, nerve growth factor, neuropeptides (immunoassay) and nerve growth factor receptors (western blots). Results. In non-diabetic rats CB1093 caused dose-dependent increases in nerve growth factor production (140 % in soleus muscle and 190 % in sciatic nerve) and a mechanical hyperalgesia in the foot. There was also increased sciatic nerve expression of neuronal NGF target gene products, substance P (16 %) and calcitonin gene-related peptide (CGRP; 52 %). Depletions of nerve growth factor, substance P and CGRP in sciatic nerves of diabetic rats were prevented by CB1093, which also increased soleus nerve growth factor concentrations to 30 % over those seen in non-diabetic rats and increased its mRNA expression in skin. The CB1093 did not affect expression of nerve growth factor receptors (trkA and p75^NTR) in dorsal root ganglia in control or diabetic rats, though the p75^NTR expression was reduced by diabetes. The mechanical hyperalgesia seen in diabetic rats treated with vehicle was not exacerbated by CB1093. Conclusion/interpretation. These findings show that in animal models of diabetes it is possible to prevent depletions of nerve growth factor and the products of its neuronal target genes by oral treatment of a highly potent inducer of NGF gene expression. Pain is a possible side-effect, though this was a function of dose and was manifest more in controls than in diabetic rats. [Diabetologia (1999) 42: 1308–1313] Received: 23 April 1999 and in revised form: 20 July 1999     

Agonist of growth hormone–releasing hormone enhances retinal ganglion cell protection induced by macrophages after optic nerve injury

Optic neuropathies are leading causes of irreversible visual impairment and blindness, currently affecting more than 100 million people worldwide. Glaucoma is a group of optic neuropathies attributed to progressive degeneration of retinal ganglion cells (RGCs). We have previously demonstrated an increase in survival of RGCs by the activation of macrophages, whereas the inhibition of macrophages was involved in the alleviation on endotoxin-induced inflammation by antagonist of growth hormone–releasing hormone (GHRH). Herein, we hypothesized that GHRH receptor (GHRH-R) signaling could be involved in the survival of RGCs mediated by inflammation. We found the expression of GHRH-R in RGCs of adult rat retina. After optic nerve crush, subcutaneous application of GHRH agonist MR-409 or antagonist MIA-602 promoted the survival of RGCs. Both the GHRH agonist and antagonist increased the phosphorylation of Akt in the retina, but only agonist MR-409 promoted microglia activation in the retina. The antagonist MIA-602 reduced significantly the expression of inflammation-related genes Il1b, Il6, and Tnf. Moreover, agonist MR-409 further enhanced the promotion of RGC survival by lens injury or zymosan-induced macrophage activation, whereas antagonist MIA-602 attenuated the enhancement in RGC survival. Our findings reveal the protective effect of agonistic analogs of GHRH on RGCs in rats after optic nerve injury and its additive effect to macrophage activation, indicating a therapeutic potential of GHRH agonists for the protection of RGCs against optic neuropathies especially in glaucoma.

Optic neuropathies can occur in common and serious ocular complications, including glaucoma, traumatic injury, inflammation, ischemia, and tumors. They share a common pathology of degeneration of retinal ganglion cells (RGCs) and axonal loss. Optic neuropathies are leading causes of visual impairment and irreversible blindness, affecting more than 100 million people worldwide (1). There is no effective treatment for the complete functional recovery in optic neuropathies. Among them, glaucoma is the top cause of irreversible impaired vision affecting 5% of the world’s population across all races and regions (2). The principle of treatment for glaucoma is to minimize progression by lowering the intraocular pressure (3). There is no therapy to date that stops or reverses optic nerve (ON) damage in glaucoma so as to cure the disease (4).Mammalian retina has low intrinsic regenerative ability, and the presence of myelin-associated inhibitors, scar formation at the injury site, and lack of trophic support further contribute to the failure in regeneration after ON injury (5–7). Research aimed at enhancing the survival of RGCs and axonal regeneration has led to potential therapeutic strategies, including exogenous supplementation of neurotrophic factors (8, 9), inhibition of the apoptotic pathway (10), counteracting the inhibitory signals associated with myelin and glial scar formation (11), deletion of Pten and Socs3 genes (12), as well as stem cell therapy (13). Clinical applications of these findings are under development.Mild ocular inflammation induced by lens injury or zymosan-induced macrophage activation has been shown to promote the survival of RGCs and axonal regeneration (14, 15). Oncomodulin is the major mediator derived from activated macrophages and neutrophils in the inflammation-induced axonal regeneration (16, 17), with the elevation of cyclic adenosine monophosphate (cAMP). However, oncomodulin does not account for all promoting effects on axonal regeneration by lens injury. There are still undiscovered factors and mechanisms involved in the inflammation-induced neural recovery after ON injury.Growth hormone (GH)–releasing hormone (GHRH) controls the secretion of GH in the anterior pituitary, and GH regulates the synthesis of insulin-like growth factor-1 (IGF1) in the liver (18). GHRH signaling also exerts extrapituitary functions, including the involvement in tumor apoptosis (19). Our previous study in endotoxin-induced anterior uveitis rat model have shown that GHRH receptor (GHRH-R) is up-regulated in the ciliary body, macrophages, and leukocytes (20). The GHRH antagonist MIA-602 can reduce the infiltration of endotoxin-induced macrophages and leukocytes into aqueous humor by inhibiting inflammatory cytokine secretion, indicating the participation of GHRH signaling in the regulation of inflammatory responses (20). Notably, we observed the expression of GHRH-R in the RGC layer of the retina. While GH should be neuroprotective against excitotoxic-induced cell death in RGCs (21), the physiological role of GHRH-R signaling in RGCs remains elusive. We previously showed that GHRH agonists regulate the PI3K/Akt and MAPK/Erk pathways (22), which are important for RGC survival. Herein we hypothesized that GHRH-R signaling could be involved in the promotion of neural recovery after ON injury induced by inflammation. In this study, we investigated the effects of GHRH agonists and antagonists on RGCs after ON injury, and its involvement in the promotion of neural repair induced by inflammation after lens injury or zymosan-induced macrophage activation.     

神经生长因子治疗脑出血的临床疗效观察

目的观察鼠神经生长因子(NGF)治疗脑出血的临床疗效。方法选择脑出血患者119例,随机分为对照组63例和NGF组56例,在常规治疗基础上,2组分别给予胞磷胆碱或鼠NGF制剂治疗2周,分别于治疗后1、2周,采用美国国立卫生研究院卒中量表(NIHSS)评分评价2组患者的神经功能缺损情况,采用Barthel指数(BI)评价患者的日常生活活动能力。结果治疗后,2组患者的NIHSS评分均较治疗前显著降低(P<0.01),BI均较治疗前显著增加(P<0.01);治疗后1周,2组NIHSS评分及BI差异无统计学意义(P>0.05);治疗后2周,NGF组患者的NIHSS评分明显低于对照组,BI明显高于对照组(P<0.01)。结论 NGF治疗可以有效缓解脑出血患者的神经功能缺损,改善患者的日常生活活动能力。     

支气管哮喘儿童外周血神经生长因子水平变化及临床意义

目的研究支气管哮喘患儿血清神经生长因子水平及临床意义。方法研究分两组,研究组72例患支气管哮喘儿童,对照组80例无喘息发作支气管肺炎患儿,抽取患儿外周血,检测其血清神经生长因子水平。结果支气管哮喘组患儿外周血神经生长因子浓度为92.6±32.5 pg/ml,轻度、中度、重度哮喘临床发作患儿血神经生长因子浓度分别为53.2±18.5 pg/ml、97.8±22.7 pg/ml、162.4±28.6 pg/ml,血清NGF水平与临床发作程度呈正相关(r=0.62;P0.05)。对照组支气管肺炎患儿外周血NGF浓度为32.8±12.6 pg/ml,低于支气管哮喘儿童外周血神经生长因子水平(P0.001)。结论支气管哮喘儿童外周血NGF明显升高,并与临床发作程度正相关。     

The insulin-like growth factor system and colorectal cancer: clinical and experimental evidence

Objective The aim of this review is to clarify the involvement of the insulinlike growth factor (IGF) system in the development of colorectal malignancy.Materials and methods Medline searches were used to identify key articles relating the IGF system with the development of colorectal cancer.Results The IGF system has been linked to colorectal malignancy by a convergence of data from epidemiological, clinical and laboratory-based sources.Conclusion Further work is needed to characterise the IGF system expression in the colon. Such clarification could lead to the identification of targets that can be manipulated for clinical advantage.     

鼠神经生长因子联合电子温针治疗糖尿病周围神经病变的疗效观察

目的观察鼠神经生长因子联合电子温针治疗糖尿病周围神经病变(Diabetic perineuropathy,DPN)的临床疗效。方法 50例DPN患者随机分为A、B 2组,A组为鼠源性神经生长因子+电子温针治疗组,B组为单纯甲钴胺治疗组,观察两组治疗前后的临床疗效、运动神经传导速度(MNCV)、感觉神经传导速度(SNCV)的改善情况、多伦多临床评分系统(Toronto clinical scoring system,TCSS)的评分情况及不良反应发生情况。结果 A组总有效率为88.00%,高于B组52.00%,差异具有统计学意义(P0.01);2组治疗前后MNCV、SNCV较治疗前均改善,差异有统计学意义(P0.05);治疗后A组改善情况优于B组,差异有统计学意义(P0.05);2组治疗前后TCSS评分均明显降低,差异有统计学意义(P0.01),治疗后A组改善情况优于B组,差异有统计学意义(P0.05)。结论鼠神经生长因子联合电子温针治疗DPN变疗效确切,是治疗DPN的一种较好的方法,值得在临床上推广应用。     

外源性神经生长因子治疗急性脑梗死疗效观察

目的 观察恩经复治疗急性脑梗死的疗效.方法 将215例急性脑梗死患者随机分为治疗组和对照组,两组基础用药相同.其中治疗组115例,除基础用药外,加外源性鼠神经生长因子（恩经复）每日1次肌注,每次9000 IU.10天为一疗程,共用两个疗程;对照组100例,仅用基础用药.使用美国国立卫生研究所脑卒中评分（NIHSS）评价神经功能恢复情况.结果 （1）治疗30天后,恩经复治疗组的NIHSS评分较对照组有明显改善（P＜0.05）;治疗组有效率（80.87%）高于对照组（67%）,差异有统计学意义（P＜0.05）.（2）两组均未发生明显不良反应.结论 恩经复治疗急性脑梗死安全有效.     

Aldose reductase in the BB rat: isolation,immunological identification and localization in the retina and peripheral nerve

Summary Aldose reductase was purified from testis of nondiabetic BB rats using DEAE cellulose, hydroxylapatite and sephadex G-100 column chromatography. The molecular weight of the isolated enzyme was found to be 36,500±1000. Antibody against the isolated enzyme was raised in rabbits. It was purified by affinity chromatography, characterised by double immunodiffusion and Western blot analysis and used to localize the enzyme in retina and in peripheral nerve of the BB rat. In the retina, aldose reductase immunoreactivity was seen in the ganglion cells, Müller cell processes, retinal pigment epithelium and in the pericytes and endothelial cells of retinal capillaries. In peripheral nerve, aldose reductase immunoreactivity was found in the paranodal cytoplasm of Schwann cells and in pericytes and endothelial cells of endoneurial capillaries.     

Effects of nerve growth factor on expression of GAP-43 in right atria after sympathectomy in diabetic rats

SUMMARY
Aim   The present study investigated the role of nerve growth factor (NGF) in the regeneration of noradrenergic nerves of right atria (following 6-hydroxydopamine; 6-OHDA, 100 mg/kg, i.p.) from non-diabetic and 8-week diabetic rats.
Results   In cryostat sections of the right atria, GAP-43 immunoreactivity was concentrated in nerve terminals, preterminal axons of the endocardium, epicardium and myocardium, as well as in nerve fibres innervating the blood vessels and ganglionic cells. In serial sections, all positive staining for GAP-43 showed immunoreactivity for the neuronal marker PGP-9.5. In untreated non-diabetic rats, the total GAP-43 immunoreactivity was reduced to 60% relative to pretreatment levels, at day 14 after 6-OHDA, as quantified by Western blotting. In diabetic rats, 6-OHDA treatment produced a marked increase in the levels of total GAP-43 at days 28 and 49. NGF treatment (1 mg/kg, s.c., 3 times/week, for 2 weeks) had no effect on the level of total GAP-43 in right atria from non-diabetic and diabetic rats before treatment with 6-OHDA. However, it normalized the reduced GAP-43 immunoreactivity observed in 6-OHDA-treated non-diabetic rats. Interestingly, NGF treatment alone produced an increase in GAP-43 phosphorylation relative to total GAP-43 in right atria from both non-diabetic (44%) and diabetic groups (42%).
Conclusions   These findings suggest that nerve terminals of the right atria retain, in the mature adult, the capacity for structural and functional plasticity. The expression of GAP-43 in right atria of control and diabetic rats was differentially affected by 6-OHDA treatment. In injured noradrenergic neurones of the right atria, NGF modified the expression of GAP-43 only in non-diabetic rats and not in diabetic rats.     

神经生长因子在原发性肝癌中的表达及其临床意义

目的 观察神经生长因子(NGF)在原发性肝癌组织中的表达及在患者血清中的含量,了解血清NGF水平与肝癌临床病理特征的关系. 方法 26例肝细胞癌及对应癌旁组织,采用半定量PCR、免疫印迹及免疫组织化学分析NGF mRNA和蛋白的表达及其分布.健康对照者、慢性肝炎、肝硬化及肝癌患者共150例,采用酶联免疫吸附法(ELISA)检测血清NGF的水平,分析其临床意义.两组之间差异的显著性比较用t检验、Mann-Whitney U检验,多组之间差异的显著性比较应用Kruskal-Wallis H检验.数据均采用SPSS 11.5统计软件处理.结果 肝癌组织中NGF在mRNA及蛋白水平的表达高于癌旁组织,NGF主要分布于肝细胞胞质.血清NGF水平:肝癌组、肝硬化组、慢性肝炎组患者和正常对照组分别为(33.86±16.11)pg/ml、(20.57±9.73)pg/ml、(13.20±6.23)pg/ml和(11.13±6.12)pg/ml,肝癌组、肝硬化组显著高于慢性肝炎组患者和正常对照组,x2值分别为39.119和12.041,P值均＜0.01.肿瘤直径大于5cm及TNM分级为Ⅲ、Ⅳ级的肝癌患者血清NGF的水平高于肿瘤直径小于5cm及TNM分级为Ⅰ、Ⅱ级的患者.结论 NGF在肝癌组织中高表达,肝癌患者血清NGF水平明显升高,与肿瘤体积和TNM分级相关,提示NGF在肝癌的发生和发展过程中可能发挥一定作用,血清NGF有助于肝癌患者的病情评估和预后估计.     

Activated hepatic stellate cells overexpress p75NTR after partial hepatectomy and undergo apoptosis on nerve growth factor stimulation.

BACKGROUND: Expression of neurotrophins (NTs) and their receptors is increased during hepatic regeneration, but their role is not well understood. METHODS: NTs and their receptors were investigated by RT-PCR and immunostaining in regenerating livers after two-thirds hepatectomy (PH) and in hepatocytes and hepatic stellate cells (HSCs) isolated from regenerating livers in mice. Induction of apoptosis after treatment with NGF and the expression of alpha-smooth muscle actin (SMA), interleukin 6 (IL-6) and hepatocyte growth factor (HGF) were also investigated in regenerating HSCs. RESULTS: Nerve growth factor (NGF) and p75 NT receptor (p75NTR) mRNA were elevated after PH, while other NTs and NT receptors showed no remarkable change. NGF was detected in regenerating hepatocytes, but not in normal hepatocytes. Regenerating HSCs expressed increased p75NTR and SMA in vivo and showed an activated phenotype and the high expression of HGF and IL-6 in vitro. Enhanced cell death was seen in HSCs, both from normal and regenerating liver, after treatment with NGF. CONCLUSIONS: Although activated HSCs may produce the factors that regulate liver regeneration, the de novo NGF production by regenerating hepatocytes may induce the death of activated HSCs via p75NTR, leading to termination of hepatic regeneration.     

Targeting growth factor supply in keratopathy treatment: comparison between maternal peripheral blood and cord blood as sources for the preparation of topical eye drops

Background

Epitheliotrophic growth factors (GF) can be supplied topically to patients with severe keratopathy through a variety of blood-derived products. We compared GF content in adult peripheral blood serum (PB-S) and cord blood serum (CB-S) as potential sources of GF. To limit inter-individual variability the assessment was performed in maternal-child pairs at the time of delivery.

Material and methods

The amounts of epidermal GF (EGF), insulin-like GF (IGF), transforming GF-beta (TGF-β), vascular endothelial GF (VEGF) in CB units collected from the umbilical vein and PB from mothers (each group n=30) were estimated by enzyme-linked immunosorbent assays. Obstetric characteristics and haematological data were recorded from the archives of the Emilia Romagna Cord Blood Bank. Statistical evaluations were performed by Wilcoxon’s test and correlations between variables were determined using Spearman’s (ρ) coefficient; p-values <0.05 were considered statistically significant.

Results

EGF, TGF-β and VEGF levels were significantly higher in CB-S than in PB-S (median 1,254.4 vs 646.0 pg/mL, 51.3 vs 38.4 μg/mL and 686.8 vs 30 pg/mL, respectively; all p<0.0001) whereas IGF content was significantly higher in PB-S than in CB-S (159.9 vs 53.5 pg/mL, respectively; p<0.0001). In CB-S, the CD34⁺ cell concentration appeared to be related to EGF, IGF and TGF-β levels whereas white blood cell count appeared to be related to EGF and TGF-β levels. VEGF levels showed no relation to the haematological parameters considered. Platelet counts were not related to GF level in either CB or PB.

Discussion

The GF content in the two blood sources was different, with CB containing larger amounts. Each GF selectively regulates cellular processes involved in corneal healing, so the use of PB or CB should be targeted to supply specific GF on the basis of the type and severity of the keratopathy.