改良聚羟乙基丙烯酸甲酯-聚甲基丙烯酸甲酯一体化人工角膜植入兔与猴角膜的实验研究(英文)

目的:探讨聚羟乙基丙烯酸甲酯(PHEMA)海绵支架材料与角膜组织的生物相容性,评价改良聚羟乙基丙烯酸甲酯(PHEMA)—聚甲基丙烯酸甲酯(PMMA)一体化人工角膜植入兔与猴角膜的初步临床效果。方法:通过两个阶段化学聚合结合车床机械切削合成改良的一体化人工角膜。实验分为两个部分完成。第1部分(A组):将PHEMA海绵边裙材料植入10只正常兔角膜板层间,术后2,4wk及2,3,4mo分别行组织病理、免疫组织化学及电镜检查,观察海绵边裙与角膜组织的生物学愈合情况。第2部分(B组):8只兔眼和2只猴眼角膜囊袋内I期植入一体化人工角膜,术后临床观察材料与组织的愈合情况;术后3mo时行Ⅱ期手术切除术眼角膜中央前板层角膜组织,暴露人工角膜中央镜柱,术后随访时间3 ～6mo,初步观察临床治疗效果。结果:1)A组接受角膜板层间边裙植入术的10只兔眼,随访期间未见并发症:组织病理学显示,PHEMA海绵边裙材料植入术后2wk始有成纤维细胞长入,术后2 ～3mo时多量成纤维细胞长入并伴有新生血管生长;免疫组织化学显示,海绵孔隙中长入的细胞和角膜基质细胞均对波形蛋白免疫反应呈阳性;电镜下可见,成纤维细胞在海绵材料间隙中生长,细胞生长状态良好,并分泌胶原和细胞外基质。2)B组接受一体化人工角膜移植术的,6只兔眼一体化人工角膜均在位,另2只兔眼I期术后有前板层角膜基质融解。接受一体化人工角膜移植术的2只猴眼,I期和Ⅱ期术后均未见明显并发症。结论:PHEMA海绵能与角膜组织良好的生物学愈合;改良PHEMA-PMMA一体化人工角膜植入术后能获得相对稳定的治疗效果。     

改良聚羟乙基丙烯酸甲酯-聚甲基丙烯酸甲酯一体化人工角膜植入兔与猴角膜的实验研究

目的：探讨聚羟乙基丙烯酸甲酯（PHEMA）海绵支架材料与角膜组织的生物相容性,评价改良聚羟乙基丙烯酸甲酯（PHEMA）一聚甲基丙烯酸甲酯（PMMA）一体化人工角膜植入龟与猴角膜的初步临床效果。方法：通过两个阶段化学聚合结合车床机械切削合成改良的一体化人工角膜。实验分为两个部分完成。第1部分（A组）：将PHEMA海绵边裙材料植入10只正常兔角膜板层间,术后2,4wk及2,3,4mo分别行组织病理、免疫组织化学及电镜检查,观察海绵边裙与角膜组织的生物学愈合情况。第2部分（B组）：8只兔眼和2只猴眼角膜囊袋内I期植入一体化人工角膜,术后临床观察材料与组织的愈合情况;术后3mo时行Ⅱ期手术切除术眼角膜中央前板层角膜组织,暴露人工角膜中央镜柱,术后随访时间3～6mo,初步观察临床治疗效果。结果：1）A组接受角膜板层间边裙植入术的10只兔眼,随访期间未见并发症：组织病理学显示,PHEMA海绵边裙材料植入术后2wk始有成纤维细胞长入,术后2～3mo时多量成纤维细胞长入并伴有新生血管生长;免疫组织化学显示,海绵孔隙中长入的细胞和角膜基质细胞均对波形蛋白免疫反应呈阳性;电镜下可见,成纤维细胞在海绵材料间隙中生长,细胞生长状态良好,并分泌胶原和细胞外基质。2）B组接受一体化人工角膜移植术的,6只兔眼一体化人工角膜均在位,男2只兔眼I期术后有前板层角膜基质融解。接受一体化人工角膜移植术的2只猴眼,I期和Ⅱ期术后均未见明显并发症。结论：PHEMA海绵能与角膜组织良好的生物学愈合;改良PHEMA-PMMA一体化人工角膜植入术后能获得相对稳定的治疗效果。     

改良聚羟乙基丙烯酸甲酯-聚甲基丙烯酸甲酯一体化人工角膜植入兔与猴角膜的研究

目的 探讨聚羟乙基丙烯酸甲酯（PHEMA）海绵支架材料与角膜组织的生物相容性,评价改良PHEMA-聚甲基丙烯酸甲酯（PMMA）一体化人工角膜植入兔与猴角膜的初步临床效果。方法通过两个阶段化学聚合结合车床机械切削合成改良的一体化人工角膜。实验分为两个部分完成。第1部分（A组）：将PHEMA海绵边裙材料植入10只正常兔角膜板层间,术后2、4周及2、3、4个月分别行组织病理、免疫组织化学及电镜检查,观察海绵边裙与角膜组织的生物学愈合情况。第2部分（B组）：8只兔眼和2只猴眼角膜囊袋内Ⅰ期植入一体化人工角膜,术后临床观察材料与组织的愈合情况;术后3个月时行Ⅱ期手术切除术眼角膜中央前板层角膜组织,暴露人工角膜中央镜柱,术后随访时间3—6个月,初步观察临床治疗效果。结果（1）A组：10只兔眼随访期间未见并发症。组织病理学显示,PHEMA海绵边裙材料植入术后2周始有成纤维细胞长入,术后2—3个月时多量成纤维细胞长入并伴有新生血管生长;免疫组织化学显示,海绵孔隙中长入的细胞和角膜基质细胞均对波形蛋白免疫反应呈阳性;电镜下可见,成纤维细胞在海绵材料间隙中生长,细胞生长状态良好,并分泌胶原和细胞外基质。（2）B组：6只兔眼的一体化人工角膜均在位,另2只兔眼Ⅰ期术后有前板层角膜基质融解。2只猴眼于Ⅰ期和Ⅱ期术后均未见明显并发症。结论PHEMA海绵能与角膜组织良好的生物学愈合;改良PHEMA—PMMA一体化人工角膜植入术后能获得相对稳定的治疗效果。（中华眼科杂志．2007,43：602-607）     

改良PHEMA-PMMA一体化人工角膜治疗兔角膜碱烧伤的实验研究

目的评价改良聚羟乙基丙烯酸甲酯（PHEMA）-聚甲基丙烯酸甲酯（PMMA）一体化人工角膜时兔角膜碱烧伤的治疗效果。方法通过两阶段化学聚合结合车床机械切削合成改良的一体化人工角膜。将PHEMA海绵边裙材料植入10只正常兔角膜板层间，另10只碱烧伤兔眼角膜囊袋内1期植入一体化PHEMA—PMMA人工角膜，术后用组织病理、免疫组织化学和电镜检查观察海绵边裙与角膜组织的生物学愈合情况。术后2个月行II期手术．术后随访观察3～6个月。结果接受板层间植入术的10只兔角膜未见任何并发症。术后2周成纤维细胞长入PHEMA海绵，术后2～3个月多量细胞长入伴有新生血管；海绵孔隙中生长细胞和角膜基质细胞波形蛋白（Vim）免疫反应阳性；电镜下，细胞在海绵材料间隙中生长状态良好，并分泌胶原和细胞外基质。接受人工角膜移植术的10只碱烧伤兔眼中，1眼1期术中角膜穿孔而被排除，另9眼Ⅱ期术后，3眼2周内人工角膜镜柱偏位，6眼观察期间人工角膜在位。结论PHEMA海绵能够与角膜组织达到良好的生物学愈合；人工角膜的片型和整体强度有待进一步改良。     

长期干燥保存角膜穿透移植实验与应用

利用干燥保存７～５７天及１６～３０个月的兔用膜，行同种异体部分穿透角膜移植后，前者３７眼中，透明愈合８眼，后者９眼中，透明愈合３眼。保存１０～５７天猴眼角膜移植后，观察的９猴（９眼）中，透明愈合３眼，半透明２眼。用干燥保存４个月～４年９个月人角膜，行挽救眼球穿透移植的６眼中，观察２６天到１年６个月，透明愈合２眼，半透明３眼。提示：在临床上，干燥保存角膜有可能作为穿透角膜移植材料应用。     

同一猴、兔眼角膜细胞成分原代培养的实验研究

目的建立从同一角膜片上分离培养角膜上皮、基质和内皮细胞的方法,为研究角膜细胞间的相互作用机制奠定基础。方法采用消化法分离培养猴眼角膜内皮细胞、上皮细胞和兔眼角膜上皮细胞．组织块培养法培养基质成纤维细胞和兔角膜内皮细胞。细胞接种于12孔培养板,并于培养不同时间行Wright染色检查细胞生长情况。结果采用消化法和组织块培养法能成功地培养猴、兔角膜上皮、基质和内皮细胞。猴、兔角膜内皮细胞培养1wk后,均能形成内皮细胞单层,细胞类似天然的六角形态．且以猴内皮细胞明显;猴角膜上皮细胞培养3～4d生长旺盛、但难以形成细胞单层,兔上皮细胞生长旺盛,1wk后已达融合状态,细胞呈膜状伸出板层伪足;猴、兔基质成纤维细胞易培养,1wk后已融合成单层细胞,细胞排列整齐,类似纤维走行样外观。结论从同一角膜材料中能分别培养出角膜3种细胞成份,消化法和组织块培养法相结合既能节省材料,又简单易行。     

Estrogen up-regulation of metalloproteinase-2 and -9 expression in rabbit lacrimal glands

Increased levels of the matrix metalloproteinases (MMPs)-2 and -9 have been found in tear fluids of patients with dry eye disease, suggesting that these MMPs may be implicated in the pathogenesis of this disease. One of the main causes of dry eye disease is lacrimal gland insufficiency. However, the contribution of the lacrimal gland (LG) to the expression and production of MMP-2 and MMP-9 in tears is not known. Since dry eye disease occurs more frequently in women, sex hormones, especially estrogens, have also been implicated in the pathogenesis of this disease. Estrogens have been shown to regulate the synthesis levels of MMP-2 and MMP-9 in several tissues, Thus, the purpose of these studies was to determine if: (1) rabbit lacrimal glands secrete MMP-2 and MMP-9; (2) MMP-2 and MMP-9 are produced by lacrimal epithelial cells and/or lacrimal lymphocytes; and (3) the expression, activity and level of these enzymes are regulated by sex hormones. Lacrimal epithelial cells (LEC) and lacrimal lymphocytes (LL) from sexually mature New Zealand White female rabbits were isolated, purified and cultured with and without 10(-6)M dihydrotestosterone (DHT) or 10(-6), 10(-8), 10(-9) and 10(-10)M 17beta-estradiol (E2). The culture supernatants were analyzed by zymography and western blotting (WB) using polyclonal anti-human MMP-2 and MMP-9 antibodies. LGs were also collected from rabbits 7 days after being sham-operated, ovariectomized (OVX), OVX treated with 4 mg/kg DHT, and OVX treated with 0.5 mg/kg of E2. LGs were collected and processed for RNA extraction as well as protein determination using WB and immunocytochemistry. The pro-forms of MMP-2 and MMP-9 were detected in primary LEC and LL culture medium by zymography and WB. Pro-MMP-2 and pro-MMP-9 were also detected at the gene and protein levels in the lacrimal glands of all four treatment groups, with the highest levels and gene expression found in the estrogen-treated group. These results suggest that both pro-MMP-2 and pro-MMP-9 are secreted by the lacrimal gland and appear to be up-regulated by estrogen. The role of the lacrimal MMPs in the pathogenesis of dry eye disease needs to be further investigated.     

Cryopreservation of rabbit and cat corneas at -18 to -24 degrees C.

A simple method of corneal cryopreservation, in which corneas were frozen at -18 to -24 degrees C, was examined. Rabbit and cat corneas were placed successively in solutions of 50% fetal calf serum in McCarey-Kaufman medium with an increasing glycerol and glucose content. They were then frozen and stored in a -20 degrees C domestic freezer. Rabbit corneas stored in this way were examined in vitro by light and scanning electron microscopy, and both rabbit and cat corneas were also assessed after orthotopic allotransplantation into adult recipient animals. Functional corneal grafts were obtained with rabbit and cat tissue that had been cryopreserved for 3-4 weeks and 1 week, respectively. Endpoint analysis (by light and scanning electron microscopy) of grafts that had survived for 50 days indicated the presence of an intact corneal endothelial monolayer. The corneal endothelium slowly degenerated as the storage time was increased. Importantly, however, the endothelium appeared to withstand the freezing and thawing processes and we conclude that it may be possible to store corneas at temperatures above -196 degrees C, without the need for complex, low-temperature cryopreservation systems.     

Distribution of fibronectin in human and rabbit corneas

In order to study the possible role of fibronectin (FN) in corneal wound healing and the relationship between FN and sensory innervation, FN was demonstrated immunohistochemically in both normal and sensorily denervated rabbit corneas and in normal or tissue-cultured human corneas. The distribution of FN was the same in the groups examined: a thin subepithelial band of FN-like immunoreactivity was seen at the level of epithelial basement membrane and at the stromal side of Descemet's membrane. Epithelial abrasions were also performed in both normal and denervated rabbit corneas. The results were compared with those obtained from organ-cultured human corneas. Following abrasion of the corneal epithelium, FN was detected in the anterior margin of the denuded stroma 18 hr after the operation in the areas where the epithelium had not healed, but not 49 hr after. Sensory denervation did not affect the distribution of FN in normal, denervated or healing rabbit cornea. It is concluded that FN is probably not controlled by sensory innervation.     

直角边缘人工晶状体预防兔后囊膜混浊的实验研究

目的探讨直角边缘人工晶状体（intraocular lens，IOL）预防后囊膜混浊（posterior capsule opacification，PCO）的作用。方法30只新西兰兔进行超声乳化晶状体摘出联合囊袋内IOL植入术后，随机植入Crane OV-55CP、Crane OV-55C、Alcon TYPE 5C 3种IOL之一。观察术后并发症和PCO情况。术后3月行光镜和透射电镜检查，观察晶状体后囊膜的形态学变化。结果术后3月Crane OV-55CP组的PCO程度比Crane OV-55C和Alcon TYPE 5C组轻（P〈0．05），各组Soemmefing环形成程度无差异（P〉0．05）。病理学检查发现Crane OV-55CP组兔赤道部增生的晶状体上皮细胞在人工晶状体的直角边缘处受到了阻挡。Crane OV-55C和Alcon TYPE 5C组大量晶状体上皮细胞迁移至后囊膜。结论直角边缘IOL延缓了兔PCO的发生、发展，是预防PCO简便安全有效的方法。     

Stromal damage in rabbit corneas exposed to CO2 laser radiation

Threshold damage to the cornea from CO2 lasers is confined to the epithelium. Exposures well above the threshold for epithelial damage produce bowl-shaped stromal wounds. Light and electron microscopy and slit-lamp photographs all show a sharp demarcation between the damaged and undamaged regions 48 hr after exposure. The micrographs also show that the damaged region is accellular. Calculations of the expected temperature increases combined with analyses of slit lamp photographs show that the wound boundary corresponds to a surface of equal peak temperature increase. Comparisons with epithelial and endothelial damage conditions suggest that stromal, endothelial and epithelial cells have essentially the same thermal damage mechanism.     

N -acetylcysteine and Taurine inhibit hyperoxia - induced cataract in rabbit lens

AIM: To investigate the efficacy of N-acetylcysteine (NAC) and Taurine (Tau) in preventing hyperoxia-induced the lens opacification and the changes of biochemical parameters on organ cultured rabbit lenses. METHODS: Twenty-four lenses from adult rabbits were divided into the control group, the hyperoxia-exposed group, the hyperoxia-exposed group containing 20mmol/L of NAC, the hyperoxia-exposed group containing 80mmol/L of Tau, respectively. The treated groups incubated with hyperoxia (PO² >80%) for 4 hours per day throughout a 7-day period. Lens transparency, histology and enzymatic activities measurements were determined after this incubation. RESULTS: Gross morphological examination of these lenses revealed some severe cortical opacification in the hyperoxia-exposed group, moderate cortical opacification in the control group and the Tau treated group. There was minimal cortical opacification in the NAC treated group. The glutathione (GSH) content and the activity of Na, K-ATPase were significantly decreased in the hyperoxia-exposed group than that of the control group, by 37.8% ( P<0.05) and 53.5% ( P<0.05), respectively. However, they were increased in the two treated groups, especially in the NAC treated group. There were no significant differences in the water-soluble protein content and the catalase and GSH reductase activities in all group lenses. CONCLUSION: Hyperoxia can induce the cortical opacification in the lens. The role of NAC in the prevention of hyperoxia-induced cataract is superior to Tau.     

Implantation of a toric poly(methyl methacrylate) intraocular lens to correct high astigmatism

A 57-year-old man experienced a decrease in visual function because of cataract formation. Corneal astigmatism was 13.4 diopters (D) because he had had a penetrating keratoplasty 27 years before. Cataract surgery was planned, and biometric data for toric intraocular lens (IOL) implantation were collected for the manufacture of a custom IOL. After phacoemulsification, a toric poly(methyl methacrylate) (PMMA) IOL of +19.0 D spherical and +12.0 D cylindrical power was implanted via a sclerocorneal tunnel incision. Three months postoperatively, corneal astigmatism was 14.3 D and best corrected visual acuity (BCVA), 20/25. Postoperative refraction (+1.5 -3.0 x 90) and BCVA remained stable for 7 months. No significant IOL rotation was observed. Implantation of a toric PMMA IOL corrected high corneal astigmatism. Toric IOL technology with high cylindrical power allows enhancement of IOL surgery.     

Spontaneous degenerative maculopathy in the monkey

Maculopathy has many varied facets. The subhuman diurnal primate demonstrates some of the changes occasionally seen in clinical practice. Examination of 574 diurnal subhuman primates, mostly older rhesus monkeys, revealed the following: drusen-like bodies in the macula and frequently elsewhere in the eye grounds of 5.9%; crescent shaped lesions suggestive of choroidal rupture; and macular and perimacular lesions associated with myopia and complicated by choroidal neovascular ingrowth.     

KGF-2治疗兔角膜中央碱烧伤的实验研究

目的　观察角质细胞生长因子 2 (KGF 2 )对实验性兔角膜中央碱烧伤的治疗效果。方法　将 2 4只兔角膜碱烧伤眼随机分成 4组 ,用KGF 2滴眼液治疗 ,以后行荧光素染色裂隙灯显微镜照相 ,照片经计算机图象系统分析 ,角膜切片HE染色 ,光镜检查。结果　 10 0 μg/mLKGF 2角膜上皮愈合率最高 ,且与剂量呈依赖性 ,各治疗组与对照组之间差异均有显著性意义 ,P <0 0 5。结论　KGF 2能够促进兔角膜碱烧伤上皮的愈合     

Beta-adrenergic stimulation of Na(+)-K(+)-2Cl(-) cotransport activity in the rabbit lens

Experimental maneuvers known to increase cellular cAMP levels evoked a stimulation in the K(+) influx across the anterior surfaces of isolated rabbit lenses, as measured by 86Rb(+) uptake. For this, the lenses were mounted in a modified Ussing-type chamber and exposed to the radiolabel under short-circuit conditions. The enhanced, cAMP-elicited flux was attributed to the basolateral Na(+)-K(+)-2Cl(-) cotransporter given its preclusion by bumetanide, a highly selective inhibitor of this symport, and the ineffectiveness of ouabain in mitigating the stimulation. The ouabain- plus bumetanide-insensitive K(+) uptake, which is about 10% of the total influx and represents passive entry of the radiolabel, was not affected by cAMP-elevating conditions. Forskolin, an activator of adenylyl cyclase; epinephrine, a non-selective adrenergic agonist; and the beta-selective agents, isoproterenol and terbutaline, were among the drugs used to elicit the increase in bumetanide-sensitive K(+) inflow. In experiments with isoproterenol, the stimulated influx evoked by the agonist was inhibited in lenses simultaneously exposed to propranolol. Other observations included that the stimulation of bumetanide-sensitive K(+) influx with forskolin was eliminated in lenses pretreated with the protein kinase inhibitors, staurosporine or H-89. However, these drugs were ineffective in preventing the increased influx produced by calyculin A, a phosphatase inhibitor, suggesting modulation of the cotransporter by at least two independent pathways. The cAMP-generating stimuli also produced an inhibition of the short-circuit current across the lens and an increase in translens resistance. These latter effects suggest that cAMP elevation also evokes an inhibition in an epithelial conductance(s) simultaneously to the stimulation of the cotransporter. As such, this study provides the first indication for the regulation of lens transport by adrenoceptors, presumably of the beta-2 subtype.     

In vivo effects of fluoroquinolones on rabbit corneas

Purpose: The use of topical fluoroquinolones to treat microbial keratitis is associated with an increased incidence of corneal perforation compared to other standard treatments. This study examined the effects of topical fluoro­quinolones on corneal collagen and keratocytes in intact rabbit corneas and corneas with an epithelial defect. Methods: Studies consisted of one group of intact corneas and one group of corneas where a 6‐mm epithelial defect was created with a surgical scrape. Within each group, eyes were randomly assigned to one of four topical medications (0.3% ciprofloxacin, 0.3% ofloxacin, fortified antibiotics (1.36% tobramycin, 5% cefrazolin) or Tears Naturale (Alcon Laboratories, Frenchs Forest, NSW, Australia). Two drops were instilled hourly for 48 h and then 2‐hourly for an additional 48 h. At 96 h the corneas were removed and processed for light microscopy, immunohistology for collagen IV, V and VI, and apoptosis staining. Results: In intact rabbit corneas there was no demonstrable difference between treatment groups. In corneas with an epithelial defect, both fluoroquinolones delayed epithelial healing when compared to fortified antibiotics or tears. Keratocyte loss was seen in all groups and was greatest in the ofloxacin group. Median stromal thickness with keratocyte loss were: ofloxacin 30%; ciprofloxacin 10%; fortified antibiotics 7.5%; and tears 15% (ofloxacin vs tears, Mann?Whitney = 16.0, P = 0.09). Keratocyte loss did not correlate with the amount of demonstrable apoptosis. Collagens IV, V and VI showed no differences between treatments. Conclusions: These results suggest that ofloxacin is potentially cytotoxic to corneal keratocytes. Such an effect could lead to the observed increased incidence of corneal per­foration in microbial keratitis.     

Effects of aFGF and bFGF on wound healing in rabbit corneas

After the debridement of the entire corneal epithelium of the rabbit eye, epithelial cells of conjunctival origin cover the denuded corneal surface. Under such experimental conditions, the rate of wound healing is considerably delayed and total regeneration is completed within 15 to 20 days, allowing evaluation of various drugs, such as the Fibroblast Growth Factor. Both acidic and basic FGF were administered topically on totally de-epithelialized rabbit eye, at three different concentrations of 1.5 and 10 Stimulation Units/50 microliters, 3 times per day. A dose-response effect was observed and in each case, acidic FGF was found to be much more potent than bFGF in increasing the rate of wound healing of the cornea. These results are correlated with a new purification procedure, avoiding acid treatment of the tissue extract. The systemic diffusion of FGF allows the contralateral eye cells to be also stimulated for mitosis and migration and to heal faster than the control eyes.     

Electron microscopic study of the endothelium of stored and cryopreserved monkey corneas

Summary Monkey corneas were either cryopreserved at a controlled rate over liquid nitrogen in a solution containing dimethylsulfoxide, sucrose and serum albumin, or were stored in moist chambers at 4°C for 48 hours. The endothelium was examined by scanning and transmission electron microscopy and the results compared. The entire endothelial layer of cells was always intact and there was little evidence of freeze-thaw-induced structural damage in the cryopreserved corneas. Mitochondrial swelling and clumping of nuclear chromatin was observed in the stored corneas.

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Zusammenfassung Hornhäute von Rhesusaffen (Macaca arctoides) wurden entweder nach der Methode von Kaufman und Capella gefrierkonserviert oder in feuchten Kammern bei +4°C für 48 Std gelagert. Anschließend wurde das Endothel elektronenmikroskopisch untersucht. Die vergleichende Auswertung ergab, daß die gesamte endotheliale Zellschicht immer intakt war. Die gefrierkonservierten Hornhäute waren in ihrer Struktur durch den Gefrierprozeß nur geringfügig geschädigt. In den bei + 4° C gelagerten Hornhäuten waren Schwellung der Mitochondrien und Verklumpung des Kernchromatins zu beobachten.

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This investigation was conducted while Dr. Waller was a Fellow in the Departments of Ophthalmology and Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin U.S.A. This work was supported in part by Deutsche Forschungsgemeinschaft Bonn-Bad Godesberg (WA 298/1) (Dr. Waller); and by a grant (EY 00625-01A1) from the United States Public Health Service-National Eye Institute, Bethesda, Maryland; and by Designated Research Funds from the Veterans Administration (Dr. Van Horn).