Immunobiology of xenogeneic cornea grafts in mouse eyes. I. Fate of xenogeneic cornea tissue grafts implanted in anterior chamber of mouse eyes

BACKGROUND: The cornea is an immune privileged tissue that, when grafted orthotopically, forms the anterior surface of the immune privileged anterior chamber. We have recently reported that allogeneic cornea fragments implanted in the anterior chamber of mouse eyes resist immune rejection, although such graft fragments are rejected outside the eye. We wished to determine the extent to which xenogeneic cornea fragments placed in the eyes of normal mice are vulnerable to immune rejection. METHODS: Guinea pig corneas, deprived surgically of epithelium, were cut into fragments and inserted into the anterior chamber of eyes of BALB/c and severe combined immune deficient (SCID) mice, adjacent to the central cornea of the recipient. The fate of the grafts was assessed clinically by biomicroscopy and histologically for 8 weeks postimplantation. RESULTS: The majority of guinea pig cornea fragments devoid of epithelium came to rest with the raw stroma adjacent to recipient endothelium. These fragments remained clear for the 8-week observation interval in both BALB/c and severe combined immune deficient mice. Clear grafts displayed viable guinea pig keratocytes and endothelial cell layers for 4 weeks. The endothelium was then replaced by murine cells by 8 weeks. A minority of guinea pig cornea fragments were oriented with donor endothelium adjacent to recipient endothelium. Although these grafts in severe combined immune deficient eyes eventually acquired an endothelial layer that faces the anterior chamber and remained clear, similar fragments in BALB/c eyes became opaque, failed to acquire a proper lining of endothelium that faces the anterior chamber, and incited an inflammatory reaction in adjacent recipient cornea. CONCLUSIONS: Immune privilege is afforded to xenografts of guinea pig cornea placed as stromal: endothelial cell fragments in the anterior chamber of mouse eyes, but only if the surface of the fragments that faces the anterior chamber is promptly covered with corneal endothelium. The possible roles of corneal endothelium in promoting immune privilege of corneal xenografts are discussed.     

Micron-resolution ranging of cornea anterior chamber by optical reflectometry.

Noncontact, high resolution measurements of anterior eye structures using optical coherence domain reflectometry are described. Distances between intraocular structures are measured by directing a beam of short coherence length light onto the eye and performing an interferometric measurement on the optical group delay of reflected signals. Measurements of corneal thickness, corneal excision depth, and anterior chamber depth are demonstrated in vitro, and the location of tissue boundaries is resolved to within +/- 2 microns. The full-width-half-maximum longitudinal resolution is 10 microns. Sensitivities to reflected signals as small as 10(-10) of the incident power are achieved by heterodyne detection.     

Pupil size in relation to ultrasonic anterior chamber depth measurements in pseudophakic eyes

The effect of pupil size on ultrasonically determined anterior-chamber depth was studied in vitro with the Storz Alpha 20/20 and with the Kretz ultrasonoscope at high and low system sensitivity. The traces were measured peak to peak and base to base; results were compared to anterior-chamber depth measurements with optical pachymetry. We conclude that when measuring anterior-chamber depth with ultrasound, the pupil must be fully dilated or reduced system sensitivity must be used and the traces measured peak to peak.     

Ultrastructural study of foetal and adult mouse renal tissue implanted in testis.

Kidneys of mice foetuses 15, 17, 19 days old, as well as kidneys of mice 1 and 4 weeks old of the pure C3H/SY species, were implanted into the right testis of 40 adult mice 1.5 to 2 months old, of the same species. The animals were sacrificed after 30 and 60 days and the evolution and development of the renal implants within the testis ware studied. The findings in the light as well as in the electron microscope, showed that the renal implants presented the histologic characters of nephroblastoma, which became clearer the more prolonged the time of the implantation was.     

Toward the survival and function of xenogeneic hepatocyte grafts.

Xenogeneic hepatocyte transplantation might offer an unobtrusive alternative to whole liver allotransplantation. Having previously found that the immune response to such grafts can be controlled by immunosuppression, we sought approaches to collection and delivery that would optimize survival and function after transplantation. Porcine hepatocytes were isolated by a 2-step collagenase technique and then: 1) used immediately; 2) stored in University of Wisconsin (UW) solution at 4 degrees C; 3) cultured in supplemented Williams E medium; or 4) cryopreserved in UW solution with 10% dimethyl sulfoxide (DMSO). The fate and function of the hepatocytes was determined after they were injected into the spleens of immunodeficient mice. Freshly isolated hepatocytes had better viability (92.2 +/- 1.9%) than hepatocytes cultured for 24 hours (78.4 +/- 6.3%), hypothermically preserved in UW solution for 24 hours (85.8 +/- 3.1%), or cryopreserved (65.0 +/- 2.6%). Freshly isolated hepatocytes secreted more albumin after transplantation than hepatocytes that were cultured, hypothermically stored, or cryopreserved. In conclusion, culture and storage profoundly compromises the function of isolated hepatocytes after transplantation. Freshly isolated hepatocytes are the preferred source for transplantation.     

Comparison of minus power anterior chamber intraocular lenses and myopic epikeratoplasty in phakic eyes

We performed a retrospective study of two sequential series of operations designed to correct high myopia in phakic eyes. The first series consisted of 21 eyes with refractive error ranging from -11.00 diopters to -21.00 D (mean -16.9 D). They received a commercially prepared, lyophilized, myopic epikeratoplasty lenticule sutured into a circular keratectomy. Seven lenticules had to be removed within the first year because of poor refractive results, corneal ulceration, or melting. The nine eyes followed from 12 to 24 months after surgery demonstrated residual refractive errors from +4.00 D to -11.25 D. We also studied an initial series of 41 eyes with preoperative myopia ranging from -9.00 D to -35.00 D (mean -15.00 D) that received a minus power anterior chamber intraocular lens. The lens had an open loop design with an anterior vault, four point angle fixation, and an optic diameter of 4.5 mm. Recovery of visual acuity was faster with the intraocular lens than with epikeratoplasty. Three intraocular lenses were removed; two because they were the wrong size and one because it was the wrong power. Two of the lenses were replaced with acceptable results. Of the 34 eyes followed for 12 months, all had a residual refractive error within +/- 2.00 D of emmetropia. During the short-term follow-up, no elevated intraocular pressure, cataract formation, or excessive endothelial damage was observed. We concluded that anterior chamber minus power intraocular lenses gave more rapid and predictable optical correction in high myopic eyes than did myopic epikeratoplasty. Longer follow-up is required before conclusions can be reached about the safety of these intraocular lenses.     

Comparative surface thrombogenicity of implanted vascular grafts.

In order to study the comparative thrombogenicity of neointimal surfaces that develop with three types of vascular graft materials (ultralightweight knitted dacron, knitted dacron external velour, and expanded Teflon), 36 female mongrel dogs had their infrarenal aortas alternately replaced with one of the three grafts. At the end of 3 or 6 months, the grafts were removed and the surface thrombogenicity of the neointimal surface was determined. Each graft was examined visually and microscopically for evidence of “healing.” At 6 months the external velour graft is lined more frequently than the other two grafts. The external velour graft has a markedly better incidence of cellular healing noted microscopically than the other two grafts at both time intervals. While the expanded Teflon has an initially lower surface thrombogenicity (probably due to the characteristics of Teflon surface), at 6 months, the velour graft has the lowest surface thrombogenicity. This is most likely due to cellular healing. Of all the completely lined grafts at both time intervals, the surface thrombogenicity of the velour grafts was most like that of the normal aorta. The velour graft appears to develop the least thrombogenic neointimal surface while becoming most frequently healed with a cellular neointimal surface.     

Damage to the corneal endothelium from anterior chamber intraocular lenses in phakic myopic eyes

The use of minus power anterior chamber intraocular lenses to correct myopia in phakic eyes has been demonstrated previously to produce a more accurate and predictable correction than corneal refractive surgery techniques. We have examined 15 such eyes by specular microscopy and 10 such eyes by Scheimpflug slit-lamp micrography. One year after surgery, in 5 (36.3%) eyes, there was a decrease of more than 20% in the central endothelial density. Paracentral endothelial damage was present in 13 eyes (86.6%). On the specular micrograph, this damage appeared as an overall decrease in the number of the cells, abnormal cell shape with marked pleomorphism, dark zones, and acellular zones that corresponded to endothelial defects. The slit-lamp anterior segment measurements showed a distance between the edges of the IOL optic and the endothelium which ranged from 0.71 to 1.50 mm. We discuss the criteria for intraocular lens removal.     

Types of anterior cervical grafts.

Anterior cervical diskectomy and fusion provide satisfactory results in most instances of cervical disk herniation and cervical spondylosis. A variety of interpositioned grafts have been described for such problems at one or two levels. Multiple level anterior decompression and fusion for patients with spondylotic myelopathy produce satisfactory results in most instances and are particularly effective when a degenerative kyphosis is present. Appropriate patient selection and attention to technical aspects of exposure, neural decompression, and graft procurement and placement directly influence the surgical outcome.     

人子宫内膜植入兔眼前房模型的研究

本实验成功地将人子宫内膜植入到１５只去卵巢新西兰雌兔的眼前房，并给每只兔外源补充雌二醇（Ｅ２）１００μｇ／ｄ和孕酮（Ｐ）８ｍｇ／ｄ。在内膜植入期间每只兔皮下注射总量为９ｍｇ的霉酚酸（ｍｙ－ｃｏｐｈｅｎｏｌｉｃａｃｉｄ）以防免疫排斥反应。在眼前房的人子宫内膜存活了２８．４４±８．６５天，兔血清Ｅ２和Ｐ的平均值分别为１６１．７１±１５．１３和３８．８８±７．４５ｐｍｏｌ／Ｌ。前房液中Ｅ２和Ｐ浓度在植入人子宫内膜前后分别为２４．８０±１６．５３和１７０９．２５±４７５．１０ｐｍｏｌ／Ｌ，２．００±０．５３和１２．２２±３．８１ｎｍｏｌ／Ｌ。树脂切片验证人子宫内膜植入兔眼前房第５天就能与虹膜附贴并有血管发生。来自巩膜的血管分布在子宫内膜周围。扫描电镜观察人子宫内膜分化成分泌细胞和纤毛细胞。植入人子宫内膜后，前房液的蛋白浓度高于植入前。可以证明该模型可用来研究人子宫内膜对药物的反应和通过对前房液的测量研究子宫内膜的分泌功能。     

The critical role of mouse CD4+ cells in the rejection of highly disparate xenogeneic pig thymus grafts

Long-term survival of fetal pig thymus (FP THY) grafts and efficient repopulation of mouse CD4+ T cells is achieved in thymectomized (ATX) B6 mice that receive T and NK cell depletion by injection of a cocktail of mAbs (GK1.5, 2.43, 30-H12, and PK136) and fetal pig thymus/liver (FP THY/LIV) grafts. The requirement for each mAb in this conditioning regimen in order to avoid the rejection of FP THY grafts has not yet been defined. In our present studies, CD4 cell-depleted ATX B6 mice and euthymic MHC class II-deficient (IIKO) mice were employed to investigate the role of mouse CD4+ cells in the rejection of FP THY grafts in vivo. After grafting FP THY/LIV to CD4+ cell-depleted ATX B6 mice, efficient repopulation of mouse CD4+ T cells was observed in the periphery. However, only two of four mice had remaining FP THY grafts by 17 weeks post-implantation, and these were of poor quality, whereas four of four T and NK cell-depleted ATX B6 mice had well-developed FP THY grafts. Furthermore, three of four FP THY/LIV-grafted, CD4+ cell-depleted ATX B6 mice rejected donor MHC-matched pig skin grafts. In contrast, three of three FP THY/LIV grafted, T and NK cell-depleted, ATX B6 mice accepted donor MHC-matched pig skin grafts, suggesting that optimal tolerance to xenogeneic pig antigens was not achieved in mice conditioned only with anti-CD4 mAb. ATX B6 mice treated with only anti-CD8 mAb rejected FP THY completely by 6 weeks post-grafting, a time when CD4+ cell-depleted ATX B6 mice had well-vascularized FP THY grafts. In addition, when euthymic IIKO mice were pre-treated with the standard conditioning regimen that includes four different mAbs, FP THY grafts survived and supported the repopulation of mouse CD4+ T cells in the periphery, while high levels of mouse CD8+ T cells developed in host thymi. These studies suggest that mouse CD4+ T cells play a critical role in the acute rejection of xenogeneic FP THY grafts. Without help from CD4+ cells, mouse CD8+ cells, NK, NK/T, and TCR(gamma/delta)+ T cells do not mediate acute rejection of FP THY grafts. Furthermore, our results suggest that other cell subsets besides CD4+ T cells play a role in the delayed rejection of highly disparate xenogeneic FP THY grafts.     

骨形态发生蛋白在异种植骨局部的免疫调节作用

目的观察骨形态发生蛋白（ＢＭＰ）在异种植骨局部对肿瘤坏死因子α（ＴＮＦα）和白细胞介素６（ＩＬ６）基因及蛋白分子表达的调节作用。方法将含有一定抗原性的牛松质骨载体与不同剂量的牛ＢＭＰ复合,植入小鼠股部肌袋,于术后５、１０、２０天取材,制备石蜡切片,采用核酸分子原位杂交和免疫组化的方法,定性定位检测植骨局部ＴＮＦα和ＩＬ６ｍＲＮＡ及蛋白分子的表达情况,通过图像分析,确定ＴＮＦα和ＩＬ６ｍＲＮＡ及蛋白分子的表达强度,分析植骨局部不同计量ＢＭＰ对ＴＮＦα和ＩＬ６表达的影响。结果植骨术后５、１０、２０天,各组植骨局部均检测到ＴＮＦα和ＩＬ６ｍＲＮＡ及蛋白分子阳性表达,以单纯载体组表达最强,高剂量ＢＭＰ组表达最弱（Ｐ＜０．０１）;牛松质骨载体中复合的ＢＭＰ对植骨局部ＴＮＦα和ＩＬ６表达有抑制作用,其表达抑制程度与ＢＭＰ呈剂量依赖关系。结论外源性ＢＭＰ在异种植骨局部对ＴＮＦα和ＩＬ６基因及蛋白分子表达有抑制作用,提示ＢＭＰ参与异种植骨局部细胞免疫调节。     

Early morphological changes induced by photodynamic therapy in amelanotic greene melanoma implanted in the anterior eye chamber of rabbits

Morphological changes induced by photodynamic therapy (PDT) in amelanotic Greene melanoma implanted in the anterior eye chamber of rabbits were followed up to 24 h after PDT to study the development of tissue and cell damage leading to necrosis. Immediately after PDT, blood circulation had stopped as shown by fluorescence angiography and light and electron microscopy. It was not restored during the observation period. The first signs of tumour tissue damage, shrinkage of tumour cells and enlargement of intracellular spaces, became apparent immediately after PDT. Tissue and cell destruction increased further, and 24 h after PDT the tumours were almost completely necrotic. The most intriguing finding, by electron microscopy, was the presence of mitochondria with fused membranes in the untreated melanoma cells and the dramatic increase of this aberration directly after PDT. Melanocytes and fibroblasts in the same regions did not exhibit these aberrant mitochondria and furthermore kept a normal fine structure after PDT. Artificially induced ischaemia led to swollen mitochondria with ballooned cristae but showed no increase in membrane fusions. PDT thus directly interferes with mitochondrial structure. Direct damage to tumour cells therefore presumably contributes to tumour necrosis.     

Neurophysiological evaluation of microsurgically implanted grafts bridging peripheral nerve defects.

Measurement of motor and sensory conduction velocities, amplitude of M-wave and sensory nerve action potentials, and coaxial needle EMG were used to evaluate function of grafts bridging defects in median, ulnar, and radial nerves in 73 patients. The results show values only moderately lower than the normal limits.