A new donor cornea harvesting technique for posterior lamellar keratoplasty

Aims: To describe a technique for posterior lamellar keratoplasty donor preparation. Methods: In an experimental study eight human donor research corneas were mounted onto an artificial anterior chamber and deep stromal pockets dissected. Four corneas were mounted in the standard endothelial side down orientation and dissected using standard instruments (group 1). Another four corneas were mounted endothelial side up and dissected using a flat spatula (group 2). Trephined lamellar graft thickness was assessed by ultrasound pachymetry. The grafts were also analysed using vital staining of the endothelium and standard histological preparation. Results: Achieved posterior graft thickness was 118 (SD 32) μm (group 1) and 92 (23) μm (group 2) (p = 0.324). Percentage of devitalised endothelial cells was 0.86% (1.48%) (group 1) and 3.9% (2.9%) (group 2) (p = 0.185). The dissections using both harvesting techniques remained in plane and were smooth. Conclusions: A blunt spatula and endothelium side up orientation on an artificial anterior chamber can be used to create posterior lamellar dissections without compromising endothelial cell number or planarity when compared to standard endothelium side down harvest

Botulinum Toxin B-Induced Mouse Model of Keratoconjunctivitis Sicca

PURPOSE. To develop a mouse model of human chronic dry eye (keratoconjunctivitis sicca [KCS]). METHODS. Under direct visualization with an operating microscope, CBA/J mice received a transconjunctival injection of saline or 1.25, 5, or 20 milliunits (mU) of botulinum toxin B (BTX-B) into the lacrimal gland. The mice were either left unstressed or were subjected to an air blower for 5 h/d, 5 d/wk in fixed temperature and humidity conditions. Tear production and corneal fluorescein staining were evaluated in all groups before injection and at several time points after. Tear production was measured with phenol red-impregnated cotton threads. Corneal fluorescein staining was photographed under cobalt blue light with a digital camera fitted with a macro lens. RESULTS. BTX-B-injected mice displayed significantly decreased tear production until the 4-week time point. Throughout all time points, the addition of environmental blower stress did not appear to alter tear production significantly. Linear regression models, used to evaluate the effects of various doses of BTX-B on tear production, showed that doses higher than 1.25 mU did not provide significantly different outcomes. After 3 days, saline-injected mice showed no corneal staining, whereas BTX-B-injected mice displayed various amounts of staining. At the early time point (day 3), there did not appear to be an additional effect of the blower on corneal fluorescein staining. However, at 1, 2, and 4 weeks, the blower stress appeared to increase the amount of corneal fluorescein staining at each BTX-B dose, although not significantly. Furthermore, at 8 to 10 weeks, in the BTX B-injected groups, corneas had persistent staining, even though tear production had already returned to normal levels. Histopathologic analyses revealed no inflammatory cell infiltration of the stroma or acini of the lacrimal glands and conjunctivae of both saline-injected and BTX-B-injected animals. CONCLUSIONS. Intralacrimal gland injection of BTX-B resulted in persistent corneal fluorescein staining within 3 days, and a significant decrease in aqueous tear production that persisted for 1 month. Intralacrimal gland injection of BTX-B suppressed lacrimation, thereby establishing a dry eye state. This animal model could be a useful tool for investigating the pathogenesis of the chronic condition KCS in humans. (Invest Ophthalmol Vis Sci. 2006;47:133-139

Modeling Chronic Graft-versus-Host Disease in MHC-Matched Mouse Strains: Genetics, Graft Composition, and Tissue Targets

Graft-versus-host disease (GVHD) remains a major complication of allogeneic hematopoietic cell transplantation. Acute GVHD (aGVHD) results from direct damage by donor T cells, whereas the biology of chronic GVHD (cGVHD) with its autoimmune-like manifestations remains poorly understood, mainly because of the paucity of representative preclinical models. We examined over an extended time period 7 MHC-matched, minor antigen-mismatched mouse models for development of cGVHD. Development and manifestations of cGVHD were determined by a combination of MHC allele type and recipient strain, with BALB recipients being the most susceptible. The C57BL/6 into BALB.B combination most closely modeled the human syndrome. In this strain combination moderate aGVHD was observed and BALB.B survivors developed overt cGVHD at 6 to 12 months affecting eyes, skin, and liver. Naïve CD4$^{+}$ cells caused this syndrome as no significant pathology was induced by grafts composed of purified hematopoietic stem cells (HSCs) or HSC plus effector memory CD4$^{+}$ or CD8$^{+}$ cells. Furthermore, co-transferred naïve and effector memory CD4$^{+}$ T cells demonstrated differential homing patterns and locations of persistence. No clear association with donor Th17 cells and the phenotype of aGVHD or cGVHD was observed in this model. Donor CD4$^{+}$ cells caused injury to medullary thymic epithelial cells, a key population responsible for negative T cell selection, suggesting that impaired thymic selection was an underlying cause of the cGVHD syndrome. In conclusion, we report for the first time that the C57BL/6 into BALB.B combination is a representative model of cGVHD that evolves from immunologic events during the early post-transplant period