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The alternative oxidase, AOX, provides a by-pass of the cytochrome segment of the mitochondrial respiratory chain when the chain is unavailable. AOX is absent from mammals, but AOX from Ciona intestinalis is benign when expressed in mice. Although non-protonmotive, so does not contribute directly to ATP production, it has been shown to modify and in some cases rescue phenotypes of respiratory-chain disease models. Here we studied the effect of C. intestinalis AOX on mice engineered to express a disease-equivalent mutant of Uqcrh, encoding the hinge subunit of mitochondrial respiratory complex III, which results in a complex metabolic phenotype beginning at 4-5 weeks, rapidly progressing to lethality within a further 6-7 weeks. AOX expression delayed the onset of this phenotype by several weeks, but provided no long-term benefit. We discuss the significance of this finding in light of the known and hypothesized effects of AOX on metabolism, redox homeostasis, oxidative stress and cell signaling. Although not a panacea, the ability of AOX to mitigate disease onset and progression means it could be useful in treatment., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest regarding the contents of this article. MSz is a shareholder in a spin-off company founded to develop AOX-based therapeutics., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

The fundamental body functions that determine maximal O 2 uptake (V̇o 2max ) have not been studied in Aqp5 -/- mice (aquaporin 5, AQP5). We measured V̇o 2max to globally assess these functions and then investigated why it was found altered in Aqp5 -/- mice. V̇o 2max was measured by the Helox technique, which elicits maximal metabolic rate by intense cold exposure of the animals. We found V̇o 2max reduced in Aqp5 -/- mice by 20%-30% compared with wild-type (WT) mice. As AQP5 has been implicated to act as a membrane channel for respiratory gases, we studied whether this is caused by the known lack of AQP5 in the alveolar epithelial membranes of Aqp5 -/- mice. Lung function parameters as well as arterial O 2 saturation were normal and identical between Aqp5 -/- and WT mice, indicating that AQP5 does not contribute to pulmonary O 2 exchange. The cause for the decreased V̇o 2max thus might be found in decreased O 2 consumption of an intensely O 2 -consuming peripheral organ such as activated brown adipose tissue (BAT). We found indeed that absence of AQP5 greatly reduces the amount of interscapular BAT formed in response to 4 wk of cold exposure, from 63% in WT to 25% in Aqp5 -/- animals. We conclude that lack of AQP5 does not affect pulmonary O 2 exchange, but greatly inhibits transformation of white to brown adipose tissue. As under cold exposure, BAT is a major source of the animals' heat production, reduction of BAT likely causes the decrease in V̇o 2max under this condition.

Current concepts regarding the biology of aging are primarily based on studies aimed at identifying factors regulating lifespan. However, lifespan as a sole proxy measure for aging can be of limited value because it may be restricted by specific pathologies. Here, we employ large-scale phenotyping to analyze hundreds of markers in aging male C57BL/6J mice. For each phenotype, we establish lifetime profiles to determine when age-dependent change is first detectable relative to the young adult baseline. We examine key lifespan regulators (putative anti-aging interventions; PAAIs) for a possible countering of aging. Importantly, unlike most previous studies, we include in our study design young treated groups of animals, subjected to PAAIs prior to the onset of detectable age-dependent phenotypic change. Many PAAI effects influence phenotypes long before the onset of detectable age-dependent change, but, importantly, do not alter the rate of phenotypic change. Hence, these PAAIs have limited effects on aging., (© 2022. The Author(s).)

Improving reproducibility and replicability in preclinical research is a widely discussed and pertinent topic, especially regarding ethical responsibility in animal research. INFRAFRONTIER, the European Research Infrastructure for the generation, phenotyping, archiving, and distribution of model mammalian genomes, is addressing this issue by developing internal quality principles for its different service areas, that provides a quality framework for its operational activities. This article introduces the INFRAFRONTIER Quality Principles in Systemic Phenotyping of genetically altered mouse models. A total of 11 key principles are included, ranging from general requirements for compliance with guidelines on animal testing, to the need for well-trained personnel and more specific standards such as the exchange of reference lines. Recently established requirements such as the provision of FAIR (Findable, Accessible, Interoperable, Reusable) data are also addressed. For each quality principle, we have outlined the specific context, requirements, further recommendations, and key references., (© 2021. The Author(s).)

Mitochondrial disorders are clinically and genetically diverse, with isolated complex III (CIII) deficiency being relatively rare. Here, we describe two affected cousins, presenting with recurrent episodes of severe lactic acidosis, hyperammonaemia, hypoglycaemia and encephalopathy. Genetic investigations in both cases identified a homozygous deletion of exons 2 and 3 of UQCRH, which encodes a structural complex III (CIII) subunit. We generated a mouse model with the equivalent homozygous Uqcrh deletion (Uqcrh -/- ), which also presented with lactic acidosis and hyperammonaemia, but had a more severe, non-episodic phenotype, resulting in failure to thrive and early death. The biochemical phenotypes observed in patient and Uqcrh -/- mouse tissues were remarkably similar, displaying impaired CIII activity, decreased molecular weight of fully assembled holoenzyme and an increase of an unexpected large supercomplex (S XL ), comprising mostly of one complex I (CI) dimer and one CIII dimer. This phenotypic similarity along with lentiviral rescue experiments in patient fibroblasts verifies the pathogenicity of the shared genetic defect, demonstrating that the Uqcrh -/- mouse is a valuable model for future studies of human CIII deficiency., (© 2021 The Authors. Published under the terms of the CC BY 4.0 license.)

Influenza during pregnancy can affect the health of offspring in later life, among which neurocognitive disorders are among the best described. Here, we investigate whether maternal influenza infection has adverse effects on immune responses in offspring. We establish a two-hit mouse model to study the effect of maternal influenza A virus infection (first hit) on vulnerability of offspring to heterologous infections (second hit) in later life. Offspring born to influenza A virus infected mothers are stunted in growth and more vulnerable to heterologous infections (influenza B virus and MRSA) than those born to PBS- or poly(I:C)-treated mothers. Enhanced vulnerability to infection in neonates is associated with reduced haematopoetic development and immune responses. In particular, alveolar macrophages of offspring exposed to maternal influenza have reduced capacity to clear second hit pathogens. This impaired pathogen clearance is partially reversed by adoptive transfer of alveolar macrophages from healthy offspring born to uninfected dams. These findings suggest that maternal influenza infection may impair immune ontogeny and increase susceptibility to early life infections of offspring., (© 2021. The Author(s).)

Using standardized guidelines in preclinical research has received increased interest in light of recent concerns about transparency in data reporting and apparent variation in data quality, as evidenced by irreproducibility of results. Although the costs associated with supporting quality through a quality management system are often obvious line items in laboratory budgets, the treatment of the costs associated with quality failure is often overlooked and difficult to quantify. Thus, general estimations of quality costs can be misleading and inaccurate, effectively undervaluing costs recovered by reducing quality defects. Here, we provide examples of quality costs in preclinical research and describe how we have addressed misconceptions of quality management implementation as only marginally beneficial and/or unduly burdensome. We provide two examples of implementing a quality management system (QMS) in preclinical experimental (animal) research environments - one in Europe, the German Mouse Clinic, having established ISO 9001 and the other in the United States, the University of Kentucky (UK), having established Good Laboratory Practice-compliant infrastructure. We present a summary of benefits to having an effective QMS, as may be useful in guiding discussions with funders or administrators to promote interest and investment in a QMS, which ultimately supports shared, mutually beneficial outcomes.

The vertebrate Scube (Signal peptide, CUB, and EGF-like domain-containing protein) family consists of three independent members, Scube1-3, which encode secreted cell surface-associated membrane glycoproteins. Limited information about the general function of this gene family is available, and their roles during adulthood. Here, we present the first Scube3 mutant mouse line (Scube3 N294K/N294K ), which clearly shows phenotypic alterations by carrying a missense mutation in exon 8, and thus contributes to our understanding of SCUBE3 functions. We performed a detailed phenotypic characterization in the German Mouse Clinic (GMC). Scube3 N294K/N294K mutants showed morphological abnormalities of the skeleton, alterations of parameters relevant for bone metabolism, changes in renal function, and hearing impairments. These findings correlate with characteristics of the rare metabolic bone disorder Paget disease of bone (PDB), associated with the chromosomal region of human SCUBE3 In addition, alterations in energy metabolism, behavior, and neurological functions were detected in Scube3 N294K/N294K mice. The Scube3 N294K/N294K mutant mouse line may serve as a new model for further studying the effect of impaired SCUBE3 gene function., (Copyright © 2016 Fuchs et al.)

Animal models resembling human mutations are valuable tools to research the features of complex human craniofacial syndromes. This is the first report on a viable dominant mouse model carrying a non-synonymous sequence variation within the endothelin receptor type A gene (Ednra c.386A>T, p.Tyr129Phe) derived by an ENU mutagenesis program. The identical amino acid substitution was reported recently as disease causing in three individuals with the mandibulofacial dysostosis with alopecia (MFDA, OMIM 616367) syndrome. We performed standardized phenotyping of wild-type, heterozygous, and homozygous Ednra Y129F mice within the German Mouse Clinic. Mutant mice mimic the craniofacial phenotypes of jaw dysplasia, micrognathia, dysplastic temporomandibular joints, auricular dysmorphism, and missing of the squamosal zygomatic process as described for MFDA-affected individuals. As observed in MFDA-affected individuals, mutant Ednra Y129F mice exhibit hearing impairment in line with strong abnormalities of the ossicles and further, reduction of some lung volumetric parameters. In general, heterozygous and homozygous mice demonstrated inter-individual diversity of expression of the craniofacial phenotypes as observed in MFDA patients but without showing any cleft palates, eyelid defects, or alopecia. Mutant Ednra Y129F mice represent a valuable viable model for complex human syndromes of the first and second pharyngeal arches and for further studies and analysis of impaired endothelin 1 (EDN1)-endothelin receptor type A (EDNRA) signaling. Above all, Ednra Y129F mice model the recently published human MFDA syndrome and may be helpful for further disease understanding and development of therapeutic interventions., Competing Interests: The authors declare that they have no conflicts of interest.

Purpose: To compare cryopreserved human corneal endothelial cells (HCECs) grown in human serum-supplemented media (HS-SM) with cryopreserved HCECs grown in fetal bovine serum-supplemented media (FBS-SM)., Methods: Three pairs of human corneas from donors aged 8, 28, and 31 years were obtained from the eye bank. From each pair, one cornea was used to start a HCEC culture using HS-SM; the other cornea was grown in FBS-SM. On reaching confluence, the six cell populations were frozen using 10% dimethyl sulfoxidecontaining medium. Thawed cells grown in HS-SM were compared with those grown in FBS-SM with respect to morphology, growth curves, immunohistochemistry, real time-reverse transcriptase polymerase chain reaction (RT-PCR) for endothelial cell markers, and detachment time., Results: No difference in morphology was observed for cells grown in the two media before or after cryopreservation. By growth curves, cell counts after thawing were similar in both media, with a slight trend toward higher cell counts in FBS-SM. Cells grown in both the media demonstrated a similar expression of endothelial cell markers when assessed by immunohistochemistry, although HCEC marker gene expression was higher in cells grown in HS-SM than in those grown in FBS-SM as assessed by RT-PCR. With FBS-SM, there was a tendency of longer detachment time and lower cell passages., Conclusions: HS-SM was similar to FBS-SM for cryopreservation of cultured HCECs as assessed by analysis of cell morphology, proliferation, and protein expression, although marker gene expression was higher in cells grown in HS-SM than in those grown in FBS-SM. Detachment time was longer with FBS-SM and in lower passages.

Glutamine, the most abundant amino acid in mammals, is critical for cell and organ functions. Its metabolism depends on the ability of cells to take up or release glutamine by transporters located in the plasma membrane. Several solute carrier (SLC) families transport glutamine, but the SLC38 family has been thought to be mostly responsible for glutamine transport. We demonstrate that despite the large number of glutamine transporters, the loss of Snat3/Slc38a3 glutamine transporter has a major impact on the function of organs expressing it. Snat3 mutant mice were generated by N-ethyl-N-nitrosurea (ENU) mutagenesis and showed stunted growth, altered amino acid levels, hypoglycemia, and died around 20 days after birth. Hepatic concentrations of glutamine, glutamate, leucine, phenylalanine, and tryptophan were highly reduced paralleled by downregulation of the mTOR pathway possibly linking reduced amino acid availability to impaired growth and glucose homeostasis. Snat3-deficient mice had altered urea levels paralleled by dysregulation of the urea cycle, gluconeogenesis, and glutamine synthesis. Mice were ataxic with higher glutamine but reduced glutamate and gamma-aminobutyric acid (GABA) levels in brain consistent with a major role of Snat3 in the glutamine-glutamate cycle. Renal ammonium excretion was lower, and the expression of enzymes and amino acid transporters involved in ammoniagenesis were altered. Thus, SNAT3 is a glutamine transporter required for amino acid homeostasis and determines critical functions in various organs. Despite the large number of glutamine transporters, loss of Snat3 cannot be compensated, suggesting that this transporter is a major route of glutamine transport in the liver, brain, and kidney.

Purpose: To evaluate the Descemet stripping automated endothelial keratoplasty (DSAEK) graft deturgescence in preservation medium after microkeratome cut using Fourier domain optical coherence tomography., Methods: The central and peripheral thickness of DSAEK grafts was measured by Fourier domain optical coherence tomography immediately after microkeratome cuts and 1, 2, 3, and 4 hours afterward. All measurements were taken when the grafts were stored in 4°C preservation medium. The hourly change in central graft thickness and graft shape (peripheral graft thicknes - central graft thickness) was calculated and tracked over time., Results: Five DSAEK grafts were measured. The average central graft thickness was 188.7 ± 44.4 μm (range, 146-255 µm) immediately after microkeratome cuts. The average central graft thickness was 147.5 ± 33.0 μm (range, 116-190 µm) after 4 hours in preservation medium (P < 0.001). The average hourly change in central graft thickness was -30.5 μm (P = 0.0051), -8.6 μm (P = 0.055), -2.0 μm (P = 0.42), and 0.0 μm (P = 0.93) at 1, 2, 3, and 4 hours, respectively, after microkeratome cuts. The average hourly change in graft shape was insignificant., Conclusions: DSAEK grafts become thinner after microkeratome cut and stabilize at approximately 2 hours. Therefore, DSAEK graft thickness should be measured at 1.5 to 3 hours after microkeratome cut.

The SLC43 family is composed of only three genes coding for the plasma membrane facilitator system l amino acid transporters LAT3 (SLC43A1; TC 2.A.1.44.1) and LAT4 (SLC43A2; TC 2.A.1.44.2), and the orphan protein EEG1 (SLC43A3; TC 2.A.1.44.3). Besides the known mechanism of transport of LAT3 and LAT4, their physiological roles still remain quite obscure. Morphants suggested a role of LAT3 in renal podocyte development in zebrafish. Expression in liver and skeletal muscle, and up-regulation by starvation suggest a role of LAT3 in the flux of branched-chain amino acids (BCAAs) from liver and skeletal muscle to the bloodstream. Finally, LAT3 is up-regulated in androgen-dependent cancers, suggesting a role in mTORC1 signaling in this type of tumors. In addition, LAT4 might contribute to the transfer of BCAAs from mother to fetus. Unfortunately, the EEG1 mouse model (EEG1(Y221∗)) described here has not yet offered a clue to the physiological role of this orphan protein., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Purpose. To evaluate Fourier-domain optical coherence tomography (FD-OCT) as an adjunct to traditional slit lamp examination of donor corneas with suspected Anterior Stromal Opacities. Methods. Seven corneas suspected of having anterior stromal opacities by slit lamp examination were evaluated with FD-OCT. Each cornea was evaluated to confirm the presence of opacity and, if present, the depth of opacity was measured. Results. The opacity depth ranged from 82  μ m to 624  μ m. The initial slit lamp impressions of five of the seven corneas were confirmed by OCT. In two corneas, the OCT findings were different from the initial slit lamp impressions. Slit lamp examination of the first cornea gave the impression of anterior stromal scarring, but OCT showed that the opacity was limited to the epithelium. Slit lamp examination of the second cornea suggested opacity limited to the epithelium, but OCT identified significant sub-Bowman's scarring. In all cases, the Eye Bank Technicians reported that the location and depth of corneal opacity were more sharply defined by OCT than by slit lamp. Conclusion. The high resolution of OCT makes it easier to determine the location of corneal opacities compared to slit lamp examinations. This enhanced visualization can improve decisions regarding transplant suitability of donor corneas.

Purpose: To evaluate the endothelial cell survival and stromal bed quality when creating deep stromal cuts with a low-pulse energy, high-frequency femtosecond laser to produce "ultrathin" tissue for Descemet stripping automated endothelial keratoplasty., Methods: Seventeen corneas were used for this study. Five corneas were cut with the laser at a depth of 420 to 500 μm to produce a tissue thickness of approximately ≤70 μm. Five corneas served as an uncut comparison group. Vital dye staining and computer digitized planimetry analysis were performed on these corneas. The 7 remaining corneas were cut for scanning electron microscopy evaluation., Results: The mean central posterior stromal thickness of cut corneas was 60.6 μm (range, 43-72 μm). Endothelial cell damage in cut and comparison corneas was 3.92% ± 2.22% (range, 1.71%-6.51%) and 4.15% ± 2.64% (range, 1.21%-7.01%), respectively (P = 0.887). Low-magnification (×12) scanning electron microscopy revealed a somewhat irregular-appearing surface with concentric rings peripherally. Qualitative grading of higher magnification (×50) central images resulted in an average score of 2.56 (between smooth and rough)., Conclusions: Ultrathin tissue for Descemet stripping automated endothelial keratoplasty can be safely prepared with minimal endothelial cell damage using a low-pulse energy, high-frequency femtosecond laser; however, the resulting stromal surface quality may not be optimal with this technique.

The uniporter TAT1 (Slc16a10) mediates the facilitated diffusion of aromatic amino acids (AAAs) across basolateral membranes of kidney, small intestine and liver epithelial cells, and across the plasma membrane of non-epithelial cells like skeletal myocytes. Its role for body AA homeostasis has now been investigated using newly generated TAT1 (Slc16a10) defective mice (tat1(-/-)). These mice grow and reproduce normally, show no gross phenotype and no obvious neurological defect. Histological analysis did not reveal abnormalities and there is no compensatory change in any tested AA transporter mRNA. TAT1 null mice, however, display increased plasma, muscle and kidney AAA concentration under both normal and high protein diet, although this concentration remains normal in the liver. A major aromatic aminoaciduria and a smaller urinary loss of all substrates additionally transported by l-type AA antiporter Lat2-4F2hc (Slc7a8) were revealed under a high protein diet. This suggests an epithelial transport defect as also shown by the accumulation of intravenously injected (123)I-2-I-l-Phe in kidney and l-[(3)H]Phe in ex vivo everted gut sac enterocytes. Taken together, these data indicate that the uniporter TAT1 is required to equilibrate the concentration of AAAs across specific membranes. For instance, it enables hepatocytes to function as a sink that controls the extracellular AAAs concentration. Additionally, it facilitates the release of AAAs across the basolateral membrane of small intestine and proximal kidney tubule epithelial cells, thereby allowing the efflux of other neutral AAs presumably via Lat2-4F2hc.

Purpose: To determine the endothelium damage resulting from the application of "dry" gentian violet (GV) stromal markings on corneas precut for Descemet stripping automated endothelial keratoplasty (DSAEK) using a Moria "S" Stamp., Methods: Five precut corneas had the stromal graft bed marked with GV using the Moria "S" stamp and 6 precut corneas were left unmarked as controls. After applying the ink to the stamp, care was taken to allow the alcohol carrier to dry for 10 seconds before applying the dry dye to the stromal surface. Tissue was then trephinated and stained with calcein AM to assess endothelial viability. Grafts were photographed and digital pixel planometry, using an established analysis technique, was used to compare the damage between the control and experimental groups., Results: The mean percent cell damage of corneas treated with GV "S" stamp (n = 5) was 8.6% (range 4.4-12.9), and it was 8.1% (range 3.9-15.1) in the DSAEK control set (n = 6). Median percent cell damage was 6.7% among GV-treated corneas and 7.4% among control corneas. The distributions were not significantly different between groups (Mann-Whitney U test = 15.0, two-tailed P = 1.0). Moreover, no "S" pattern of damage was seen in any study eye., Conclusions: There were no significant differences in endothelial damage between the 2 groups. GV stromal markings may be applied without undue damage to the endothelium using the dry-ink technique described.

Purpose: To use excimer laser smoothing passes to reshape Descemet-stripping automated endothelial keratoplasty (DSAEK) endothelial grafts and to evaluate the effect on the donor endothelium., Methods: The stromal surface of microkeratome-cut DSAEK grafts was smoothed using excimer laser smoothing passes with masking fluid. Excimer laser hyperopic ablation was used to improve the uniformity of graft thickness within the optical zone. Fourier-domain optical coherence tomography was used to measure endothelial graft pachymetry, plan ablations, and evaluate donor contour. Vital dye staining was performed to assess endothelial cell damage. Scanning electron microscopy images of stromal surfaces were graded on a 5-point scale by masked observers to evaluate surface roughness., Results: Four grafts underwent excimer laser smoothing. Vital dye staining showed no endothelial damage. Microkeratome-cut surfaces treated with laser smoothing (mean grade = 2.04) were smoother than nonsmoothed microkeratome-cut surfaces (mean grade = 4.07; P < 0.01), surfaces that underwent dry laser ablation (mean grade = 3.63; P < 0.01) and manually dissected interfaces (mean grade = 4.75; P < 0.0001). No difference was observed between stromal beds created by peeling Descemet membrane (mean grade = 1.64) compared with surfaces produced by microkeratome cutting followed by laser smoothing (mean grade = 2.04; P = 0.14). One graft underwent combined excimer smoothing and peripheral hyperopic ablation. The center-periphery thickness difference was 15 μm before ablation and 4 μm afterward., Conclusions: Laser smoothing passes can be used to improve the contour and smoothness of DSAEK grafts without damaging donor endothelial cells. Clinical trials are needed to determine whether reshaping donors using excimer laser can deliver improved visual outcomes after DSAEK.

Purpose: To compare the quality of corneal tissue after 2 different procurement techniques, whole-globe enucleation and in situ excision., Design: Cross-sectional study., Methods: Data in the interval 2005 to 2008 were collected from the Lions Eye Bank of Oregon. A total of 3618 eyes underwent whole-globe enucleation (enucleation group) and 2048 eyes were subject to in situ excision (in situ group). Endothelial cell density (ECD) and death-to-preservation time (D-to-P) were analyzed. Grading scores ranging from 0 (excellent) to 4 (unacceptable for transplantation) were used to evaluate the epithelium, stroma, Descemet fold, and overall endothelium. The incidences of ECD less than 2000 cells/mm(2) (ECD <2000), primary graft failure (PGF), and postoperative infection were investigated., Results: ECD was 2726 +/- 419 cells/mm(2) in the enucleation and 2645 +/- 395 cells/mm(2) in the in situ group (P < .001). D-to-P was 9.81 +/- 3.56 hours and 8.90 +/- 3.65 hours, respectively (P < .001). The mean grade of the stroma was 1.44 +/- 0.51 in the enucleation group and 1.50 +/- 0.56 in the in situ group (P = .001). The mean grades of overall endothelium were 1.51 +/- 0.72 and 1.58 +/- 0.69, respectively (P < .001). The incidence of ECD <2000 was 2.38% in the enucleation group and 2.39% in the in situ group (P > .999), PGF rates were 0.72% and 0.68% (P > .999), and postoperative infection levels were 0.14% and 0.39% (P = .080)., Conclusion: Although there were minor differences in parameters related to the endothelium, in situ excision seemed equivalent to whole-globe enucleation when various parameters were evaluated., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

Purpose: The purpose of this study was to evaluate outcomes of Descemet's stripping automated endothelial keratoplasty (DSAEK) using anterior stromal flawed (ASF) donor corneas that were unsuitable for use in full-thickness penetrating keratoplasty as a result of stromal scars, pterygia, or previous corneal refractive surgery and to compare results with DSAEK using standard tissue., Methods: We conducted a review of our initial 42 (19 with 6-month follow up) consecutive DSAEK surgeries using ASF tissue compared with 357 (199 with 6-month follow up) time-matched controls using standard tissue. Intraoperative and perioperative complications, including dislocations and primary graft failures, were compared. Six-month best spectacle-corrected vision, incidence of rejection episodes, postoperative refractive astigmatism, keratometric values, pre- and postoperative topography-derived surface asymmetry index, and surface regularity index were compared., Results: One surgeon-cut ASF tissue was perforated before surgery and was discarded. No surgeon-cut standard tissue was perforated. No intraoperative complications and no episodes of primary graft failure or pupillary block glaucoma occurred in either group. One (2.4%) postoperative graft dislocation and one (5.2%) graft rejection episode occurred in the study group. There were 10 (2.8%) dislocations and 8 (2.2%) graft rejection in the controls. A statistically similar significant improvement in best spectacle-corrected vision occurred in both groups. Corneal topography, pachymetry, and manifest astigmatism were not significantly different between groups., Conclusion: Postoperative results of DSAEK using donor tissue excluded from use in penetrating keratoplasty as a result of stromal flaws are equivalent to results using standard donor tissue. Central corneal thickness measurements should be performed before cutting to avoid tissue perforation. The use of ASF tissue for DSAEK will expand the cornea donor pool.

Purpose: To determine the acute endothelial cell damage from trephination and tissue insertion in endothelial keratoplasty (EK) surgery. The influence of insertion technique (forceps insertion vs "pull-through" insertion) of donor tissue and incision size (3 vs 5 mm length) was assessed., Methods: Forty precut 8.-mm-diameter donor posterior buttons were used in this study. Thirty-five buttons were inserted through a limbal incision of either 3 or 5 mm length into the anterior chamber of cadaver eyes and then removed through an open sky technique without further trauma. Five buttons that were trephined but not inserted served as a control group. Vital dye staining and computer digitized planimetry were used to analyze the tissue and quantify the total damaged area over the entire endothelial surface. Five buttons for each of 7 insertion techniques were used. The 8 tissue groups evaluated were as follows: group 1: control group of trephination only, with no insertion; group 2: forceps with folded tissue through 5-mm incision; group 3: suture pull through of nonfolded tissue through a 5-mm incision; group 4: forceps pull through of Busin glide folded tissue through a 5-mm incision; group 5: forceps with folded tissue through a 3-mm incision; group 6: suture pull through with folded tissue through a 3-mm incision; group 7: suture pull through with nonfolded tissue through a 3-mm incision; and group 8: forceps pull through of Busin glide folded tissue through a 3-mm incision., Results: The control group demonstrated 9% +/- 2% peripheral cell damage from simple trephination of the tissue but without insertion. In the 5-mm incision surgeries, forceps insertion (group 2) caused 18% +/- 3% loss, suture pull-through insertion (group 3) caused 18% +/- 2% loss, and Busin glide pull through (group 4) caused 20% +/- 5% loss. There were no significant differences in damage between any of the 5-mm incision group techniques (P > 0.99). In the 3-mm incision surgeries, forceps insertion (group 5) caused a 30% +/- 3% loss, pull through with folded tissue (group 6) caused 30% +/- 5% loss, pull through with nonfolded tissue (group 7) caused 56% +/- 4% loss, and Busin glide pull through (group 8) caused a 28%+/- 5% loss. There was no difference in damage among the 3-mm groups (P > 0.96), with the exception of group 7 where pulling the unfolded tissue through a 3-mm incision was significantly worse than all other techniques (P < 0.001). There was significantly greater cell area damage in the 3-mm groups (36%) than in the 5-mm groups (19%) (P <0.001). Large patterns of striae with cell loss were seen in the 3-mm groups emanating from the peripheral traction site, regardless of whether the traction to pull the tissue through the incision and into the chamber was generated by a suture or cross-chamber forceps. Direct forceps insertion caused circular patterns of injury at the tip compression site regardless of incision size, but this damage was multiplied and exacerbated by insertion through a smaller incision., Conclusions: Smaller size (3 mm) incisions for EK surgery result in greater acute endothelial area damage than larger size (5 mm) incisions. Pull-through insertion techniques through a 5-mm incision seem equivalent in the amount of induced area damage to that of forceps insertion. Compressive injury from the incision appeared less when the tissue was folded than when not folded. Insertion with any technique through a 3-mm incision resulted in larger areas of endothelial damage. All these iatrogenic death zones outside the central endothelial area would be missed clinically by standard early specular microscopy after EK surgery.

Purpose: We developed a simple, practical, and inexpensive technique to analyze areas of endothelial cell loss and/or damage over the entire corneal area after vital dye staining by using a readily available, off-the-shelf, consumer software program, Adobe Photoshop. The purpose of this article is to convey a method of quantifying areas of cell loss and/or damage., Methods: Descemet-stripping automated endothelial keratoplasty corneal transplant surgery was performed by using 5 precut corneas on a human cadaver eye. Corneas were removed and stained with trypan blue and alizarin red S and subsequently photographed. Quantitative assessment of endothelial damage was performed by using Adobe Photoshop 7.0 software., Results: The average difference for cell area damage for analyses performed by 1 observer twice was 1.41%. For analyses performed by 2 observers, the average difference was 1.71%. Three masked observers were 100% successful in matching the randomized stained corneas to their randomized processed Adobe images., Conclusions: Vital dye staining of corneal endothelial cells can be combined with Adobe Photoshop software to yield a quantitative assessment of areas of acute endothelial cell loss and/or damage. This described technique holds promise for a more consistent and accurate method to evaluate the surgical trauma to the endothelial cell layer in laboratory models. This method of quantitative analysis can probably be generalized to any area of research that involves areas that are differentiated by color or contrast.

Purpose: To evaluate the use of corneal donor tissue deemed unsuitable for full-thickness penetrating keratoplasty (PK) for use in deep lamellar endothelial keratoplasty (DLEK) and to compare postoperative results to those of DLEK surgery using donor tissue that is suitable for PK., Methods: Small-incision DLEK surgery was performed using 39 donor corneas unsuitable for PK. Thirty-five donors had anterior scars or opacities, 3 donors had pterygia within the 8-mm zone, and 1 had prior LASIK. All donor preparation was completed by manual stromal dissection. The DLEK surgical and postoperative courses were reviewed. Preoperative and 6-month postoperative results of this study group were compared with a control group consisting of the first 55 consecutive small-incision DLEK patients receiving donor corneas that had no criteria excluding them from use in PK. Four eyes in the study group and 1 eye in the control group had the confounding variables of the presence of an anterior-chamber lens or surgical vitrectomy with macular disease in the recipient eye., Results: There was no significant difference in preoperative measurements of best spectacle-corrected visual acuity (BSCVA; P = 0.372), donor endothelial cell density (ECD; P = 0.749), or corneal topography [surface regularity index (SRI), P = 0.485; or surface asymmetry index (SAI), P = 0.154] between the 2 groups. For the patients receiving corneas deemed unacceptable for PK, at 6 months after surgery, the vision (P = 0.002) and corneal topography measurements improved significantly from before surgery (SRI, P < 0.001; SAI, P < 0.001), and there was no significant change in refractive astigmatism (P = 0.240). There was a significant difference in the vision at 6 months postoperatively between the overall study group and the control group, with the mean vision of the study group at 20/56 and the control group at 20/43 (P = 0.015). If eyes with known cystoid macular edema (CME) and vitrectomy are removed from each group, there is no significant difference in vision at 6 months between the study group and the control group (P = 0.110), with the average BSCVA of those receiving donor corneas unsuitable for PK equal to 20/48 (range, 20/25-20/200) and the average vision for those receiving PK-acceptable donor tissue equal to 20/43 (range, 20/20-20/80). The 6-month average refractive astigmatism of the study group was 1.12 +/- 0.99 D (range, 0.00-4.00 D), and the average endothelial cell count was 2064 +/- 396 cells/mm(2) (range, 1208-2957 cells/mm(2)). There was no significant difference in 6-month postoperative endothelial cell count (P = 0.443), refractive astigmatism (P = 0.567), or corneal topography (SRI, P = 0.332; SAI, P = 0.110) in study patients who received corneas unsuitable for PK compared with control patients who received corneas suitable for PK., Conclusions: Endothelial keratoplasty such as DLEK surgery with manual donor preparation broadens the donor pool by enabling corneas that cannot be used for PK to be used for selective endothelial transplantation without deleterious postoperative results.

We sought to identify associations of basal metabolic rate (BMR) with morphological traits in laboratory mice. In order to expand the body mass (BM) range at the intra-strain level, and to minimize relevant genetic variation, we used male and female wild type mice (C3HeB/FeJ) and previously unpublished ENU-induced dwarf mutant littermates (David mice), covering a body mass range from 13.5 g through 32.3 g. BMR was measured at 30 degrees C, mice were killed by means of CO(2 )overdose, and body composition (fat mass and lean mass) was subsequently analyzed by dual X-ray absorptiometry (DEXA), after which mice were dissected into 12 (males) and 10 (females) components, respectively. Across the 44 individuals, 43% of the variation in the basal rates of metabolism was associated with BM. The latter explained 47% to 98% of the variability in morphology of the different tissues. Our results demonstrate that sex is a major determinant of body composition and BMR in mice: when adjusted for BM, females contained many larger organs, more fat mass, and less lean mass compared to males. This could be associated with a higher mass adjusted BMR in females. Once the dominant effects of sex and BM on BMR and tissue mass were removed, and after accounting for multiple comparisons, no further significant association between individual variation in BMR and tissue mass emerged.