Indicators of replicative damage in equine tendon fibroblast monolayers

<p>Background: Superficial digital flexor tendon (SDFT) injuries of horses usually follow cumulative matrix microdamage; it is not known why the reparative abilities of tendon fibroblasts are overwhelmed or subverted. Relevant in vitro studies of this process require fibroblasts not already responding to stresses caused by the cell culture protocols. We investigated indicators of replicative damage in SDFT fibroblast monolayers, effects of this on their reparative ability, and measures that can be taken to reduce it.</p> <p>Results: We found significant evidence of replicative stress, initially observing consistently large numbers of binucleate (BN) cells. A more variable but prominent feature was the presence of numerous gammaH2AX (γH2AX) puncta in nuclei, this being a histone protein that is phosphorylated in response to DNA double-stranded breaks (DSBs). Enrichment for injury detection and cell cycle arrest factors (p53 (ser15) and p21) occurred most frequently in BN cells; however, their numbers did not correlate with DNA damage levels and it is likely that the two processes have different causative mechanisms. Such remarkable levels of injury and binucleation are usually associated with irradiation, or treatment with cytoskeletal-disrupting agents.</p> <p>Both DSBs and BN cells were greatest in subconfluent (replicating) monolayers. The DNA-damaged cells co-expressed the replication markers TPX2/repp86 and centromere protein F. Once damaged in the early stages of culture establishment, fibroblasts continued to express DNA breaks with each replicative cycle. However, significant levels of cell death were not measured, suggesting that DNA repair was occurring. Comet assays showed that DNA repair was delayed in proportion to levels of genotoxic stress.</p> <p>Conclusions: Researchers using tendon fibroblast monolayers should assess their “health” using γH2AX labelling. Continued use of early passage cultures expressing initially high levels of γH2AX puncta should be avoided for mechanistic studies and ex-vivo therapeutic applications, as this will not be resolved with further replicative cycling. Low density cell culture should be avoided as it enriches for both DNA damage and mitotic defects (polyploidy). As monolayers differing only slightly in baseline DNA damage levels showed markedly variable responses to a further injury, studies of effects of various stressors on tendon cells must be very carefully controlled.</p&gt

Paradigm shifts in understanding equine laminitis

Laminitis, one of the most debilitating conditions of all equids, is now known to be the result of several systemic disease entities. This finding, together with other recent developments in the field of laminitis research, have provoked a rethink of our clinical and research strategies for this condition. First, laminitis is now considered to be a clinical syndrome associated with systemic disease (endocrine disease, sepsis or systemic inflammatory response syndrome, SIRS) or altered weight bearing rather than being a discrete disease entity. Next, laminitis associated with endocrine disease (endocrinopathic laminitis) is now believed to be the predominant form in animals presenting (primarily) for lameness. Third, the designation of laminitis as a primary and severe basement membrane pathology now requires revision. Instead, current data now proposes a variable subclinical phase associated with gross changes in the hoof capsule, with stretching and elongation of the lamellar cells an early and key event in the pathophysiology. These findings have fuelled new mechanistic hypotheses and research directions that will be discussed, together with their implications for future clinical management. (C) 2017 The Authors. Published by Elsevier Ltd.Peer reviewe

Transverse sectioning in the evaluation of skin biopsy specimens from alopecic dogs.

Transverse sectioning of skin biopsy specimens has revolutionised assessment of human alopecia by demonstration of every hair in each specimen, allowing quantitative evaluation of follicular activity. Since only vertical sectioning is performed routinely in veterinary laboratories, we aimed to determine whether transverse sectioning was a valuable technique in assessment of canine alopecia. Paired vertical and transverse sections of biopsy specimens from 31 alopecic dogs were examined independently in triplicate in random order and blinded to previous diagnosis using a standard check-list proforma. Assessments of key features (follicular activity [anagen/telogen], infundibular hyperkeratosis, sebaceous gland abnormalities, pigment clumping, dermal inflammation) by each sectioning method were compared. In the 31 cases, (atrophic [n = 13], dysplastic [n = 12], inflammatory diseases [n = 6]), follicular inactivity scores (median, [lower-upper quartile]) in transverse sections significantly exceeded those in vertical sections (transverse 4 [3-5], vertical 3 [2-4]). Agreement between the two sectioning planes was moderate for infundibular hyperkeratosis (kappa = 0.5210) and dermal inflammation (0.4351), fair for sebaceous gland abnormalities (0.3966) and pigment clumping (0.2197), but slight for follicular activity (0.1041). Vertical sectioning demonstrated diagnostically important epidermal pathology (n = 2) and dermal thinning (n = 3) whereas transverse sectioning enhanced assessment of hair growth phase (n = 11), follicular structure and architecture (n = 11), and focal luminal or mural folliculitides (n = 3). Transverse sectioning confers significant benefits and complements traditional vertical sectioning in the histological assessment of canine hair follicle diseases, particularly when subtle abnormalities comprise distorted compound follicle architecture, hair cycle arrest or when relatively few adnexal structures are affected. [Abstract copyright: © 2020 The Authors. Journal of Small Animal Practice published by John Wiley & Sons Ltd on behalf of British Small Animal Veterinary Association.

The Consequences of Remote and Hybrid Instruction During the Pandemic

Using testing data from 2.1 million students in 10,000 schools in 49 states (plus D.C.), we investigate the role of remote and hybrid instruction in widening gaps in achievement by race and school poverty. We find that remote instruction was a primary driver of widening achievement gaps. Math gaps did not widen in areas that remained in-person (although there was some widening in reading gaps in those areas). We estimate that high-poverty districts that went remote in 2020-21 will need to spend nearly all their federal aid on academic recovery to help students recover from pandemic-related achievement losses

Intraobserver Repeatability and Interobserver Reproducibility of Foveal Cone Density Measurements in CNGA3- and CNGB3-Associated Achromatopsia

PURPOSE: To examine repeatability and reproducibility of foveal cone density measurements in patients with CNGA3- and CNGB3-associated achromatopsia (ACHM) using split-detection adaptive optics scanning light ophthalmoscopy (AOSLO). METHODS: Thirty foveae from molecularly confirmed subjects with ACHM, half of whom harbored disease-causing variants in CNGA3 and half in CNGB3, underwent nonconfocal split-detection AOSLO imaging. Cone photoreceptors within the manually delineated rod-free zone were manually identified twice by two independent observers. The coordinates of the marked cones were used for quantifying foveal cone density. Cone density and difference maps were generated to compare cone topography between trials. RESULTS: We observed excellent intraobserver repeatability in foveal cone density estimates, with intraclass correlation coefficients (ICCs) ranging from 0.963 to 0.991 for CNGA3 and CNGB3 subjects. Interobserver reproducibility was also excellent for both CNGA3 (ICC = 0.952; 95% confidence interval [CI], 0.903–1.0) and CNGB3 (ICC = 0.968; 95% CI, 0.935–1.0). However, Bland-Altman analysis revealed bias between observers. CONCLUSIONS: Foveal cone density can be measured using the described method with good repeatability and reproducibility both for CNGA3- and CNGB3-associated ACHM. Any degree of bias observed among the observers is of uncertain clinical significance but should be evaluated on a study-specific basis. TRANSLATIONAL RELEVANCE: This approach could be used to explore disease natural history, as well as to facilitate stratification of patients and monitor efficacy of interventions for ongoing and upcoming ACHM gene therapy trials

Characterization of Retinal Structure in ATF6-Associated Achromatopsia.

PurposeMutations in six genes have been associated with achromatopsia (ACHM): CNGA3, CNGB3, PDE6H, PDE6C, GNAT2, and ATF6. ATF6 is the most recent gene to be identified, though thorough phenotyping of this genetic subtype is lacking. Here, we sought to test the hypothesis that ATF6-associated ACHM is a structurally distinct form of congenital ACHM.MethodsSeven genetically confirmed subjects from five nonconsanguineous families were recruited. Foveal hypoplasia and the integrity of the ellipsoid zone (EZ) band (a.k.a., IS/OS) were graded from optical coherence tomography (OCT) images. Images of the photoreceptor mosaic were acquired using confocal and nonconfocal split-detection adaptive optics scanning light ophthalmoscopy (AOSLO). Parafoveal cone and rod density values were calculated and compared to published normative data as well as data from two subjects harboring CNGA3 or CNGB3 mutations who were recruited for comparative purposes. Additionally, nonconfocal dark-field AOSLO images of the retinal pigment epithelium were obtained, with quantitative analysis performed in one subject with ATF6-ACHM.ResultsFoveal hypoplasia was observed in all subjects with ATF6 mutations. Absence of the EZ band within the foveal region (grade 3) or appearance of a hyporeflective zone (grade 4) was seen in all subjects with ATF6 using OCT. There was no evidence of remnant foveal cone structure using confocal AOSLO, although sporadic cone-like structures were seen in nonconfocal split-detection AOSLO. There was a lack of cone structure in the parafovea, in direct contrast to previous reports.ConclusionsOur data demonstrate a near absence of cone structure in subjects harboring ATF6 mutations. This implicates ATF6 as having a major role in cone development and suggests that at least a subset of subjects with ATF6-ACHM have markedly fewer cellular targets for cone-directed gene therapies than do subjects with CNGA3- or CNGB3-ACHM

Using a runway paradigm to assess the relative strength of rats' motivations for enrichment objects

Laboratory animals should be provided with enrichment objects in their cages; however, it is first necessary to test whether the proposed enrichment objects provide benefits that increase the animals’ welfare. The two main paradigms currently used to assess proposed enrichment objects are the choice test, which is limited to determining relative frequency of choice, and consumer demand studies, which can indicate the strength of a preference but are complex to design. Here, we propose a third methodology: a runway paradigm, which can be used to assess the strength of an animal’s motivation for enrichment objects, is simpler to use than consumer demand studies, and is faster to complete than typical choice tests. Time spent with objects in a standard choice test was used to rank several enrichment objects in order to compare with the ranking found in our runway paradigm. The rats ran significantly more times, ran faster, and interacted longer with objects with which they had previously spent the most time. It was concluded that this simple methodology is suitable for measuring rats’ motivation to reach enrichment objects. This can be used to assess the preference for different types of enrichment objects or to measure reward system processes

Assessment of murine collagen-induced arthritis by longitudinal non-invasive duplexed molecular optical imaging

In the present study we evaluated the use of four commercially available fluorescent probes to monitor disease activity in murine CIA and its suppression during glucocorticoid therapy.  Arthritis was induced in male DBA/1 mice by immunization with type II collagen in Complete Freund's Adjuvant, followed by a boost of collagen in PBS. Four fluorescent probes from PerkinElmer in combination [ProSense 750 fluorescent activatable sensor technology (FAST) with Neutrophil Elastase 680 FAST and MMPSense 750 FAST with CatK 680 FAST] were used to monitor disease development from day 5 through to day 40 post-immunization. Fluorescence generated in vivo by the probes was correlated with clinical and histological score and paw measurements.  The fluorescence intensity emitted by each probe was shown to correlate with the conventional measurements of disease. The highest degree of correlation was observed with ProSense 750 FAST in combination with Neutrophil Elastase 680 FAST; these probes were then used to successfully assess CIA suppression during dexamethasone treatment.  We have demonstrated that longitudinal non-invasive duplexed optical fluorescence imaging provides a simple assessment of arthritic disease activity within the joints of mice following the induction of CIA and may represent a powerful tool to monitor the efficacy of drug treatments in preclinical studies