Insulin and the insulin-like growth factor system : a novel theory for hyperinsulinemic laminitis pathogenesis

Research into hyperinsulinemic laminitis has progressed significantly in recent years with the use of the prolonged-euglycemic, hyperinsulinemic clamp (p-EHC). Previous investigations of laminitis pathophysiology have focused on digital vascular dysfunction, inflammation, altered glucose metabolism within the lamellae, and lamellar basement membrane breakdown by metalloproteinases. The etiopathogenesis of laminitis occurring in association with hyperinsulinemia is yet to be fully characterized, but it may not involve these mechanisms. Insulin stimulates cellular proliferation and can also affect other body systems, such as the insulin-like growth factor (IGF) system. Insulin-like growth factor-1 (IGF-1) is structurally homologous to insulin and, like insulin, binds with strong affinity to a specific tyrosine kinase receptor on the cell surface to produce its effects, which include promoting cell proliferation. Receptors for IGF-1 (IGF-1R) are present in the lamellar epidermis. An alternative theory for the pathogenesis of hyperinsulinemic laminitis is that uncontrolled cell proliferation, mediated through both the insulin receptor (InsR) and IGF-1R, leads to lengthening, weakening, and failure of the lamellae. An analysis of the proliferative activity of lamellar epidermal cells during the developmental and acute phases of hyperinsulinemic laminitis, and lamellar gene expression of the InsR and IGF-1R was undertaken.

Nutritional analysis of gastric contents and body condition score at a single time point in feral horses in Australia

Objective: To determine the impact of a free-choice diet on nutritional intake and body condition of feral horses. Animals: Cadavers of 41 feral horses from 5 Australian locations. Procedures: Body condition score (BCS) was determined (scale of 1 to 9), and the stomach was removed from horses during postmortem examination. Stomach contents were analyzed for nutritional variables and macroelement and microelement concentrations. Data were compared among the locations and also compared with recommended daily intakes for horses. Results: Mean BCS varied by location; all horses were judged to be moderately thin. The BCS for males was 1 to 3 points higher than that of females. Amount of protein in the stomach contents varied from 4.3% to 14.9% and was significantly associated with BCS. Amounts of water-soluble carbohydrate and ethanol-soluble carbohydrate in stomach contents of feral horses from all 5 locations were higher than those expected for horses eating high-quality forage. Some macroelement and microelement concentrations were grossly excessive, whereas others were grossly deficient. There was no evidence of ill health among the horses. Conclusions and Clinical Relevance: Results suggested that the diet for several populations of feral horses in Australia appeared less than optimal. However, neither low BCS nor trace mineral deficiency appeared to affect survival of the horses. Additional studies on food sources in these regions, including analysis of water-soluble carbohydrate, ethanol-soluble carbohydrate, and mineral concentrations, are warranted to determine the provenance of such rich sources of nutrients. Determination of the optimal diet for horses may need revision.

Evaluation of primary epidermal lamellar density in the forefeet of near-term fetal Australian feral and domesticated horses

Objective: To investigate the density of the primary epidermal lamellae (PEL) around the solar circumference of the forefeet of near-term fetal feral and nonferal (ie, domesticated) horses. Sample: Left forefeet from near-term Australian feral (n = 14) and domesticated (4) horse fetuses. Procedures: Near-term feral horse fetuses were obtained from culled mares within 10 minutes of death; fetuses that had died in utero 2 weeks prior to anticipated birth date and were delivered from live Thoroughbred mares were also obtained. Following disarticulation at the carpus, the left forefoot of each fetus was frozen during dissection and data collection. In a standard section of each hoof, the stratum internum PEL density was calculated at the midline center (12 o'clock) and the medial and lateral break-over points (11 and 1 o'clock), toe quarters (10 and 2 o'clock), and quarters (4 and 6 o'clock). Values for matching lateral and medial zones were averaged and expressed as 1 density. Density differences at the 4 locations between the feral and domesticated horse feet were assessed by use of imaging software analysis. Results: In fetal domesticated horse feet, PEL density did not differ among the 4 locations. In fetal feral horse feet, PEL density differed significantly among locations, with a pattern of gradual reduction from the dorsal to the palmar aspect of the foot. The PEL density distribution differed significantly between fetal domesticated and feral horse feet. Conclusions and Clinical Relevance: Results indicated that PEL density distribution differs between fetal feral and domesticated horse feet, suggestive of an adaptation of feral horses to environment challenges.

Sole depth and weight-bearing characteristics of the palmar surface of the feet of feral horses and domestic Thoroughbreds

Objective: To determine solar load-bearing structures in the feet of feral horses and investigate morphological characteristics of the sole in feral horses and domestic Thoroughbreds. Sample: Forelimbs from cadavers of 70 feral horses and 20 domestic Thoroughbreds in Australia. Procedures: Left forefeet were obtained from 3 feral horse populations from habitats of soft substrate (SS [n = 10 horses]), hard substrate (HS [10]), and a combination of SS and HS (10) and loaded in vitro. Pressure distribution was measured with a pressure plate. Sole depth was measured at 12 points across the solar plane in feet obtained from feral horses from SS (n = 20 horses) and HS (20) habitats and domestic Thoroughbreds (20). Results: Feet of feral horses from HS habitats loaded the periphery of the sole and hoof wall on a flat surface. Feral horses from HS or SS habitats had greater mean sole depth than did domestic Thoroughbreds. Sole depth was greatest peripherally and was correlated with the loading pattern. Conclusions and Clinical Relevance: The peripheral aspect of the sole in the feet of feral horses had a load-bearing function. Because of the robust nature of the tissue architecture, the hoof capsule of feral horses may be less flexible than that of typical domestic horses. The application of narrow-web horseshoes may not take full advantage of the load-bearing and force-dissipating properties of the peripheral aspect of the sole. Further studies are required to understand the effects of biomechanical stimulation on the adaptive responses of equine feet.

Distances travelled by feral horses in ‘outback’ Australia

Reasons for performing study: The distance travelled by Australian feral horses in an unrestricted environment has not previously been determined. It is important to investigate horse movement in wilderness environments to establish baseline data against which the movement of domestically managed horses and wild equids can be compared. Objectives: To determine the travel dynamics of 2 groups of feral horses in unrestricted but different wilderness environments. Methods: Twelve feral horses living in 2 wilderness environments (2000 vs. 20,000 km2) in outback Australia were tracked for 6.5 consecutive days using custom designed, collar mounted global positioning systems (GPS). Collars were attached after darting and immobilising the horses. The collars were recovered after a minimum of 6.5 days by re-darting the horses. Average daily distance travelled was calculated. Range use and watering patterns of horses were analysed by viewing GPS tracks overlaid on satellite photographs of the study area. Results: Average distance travelled was 15.9 ± 1.9 km/day (range 8.1–28.3 km/day). Horses were recorded up to 55 km from their watering points and some horses walked for 12 h to water from feeding grounds. Mean watering frequency was 2.67 days (range 1–4 days). Central Australian horses watered less frequently and showed a different range use compared to horses from central Queensland. Central Australian horses walked for long distances in direct lines to patchy food sources whereas central Queensland horses were able to graze close to water sources and moved in a more or less circular pattern around the central water source. Conclusions: The distances travelled by feral horses were far greater than those previously observed for managed domestic horses and other species of equid. Feral horses are able to travel long distances and withstand long periods without water, allowing them to survive in semi-arid conditions.

Morphometry and abnormalities of the feet of Kaimanawa feral horses in New Zealand

Objective: The present study investigated the foot health of the Kaimanawa feral horse population and tested the hypotheses that horses would have a large range of foot morphology and that the incidence of foot abnormality would be significantly high. Procedures: Abnormality was defined as a variation from what the two veterinarian assessors considered as optimal morphology and which was considered to impact negatively on the structure and/or function of the foot. Fifteen morphometric variables were measured on four calibrated photographic views of all four feet of 20 adult Kaimanawa feral horses. Four morphometric variables were measured from the lateromedial radiographs of the left forefoot of each horse. In addition, the study identified the incidence of gross abnormality observed on the photographs and radiographs of all 80 feet. Results: There was a large variation between horses in the morphometric dimensions, indicating an inconsistent foot type. Mean hoof variables were outside the normal range recommended by veterinarians and hoof care providers; 35% of all feet had a long toe conformation and 15% had a mediolateral imbalance. Abnormalities included lateral (85% of horses) and dorsal (90% of horses) wall flares, presence of laminar rings (80% of horses) and bull-nose tip of the distal phalanx (75% of horses). Both hypotheses were therefore accepted. Conclusions: The Kaimanawa feral horse population demonstrated a broad range of foot abnormalities and we propose that one reason for the questionable foot health and conformation is lack of abrasive wearing by the environment. In comparison with other feral horse populations in Australia and America there may be less pressure on the natural selection of the foot of the Kaimanawa horses by the forgiving environment of the Kaimanawa Ranges. Contrary to popular belief, the feral horse foot type should not be used as an ideal model for the domestic horse foot.

Limbal mesenchymal stromal cells (L-MSC) display immunosuppressive properties across donor and species boundaries

Purpose: We have evaluated the immunosuppressive properties of L-MSC with the view to using these cells in allogeneic cell therapies for corneal disorders. We hypothesized that L-MSC cultures would suppress T-cell activation, in a similar way to those established from human bone marrow (BM-MSC). Methods: MSC cultures were established from the limbal stroma of cadaveric donor eye tissue (up to 1 week postmortem) using either conventional serum-supplemented growth medium or a commercial serum-free medium optimized for bone marrow derived MSC (MesenCult-XF system). The MSC phenotype was examined by flow cytometry according to current and emerging markers for human MSC. Immunosuppressive properties were assessed using a mixed lymphocyte reaction (MLR) assay, whereby the white cell fraction from two immunologically incompatible blood donors are cultured together in direct contact with growth arrested MSC. T-cell activation (proliferation) was measured by uptake of tritiated thymidine. Human L-MSC were tested in parallel with human BM-MSC and rabbit L-MSC. Human and rabbit L-MSC were also tested for their ability to stimulate the growth of limbal epithelial (LE) cells in colony formation assays (for both human as well as rabbit LE cells). Results: L-MSC cultures were >95% negative for CD34, CD45 and HLA-DR and positive for CD73, CD90, CD105 and HLA-ABC. Modest levels (30%) of CD146 expression were observed for L-MSC cultures grown in serum-supplemented growth medium, but not those grown in MesenCult-XF. All MSC cultures derived from both human and rabbit tissue suppressed T-cell activation to varying degrees according to culture technique and species (MesenCult-XF >> serum-fed cultures, rabbit L-MSC >> human L-MSC). All L-MSC stimulated colony formation by LE cells irrespectively of the combination of cell species used. Conclusions: L-MSC display immunosuppressive qualities, in addition to their established non-immunogenic cell surface marker profile, and stimulate LE cell growth in vitro across species boundaries. These results support the potential use of allogeneic or even xenogeneic L-MSC in the treatment of corneal disorders.

Development of an ultra-thin fibroin membrane for RPE cell transplantation

Purpose: One of the challenges associated with cell-based therapies for repairing the retina is the development of suitable materials on which to grow and transplant retinal cells. Using the ARPE-19 cell line, we have previously demonstrated the feasibility of growing RPE-derived cells on membranes prepared from the silk protein fibroin. The present study was aimed at developing a porous, ultra-thin fibroin membrane that might better support development of apical-basal polarity in culture, and to extend this work to primary cultures of human RPE cells. Methods: Ultra-thin fibroin membranes were prepared using a highly polished casting table coated with Topas® (a cyclic olefin copolymer) and a 1:0.03 aqueous solution of fibroin and PEO (Mv 900 000 g/mol). Following drying, the membranes were water annealed to make them water-stable, washed in water to remove PEO, sterilised by treatment with 95% ethanol, and washed extensively in saline. Primary cultures containing human RPE cells were established from donor posterior eye cups and maintained in DMEM/F12 medium supplemented with 10% fetal bovine serum and antibiotics. First passage cultures were seeded onto fibroin membranes pre-coated with vitronectin and grown for 6 weeks in medium supplemented with 1% serum. Comparative cultures were established on porous 1.0 µm pore PET membrane (Millipore) and using ARPE-19 cells. Results: The fibroin membranes displayed an average thickness of 3 µm and contained numerous dimples/pore-like structures of up to 3-5 µm in diameter. The primary cultures predominantly contained pigmented epithelial cells, but mesenchymal cells (presumed fibroblasts) were also often present. Passaged cultures appeared to attach equally well to either fibroin or PET membranes. Over time cells on either material adopted a more cobblestoned morphology. Conclusions: Progress has been made towards developing a porous ultra-thin fibroin membrane that supports cultivation of RPE cells. Further studies are required to determine the degree of membrane permeability and RPE polarity.

Fibroin-based materials support cultivation of limbal stromal cells

Purpose: The silk protein fibroin provides a potential substrate for use in ocular tissue reconstruction. We have previously demonstrated that transparent membranes produced from fibroin support cultivation of human limbal epithelial cells (Tissue Eng A. 14(2008)1203-11). We presently extend this body of work to studies of human limbal stromal cell (HLS) growth on fibroin in the presence and absence of serum. Methods: Primary cultures of HLS cells were established in DMEM/F12 medium supplemented with either 10% fetal bovine serum (FBS) or 2% B27 supplement. Defined keratinocyte serum-free medium (DK-SFM, Invitrogen) was also tested. The resulting cultures were analysed by flow cytometry for expression of CD34, CD90, CD45, and CD141. Cultures grown under each condition were subsequently passaged either onto transparent fibroin membranes prepared from purified fibroin or within 3D scaffolds prepared from partially-solubilised fibroin. Results: HLS cultures were successfully established under each condition, but grew more slowly and passaged poorly in the absence of serum. Cultures grown in 10% FBS were <0.5% CD34+ (keratocytes) and >97% CD90+ (fibroblasts). Cultures established in 2% B27 formed floating spheres and contained >8% CD34+ cells and reduced CD90 expression. Cultures established in DK-SFM displayed traces of epithelial cell growth (CD141), but mostly consisted of CD90+ cells with <1% CD34+ cells. Cells of bone marrow lineage (CD45) were rarely observed under any conditions. Cultures grown in 10% FBS were able to adhere to and proliferate on silk fibroin 3-D scaffolds and transparent films while those grown serum-free could not. Adhesion of HLS cells to fibroin was initially poorer than that displayed on tissue culture plastic. Conclusions: HLS cultures containing cells of predominantly fibroblast lineage can be grown on fibroin-based materials, but this process is dependent upon additional ECM factors such as those provided by serum.

Reliability and validity of conjunctival ultraviolet autofluorescence measurement

BACKGROUND: Conjunctival ultraviolet autofluorescence (UVAF) photography was developed to detect and characterise pre-clinical sunlight-induced UV damage. The reliability of this measurement and its relationship to outdoor activity are currently unknown. METHODS: 599 people aged 16-85 years in the cross-sectional Norfolk Island Eye Study were included in the validation study. 196 UVAF individual photographs (49 people) and 60 UVAF photographs (15 people) of Norfolk Island Eye Study participants were used for intra- and inter-observer reliability assessment, respectively. Conjunctival UVAF was measured using UV photography. UVAF area was calculated using computerised methods by one grader on two occasions (intra-observer analysis) or two graders (inter-observer analysis). Outdoor activity category, during summer and winter separately, was determined with a UV questionnaire. Total UVAF equalled the area measured in four conjunctival areas (nasal/temporal conjunctiva of right and left eyes). RESULTS: Intra-observer (ρ_c=0.988, 95% CI 0.967 to 0.996, p<0.001), and inter-observer concordance correlation coefficients (ρ_c=0.924, 95% CI 0.870 to 0.956, p<0.001) of total UVAF exceeded 0.900. When grouped according to 10 mm(2) total UVAF increments, intra- and inter-observer reliability was very good (κ=0.81) and good (κ=0.71), respectively. Increasing time outdoors was strongly with increasing total UVAF in summer and winter (p(trend) <0.001). CONCLUSION: Intra- and inter-observer reliability of conjunctival UVAF is high. In this population, UVAF correlates strongly with the authors' survey-based assessment of time spent outdoors.

The association between time spent outdoors and myopia using a novel biomarker of outdoor light exposure

PURPOSE: We sought to determine whether conjunctival ultraviolet autofluorescence (UVAF), a biomarker of outdoor light exposure, is associated with myopia. METHODS: We performed a cross-sectional study on Norfolk Island and recruited individuals aged ≥ 15 years. Participants completed a sun-exposure questionnaire and underwent non-cycloplegic autorefraction. Conjunctival UVAF used a specially adapted electronic flash system fitted with UV-transmission filters (transmittance range 300-400 nm, peak 365 nm) as the excitation source. Temporal and nasal conjunctival UVAF was measured in both eyes using computerized photographic analysis with the sum referred to as "total UVAF." RESULTS: In 636 participants, prevalence of myopia decreased with an increasing quartile of total UVAF (P(trend) = 0.002). Median total UVAF was lower in subjects with myopia (spherical equivalent [SE] ≤ -1.0 diopter [D]) than participants without myopia: 16.6 mm(2) versus 28.6 mm(2), P = 0.001. In the multivariable model that adjusted for age, sex, smoking, cataract, height and weight, UVAF was independently associated with myopia (SE ≤ -1.0 D): odds ratio (OR) for total UVAF (per 10 mm(2)) was 0.81, 95% confidence interval (CI) 0.69 to 0.94, P = 0.007. UVAF was also significantly associated with myopia when analysis was restricted to subjects <50 years, and in moderate-severe myopia (SE ≤ -3.0 D). Prevalence of myopia decreased with increasing time outdoors (P(trend) = 0.03), but time outdoors was not associated with myopia on multivariable analysis. CONCLUSIONS: Study authors identified a protective association between increasing UVAF and myopia. The protective association of higher UVAF against myopia was stronger than that of increased levels of time spent outdoors as measured by this study's questionnaire. Future studies should investigate the association between UVAF and incident myopia, and its relationship to myopic progression.

Prevalence and predictors of refractive error in a genetically isolated population : the Norfolk Island Eye Study

BACKGROUND: We aimed to determine the prevalence and associations of refractive error on Norfolk Island. DESIGN: Population-based study on Norfolk Island, South Pacific. PARTICIPANTS: All permanent residents on Norfolk Island aged ≥ 15 years were invited to participate. METHODS: Patients underwent non-cycloplegic autorefraction, slit-lamp biomicroscope examination and biometry assessment. Only phakic eyes were analysed. MAIN OUTCOME MEASURES: Prevalence and multivariate associations of refractive error and myopia. RESULTS: There were 677 people (645 right phakic eyes, 648 left phakic eyes) aged ≥ 15 years were included in this study. Mean age of participants was 51.1 (standard deviation 15.7; range 15-81). Three hundred and seventy-six people (55.5%) were female. Adjusted to the 2006 Norfolk Island population, prevalence estimates of refractive error were as follows: myopia (mean spherical equivalent ≥ -1.0 D) 10.1%, hypermetropia (mean spherical equivalent ≥ 1.0 D) 36.6%, and astigmatism 17.7%. Significant independent predictors of myopia in the multivariate model were lower age (P < 0.001), longer axial length (P < 0.001), shallower anterior chamber depth (P = 0.031) and increased corneal curvature (P < 0.001). Significant independent predictors of refractive error were increasing age (P < 0.001), male gender (P = 0.009), Pitcairn ancestry (P = 0.041), cataract (P < 0.001), longer axial length (P < 0.001) and decreased corneal curvature (P < 0.001). CONCLUSIONS: The prevalence of myopia on Norfolk Island is lower than on mainland Australia, and the Norfolk Island population demonstrates ethnic differences in the prevalence estimates. Given the significant associations between refractive error and several ocular biometry characteristics, Norfolk Island may be a useful population in which to find the genetic basis of refractive error.

Immunosuppressive properties of mesenchymal stromal cell cultures derived from the limbus of human and rabbit corneas

Background aims Mesenchymal stromal cells (MSCs) cultivated from the corneal limbus (L-MSCs) provide a potential source of cells for corneal repair. In the present study, we investigated the immunosuppressive properties of human L-MSCs and putative rabbit L-MSCs to develop an allogeneic therapy and animal model of L-MSC transplantation. Methods MSC-like cultures were established from the limbal stroma of human and rabbit (New Zealand white) corneas using either serum-supplemented medium or a commercial serum-free MSC medium (MesenCult-XF Culture Kit; Stem Cell Technologies, Melbourne, Australia). L-MSC phenotype was examined by flow cytometry. The immunosuppressive properties of L-MSC cultures were assessed using mixed leukocyte reactions. L-MSC cultures were also tested for their ability to support colony formation by primary limbal epithelial (LE) cells. Results Human L-MSC cultures were typically CD34−, CD45− and HLA-DR− and CD73+, CD90+, CD105+ and HLA-ABC+. High levels (>80%) of CD146 expression were observed for L-MSC cultures grown in serum-supplemented medium but not cultures grown in MesenCult-XF (approximately 1%). Rabbit L-MSCs were approximately 95% positive for major histocompatibility complex class I and expressed lower levels of major histocompatibility complex class II (approximately 10%), CD45 (approximately 20%), CD105 (approximately 60%) and CD90 (<10%). Human L-MSCs and rabbit L-MSCs suppressed human T-cell proliferation by up to 75%. Conversely, L-MSCs from either species stimulated a 2-fold to 3-fold increase in LE cell colony formation. Conclusions L-MSCs display immunosuppressive qualities in addition to their established non-immunogenic profile and stimulate LE cell growth in vitro across species boundaries. These results support the potential use of allogeneic L-MSCs in the treatment of corneal disorders and suggest that the rabbit would provide a useful pre-clinical model.

Diurnal rhythm and effects of feeding, exercise and recombinant equine growth hormone on serum insulin concentrations in the horse

Reasons for performing the study As growth hormone increases lean body mass, it could be a therapy for obese horses. However, growth hormone use induces hyperinsulinaemia in some species, so further investigation is warranted. Objectives To investigate the effects of feeding, exercise and growth hormone therapy on basal insulin concentrations in healthy horses. Study design In vivo experimental study. Methods Blood samples were obtained every 30 min from 12 geldings over 24 h, to establish basal serum insulin concentrations, before they underwent a 3-week exercise programme. Horses were allocated into 2 groups and exercised for another 4 weeks. Group A received daily i.m. injections of recombinant equine growth hormone; 5 mg/day for 5 days, then 12.5 mg/day for 16 days. Blood samples were taken daily before feeding. Insulin vs. time area under curve of Groups A and B were compared using a Student's unpaired t test. Results Horses demonstrated insulin peaks within 2 h of feeding of 577 ± 108.3 pmol/l at 09.30 h and 342.4 ± 75.7 pmol/l at 17.30 h, despite receiving the same meal. The nadir was between midnight and 07.30 h. Exercise had no effect on basal insulin concentrations prior to equine growth hormone administrations. The equine growth hormone injections increased serum insulin concentrations (P = 0.01) within Group A, from 44.4 ± 15.3 pmol/l initially to 320.9 ± 238.2 pmol/l by Day 12. Exogenous growth hormone caused variable hyperinsulinaemia, which was alleviated once equine growth hormone administration ceased. Conclusions Single serum samples taken prior to the morning meal provide basal insulin concentrations. Exercise did not change basal insulin concentrations. However, equine growth hormone injections increased basal insulin concentrations, which were not ameliorated by exercise. Potential relevance This therapy is not recommended to address obesity in insulin-resistant equids.