Undetectable vitamin D3 in equine skin irradiated with ultraviolet light.

Vitamin D requirements for most animals are expected to be fulfilled through daily exposure of the skin to solar ultraviolet B radiation. The synthesis of vitamin D3 in skin depends on different factors including melanin pigmentation, the amount of UVB radiation reaching the skin, type of clothing/hair coat, latitude and altitude, season, and time of day. Alternatively vitamin D2 may be obtained from UVB irradiated pasture species. Recent studies have shown that in unsupplemented grazing horses 25-hydroxyvitamin D2 is the predominant form of vitamin D in plasma, and that 25OHD3 is undetectable suggesting horses may rely on diet to obtain vitamin D. In order to mimic the natural environment of skin to sunlight exposure, five equine and two ovine devitalized skin samples were irradiated with 5 J/cm2 of UVB light followed by measurement of 7-dehydrocholesterol (7-DHC) and vitamin D3 concentrations using reverse-phase high pressure liquid chromatography (HPLC). HPLC revealed the presence of 7-DHC in the skin of both horses and sheep. Vitamin D3 was undetectable in both ovine and equine skin prior to irradiation, but after irradiation with UVB light, ovine skin showed an increase in vitamin D3 concentration (mean 0.16 ± 0.07 µg/g), whereas vitamin D3 was undetectable in equine skin. These results provide additional evidence that horses make negligible quantities of vitamin D3 in their skin after exposure to UVB light and may therefore rely on their diet as a primary source of vitamin D.

Short-duration hypothermia completed prior to reperfusion prevents intracranial pressure elevation following ischaemic stroke in rats.

Reperfusion therapies re-establish blood flow after arterial occlusion and improve outcome for ischaemic stroke patients. Intracranial pressure (ICP) elevation occurs 18-24 h after experimental stroke. This elevation is prevented by short-duration hypothermia spanning the time of reperfusion. We aimed to determine whether hypothermia-rewarming completed prior to reperfusion, also prevents ICP elevation 24 h post-stroke. Transient middle cerebral artery occlusion was performed on male outbred Wistar rats. Sixty-minute hypothermia to 33 °C, followed by rewarming was induced prior to reperfusion in one group, and after reperfusion in another group. Normothermia controls received identical anaesthesia protocols. ΔICP from pre-stroke to 24 h post-stroke was measured, and infarct volumes were calculated. Rewarming pre-reperfusion prevented ICP elevation (ΔICP = 0.3 ± 3.9 mmHg vs. normothermia ΔICP = 5.2 ± 2.1 mmHg, p = 0.02) and reduced infarct volume (pre-reperfusion = 78.6 ± 23.7 mm3 vs. normothermia = 125.1 ± 44.3 mm3, p = 0.04) 24 h post-stroke. There were no significant differences in ΔICP or infarct volumes between hypothermia groups rewarmed pre- or post-reperfusion. Hypothermia during reperfusion is not necessary for prevention of ICP rise or infarct volume reduction. Short-duration hypothermia may be an applicable early treatment strategy for stroke patients prior to- during-, and after reperfusion therapy.

Ultra-Short Duration Hypothermia Prevents Intracranial Pressure Elevation Following Ischaemic Stroke in Rats.

There is a transient increase in intracranial pressure (ICP) 18-24 h after ischaemic stroke in rats, which is prevented by short-duration hypothermia using rapid cooling methods. Clinical trials of long-duration hypothermia have been limited by feasibility and associated complications, which may be avoided by short-duration cooling. Animal studies have cooled faster than is achievable in patients. We aimed to determine whether gradual cooling at a rate of 2°C/h to 33°C or 1°C/h to 34.5°C, with a 30 min duration at target temperatures, prevented ICP elevation and reduced infarct volume in rats. Transient middle cerebral artery occlusion was performed, followed by gradual cooling to target temperature. Hypothermia to 33°C prevented significant ICP elevation (hypothermia ΔICP = 1.56 ± 2.26 mmHg vs normothermia ΔICP = 8.93 ± 4.82 mmHg; p = 0.02) and reduced infarct volume (hypothermia = 46.4 ± 12.3 mm3 vs normothermia = 85.0 ± 17.5 mm3; p = 0.01). Hypothermia to 34.5°C did not significantly prevent ICP elevation or reduce infarct volume. We showed that gradual cooling to 33°C, at cooling rates achievable in patients, had the same ICP preventative effect as traditional rapid cooling methods. This suggests that this paradigm could be translated to prevent delayed ICP rise in stroke patients.

Altered Cerebrospinal Fluid Clearance and Increased Intracranial Pressure in Rats 18 h After Experimental Cortical Ischaemia.

Oedema-independent intracranial pressure (ICP) rise peaks 20-22-h post-stroke in rats and may explain early neurological deterioration. Cerebrospinal fluid (CSF) volume changes may be involved. Cranial CSF clearance primarily occurs via the cervical lymphatics and movement into the spinal portion of the cranio-spinal compartment. We explored whether impaired CSF clearance at these sites could explain ICP rise after stroke. We recorded ICP at baseline and 18-h post-stroke, when we expect changes contributing to peak ICP to be present. CSF clearance was assessed in rats receiving photothrombotic stroke or sham surgery by intraventricular tracer infusion. Tracer concentration was quantified in the deep cervical lymph nodes ex vivo and tracer transit to the spinal subarachnoid space was imaged in vivo. ICP rose significantly from baseline to 18-h post-stroke in stroke vs. sham rats [median = 5 mmHg, interquartile range (IQR) = 0.1-9.43, n = 12, vs. -0.3 mmHg, IQR = -1.9-1.7, n = 10], p = 0.03. There was a bimodal distribution of rats with and without ICP rise. Tracer in the deep cervical lymph nodes was significantly lower in stroke with ICP rise (0 µg/mL, IQR = 0-0.11) and without ICP rise (0 µg/mL, IQR = 0-4.47) compared with sham rats (4.17 µg/mL, IQR = 0.74-8.51), p = 0.02. ICP rise was inversely correlated with faster CSF transit to the spinal subarachnoid space (R = -0.59, p = 0.006, Spearman's correlation). These data suggest that reduced cranial clearance of CSF via cervical lymphatics may contribute to post-stroke ICP rise, partially compensated via increased spinal CSF outflow.

Short-Duration Hypothermia Induction in Rats using Models for Studies examining Clinical Relevance and Mechanisms.

Therapeutic hypothermia (TH) is a powerful neuroprotective strategy that has provided robust evidence for neuroprotection in pre-clinical studies of neurological disorders. Despite strong pre-clinical evidence, TH has not shown efficacy in clinical trials of most neurological disorders. The only successful trials employing therapeutic hypothermia were related to cardiac arrest in adults and hypoxic ischemic injury in neonates. Further investigations into the parameters of its use, and study design comparisons between pre-clinical and clinical studies, are warranted. This article demonstrates two methods of short-duration hypothermia induction. The first method allows for rapid hypothermia induction in rats using ethanol spray and fans. This method works by cooling the skin, which has been less commonly used in clinical trials and may have different physiological effects. Cooling is much more rapid with this technique than is achievable in human patients due to differences in surface area to volume ratio. Along with this, a second method is also presented, which allows for a clinically achievable cooling rate for short-duration hypothermia. This method is easy to implement, reproducible and does not require active skin cooling.

Expression of Phosphatonin-Related Genes in Sheep, Dog and Horse Kidneys Using Quantitative Reverse Transcriptase PCR.

The aim of this preliminary study was to determine the relative expression of phosphatonin pathway-related genes in normal dog, sheep and horse kidneys and to explore the relationships between the different genes. Kidneys were collected post-mortem from 10 sheep, 10 horses and 8 dogs. RNA was extracted, followed by reverse transcriptase quantitative polymerase chain reaction for fibroblast growth factor receptor 1 IIIc (FGFR1IIIC), sodium-phosphate co-transporter (NPT) 1 (SLC17A1), NPT2a (SLC34A1), NPT2c (SLC34A3), parathyroid hormone 1 receptor (PTH1R), klotho (KL), vitamin D receptor (VDR), 1a-hydroxylase (CYP27B1) and 24-hydroxylase (CYP24A1). NPT2a was highly expressed in the dog kidneys, compared with those of the horses and sheep. NPT1 had greatest expression in horses and sheep, although the three different NPTs all had relatively similar expression in sheep. There was little variability in FGFR1IIIc expression, particularly in the dogs and horses. FGFR1IIIc expression was negatively correlated with NPT genes (except NPT2a in sheep), while NPT genes were all positively correlated with each other. Unexpectedly, klotho was positively correlated with NPT genes in all three species. These results provide the basis for further research into this important regulatory system. In particular, species differences in phosphatonin gene expression should be considered when considering the pathogenesis of chronic kidney disease.

Evaluation of housekeeping genes for quantitative gene expression analysis in the equine kidney.

Housekeeping genes (HKGs) are used as internal controls for normalising and calculating the relative expression of target genes in RT-qPCR experiments. There is no unique universal HKG and HKGs vary among organisms and tissues, so this study aimed to determine the most stably expressed HKGs in the equine kidney. The evaluated HKGs included 18S ribosomal RNA (18S), 28S ribosomal RNA (28S), ribosomal protein L32 (RPL32), ß-2-microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), succinate dehydrogenase complex (SDHA), zeta polypeptide (YWHAZ), and hypoxanthine phosphoribosyltransferase 1 (HPRT1). The HKGs expression stability data were analysed with two software packages, geNorm and NormFinder. The lowest stability values for geNorm suggests that YWHAZ and HPRT1 would be most optimal (M=0.31 and 0.32, respectively). Further, these two genes had the best pairwise stability value using NormFinder (geNorm V=0.085). Therefore, these two genes were considered the most useful for RT-qPCR studies in equine kidney.

Evaluation and Comparison of Vitamin D Responsive Gene Expression in Ovine, Canine and Equine Kidney.

The aim of this study was to determine the relative abundance and relationship of vitamin D responsive and calcium transporting transcripts (TRPV5, TRPV6, calD9k, calD28k, PMCA, NCX1, CYP27B1, CYP24A1, and VDR) in ovine, canine and, equine kidney using quantitative real-time PCR (RT-qPCR), and then perform a comparison between the three species. Renal tissue samples were harvested post-mortem from 10 horses, 10 sheep, and five dogs. Primers were designed for each gene. For each sample total RNA was extracted, cDNA synthesised, and RT-qPCR was performed. RT-qPCR data were normalised and statistical comparison was performed. Due to their consistent correlation with each other in each species, TRPV6, calD9k/calD28k, and PMCA appeared to be the main pathways involved in active transepithelial calcium transport in the kidney of sheep, dogs and horses. The results indicate that all of the studied genes were expressed in the renal tissue of studied species, although the expression levels and correlation of transcripts with each other were different from species to species. All vitamin D responsive and calcium transporting transcripts were highly correlated with VDR in equine kidney, but not in sheep and dogs. The CYP27B1 and CYP24A1 mRNAs showed a different renal expression pattern and correlation in horses compared with sheep and dogs. Given the high urinary calcium concentration and low serum 1,25(OH)2D concentration in horses, it could be expected that CYP27B1 expression would be lower than CYP24A1 in the horse, and this did not appear to be the case. The findings suggest that despite low serum vitamin D concentrations, vitamin D still plays a significant role in calcium metabolism in horses, especially given the strong correlations between VDR and vitamin D responsive transcripts in these animals.