Vitamin D and calcium metabolism in horses in New Zealand : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Veterinary Science at Institute of Veterinary, Animal and Biomedical Sciences (IVABS), Massey University, Manawatū, New Zealand

The physiology of vitamin D in horses has not been studied in great depth. Few studies on vitamin D metabolites (25OHD2, 25OHD3, and 1,25(OH)2D) and their relationship to other serum analytes exist. In addition, some studies suggest that equine vitamin D physiology may be different from other species. This thesis aimed to investigate aspects of vitamin D metabolism in horses. The effect of blanketing on vitamin D synthesis and its relationship with other analytes involved in calcium homeostasis, including vitamin D metabolites (25-hydroxyvitamin D2 (25OHD2), and 25-hydroxyvitamin D3 (25OHD3), 1,25-dihydroxyvitamin D (1,25(OH)2D)), ionised calcium (iCa), total calcium (tCa), phosphorus (P), total magnesium (tMg) and parathyroid hormone (PTH) were studied in horses. Regardless of blanketing, 25OHD3 was undetectable in equine serum and 25OHD2 was the main form of 25OHD in circulation. A strong seasonal variation in serum 25OHD2, 1,25(OH)2D, iCa, tCa, P, tMg and PTH concentrations was detected, although no differences were seen between horses that were blanketed and those that were not. The circadian rhythms of serum vitamin D metabolites, iCa, tCa, P, tMg, and PTH concentrations in horses was studied over 48 h on the summer and winter solstices. A significant difference was seen between the serum concentrations of studied analytes between solstices, with no rhythm detected in winter. An in vivo study suggested that equine skin may be unable to convert 7-dehydrocholesterol (7-DHC) to vitamin D3 after exposure to ultraviolet B (UVB) light. Quantitative PCR was performed on equine kidney to study the expression of vitamin D responsive and calcium transporting genes, which were then compared to genes in sheep and dogs. The results suggested that TRPV6, calD9k /calD28k, and PMCA were the main calcium transporting pathways in the kidney of these species, and there was a high correlation between VDR and other studied genes. It was concluded that 25OHD2 is the main metabolic precursor for 1,25(OH)2D and should be considered the best available index of vitamin D status in unsupplemented horses, and that horses most likely rely on diet as their primary source of vitamin D

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

Abstract 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

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

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

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

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

<div><p>The aim of this study was to determine the relative abundance and relationship of vitamin D responsive and calcium transporting transcripts (TRPV5, TRPV6, calD_9k, calD_28k, 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, calD_9k/calD_28k, 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.</p></div