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Keywords:

  • horse;
  • laminitis;
  • hypothermia;
  • cryotherapy;
  • inflammation;
  • cytokines

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Authors' declaration of interests
  8. Source of funding
  9. Acknowledgements
  10. Manufacturers' addresses
  11. References

Reasons for performing study: The pathophysiological events inhibited by prophylactic digital hypothermia that result in reduction of the severity of acute laminitis are unknown.

Objectives: To determine if digital hypothermia inhibits lamellar inflammatory signalling during development of oligofructose (OF) induced laminitis.

Methods: Fourteen Standardbred horses were given 10 g/kg bwt OF by nasogastric tube with one forelimb (CRYO) continuously cooled by immersion in ice and water and one forelimb (NON-RX) at ambient temperature. Lamellae were harvested prior to the onset of lameness (24 h post OF administration, DEV group, n = 7) or at the onset of lameness (OG1 group, n = 7). Lamellar mRNA was purified and cDNA produced for real time-quantitative PCR analysis of mRNA concentrations of cytokines (IL-6, IL-1β, IL-10), chemokines (CXCL1, CXCL6, CXCL8/IL-8, MCP-1, MCP-2), cell adhesion molecules (ICAM-1, E-selectin), COX-2 and 3 housekeeping genes. Data were analysed (NON-RX vs. CRYO, NON-RX vs. archived control [CON, n = 7] lamellar tissue) using nonparametric tests.

Results: Compared with CON, the OG1 NON-RX had increased (P<0.05) lamellar mRNA concentrations of all measured mediators except IL-10, IL-1β and MCP-1/2, whereas only CXCL8 was increased (P<0.05) in DEV NON-RX. Within the OG1 group, CRYO limbs (compared with NON-RX) had decreased (P<0.05) mRNA concentrations of the majority of measured inflammatory mediators (no change in MCP-1 and IL-10). Within the DEV group, mRNA concentrations of CXCL-1, ICAM-1, IL-1β, CXCL8 and MCP-2 were decreased (P<0.05) and the anti-inflammatory cytokine IL-10 was increased (compared with NON-RX limbs; P<0.05).

Conclusions: Digital hypothermia effectively blocked early lamellar inflammatory events likely to play an important role in lamellar injury including the expression of chemokines, proinflammatory cytokines, COX-2 and endothelial adhesion molecules.

Potential relevance: This study demonstrates a potential mechanism by which hypothermia reduces the severity of acute laminitis, and may help identify molecular targets for future laminitis intervention.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Authors' declaration of interests
  8. Source of funding
  9. Acknowledgements
  10. Manufacturers' addresses
  11. References

Laminitis may develop secondary to primary diseases that are characterised by systemic inflammation, particularly Gram-negative sepsis (Parsons et al. 2007). Recently, the central role of inflammation in the pathogenesis of acute laminitis has been highlighted experimentally (Fontaine et al. 2001; Waguespack et al. 2004a,b; Black et al. 2006; Blikslager et al. 2006; Belknap et al. 2007; Black 2009; Faleiros et al. 2009a,b). Early upregulation of inflammatory cytokines, chemokines and other mediators, as well as an influx of leucocytes into the lamellar tissue has led to the suggestion that the lamellar failure of laminitis may be akin to the organ injury and/or failure that occurs as a result of sepsis/systemic inflammatory response syndrome (SIRS) (Blikslager et al. 2006; Belknap et al. 2007). Much of this evidence comes from experimental laminitis induction utilising black walnut extract (BWE), a unique model characterised by rapid, multisystemic, neutrophil margination and activation. There is, however, recent evidence that inflammation also plays a key role in laminitis induced by alimentary carbohydrate (CHO) overload, an experimental model that appears to more closely mimic naturally occurring sepsis in the horse. Similar lamellar inflammatory events, including inflammatory signalling and leucocyte accumulation, occur during CHO laminitis induction albeit somewhat delayed and closer to the onset of lameness (Belknap et al. 2007; Leise et al. 2011).

Maintaining a state of digital hypothermia during the development of laminitis has been demonstrated to reduce the severity of lamellar damage in the experimental alimentary CHO overload model utilising oligofructose (OF) (van Eps and Pollitt 2004, 2009). It has been suggested that hypothermia may protect lamellar tissue by: reducing delivery of haematogenous ‘trigger factors’; reducing protease enzyme activity; reducing metabolic energy requirements during ischaemia or inhibiting inflammation (van Eps and Pollitt 2004). The application of cryotherapy causes analgesia, hypometabolism and a vascular response (predominantly vasoconstriction) (Swenson et al. 1996). Hypothermia is also reported to exert a profound anti-inflammatory effect in other species through reduced production and activity of proinflammatory cytokines (IL-1β, IL-2, IL-6 and CXCL8) (Westermann et al. 1999; Lim et al. 2003; Webster et al. 2009), increased production of anti-inflammatory cytokines (IL-10) (Lim et al. 2003; Scumpia et al. 2004), reduced rolling and adhesion ofleucocytes (Kamler et al. 2005; Prandini et al. 2005) and reduced production of oxygen radicals by polymorphonuclear leucocytes (Novack et al. 1996). Recently, the profound anti-inflammatory effect of hypothermia on end-organ damage in models of sepsis and SIRS has been examined (Lim et al. 2003; Scumpia et al. 2004; Chin et al. 2007; Fujimoto et al. 2008). Pre-emptive hypothermia (10°C below normal) markedly reduced the severity of acute lung injury in a rat model of sepsis by reducing neutrophil emigration, inhibiting proinflammatory cytokine activity and increasing anti-inflammatory cytokine activity (Lim et al. 2003). In a subsequent study, less profound hypothermia (5°C below normal) applied even after the lung was primed with neutrophilic inflammation, also decreased the severity of acute lung injury, suggesting a therapeutic role for hypothermia beyond its preventive effect (Chin et al. 2007). We hypothesised that digital hypothermia would have a similar inhibitory effect on inflammatory events in the lamellae during the early stages of laminitis. To investigate this, the mRNA concentration of a range of cytokines (IL-6, IL-1β, IL-10), chemokines (CXCL1, CXCL6, CXCL8/IL-8, MCP-1, MCP-2), cell adhesion molecules (ICAM-1, E-selectin) and COX-2 was quantified in the lamellar tissue of cryotherapy-treated and untreated feet from horses undergoing experimental laminitis induction with OF.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Authors' declaration of interests
  8. Source of funding
  9. Acknowledgements
  10. Manufacturers' addresses
  11. References

The project was approved by the University of Queensland Animal Ethics Committee (AEC) that monitors compliance with the Animal Welfare Act (2001) and The Code of Practice for the care and use of animals for scientific purposes (current edition). All animals were monitored continuously by the investigators.

Animals, laminitis induction and cryotherapy procedures

Fourteen Standardbred horses (10 geldings and 4 mares) aged 4–11 years, with no lameness and no gross or radiographic abnormalities of the feet, were housed and fed in stables for 4 weeks prior to the experiment. The horses were divided into 2 groups and prepared separately for Experiment 1 (n = 8) and Experiment 2 (n = 6). Laminitis induction was by alimentary overload with OF as previously described (van Eps and Pollitt 2006). Immediately after nasogastric administration of the bolus dose (10 g/kg bwt of OF), each horse was confined to stocks, with one of the forelimbs continuously cooled (CRYO) and the other (NON-RX) forelimb maintained at ambient temperature for the duration of the experiment. Cooling was achieved by placing the CRYO limb in a rubber boot (Bigfoot Ice boot)1 containing a mixture of 50% cubed ice and 50% water, to a level just below the carpus, as previously described (van Eps and Pollitt 2004). Forelimb hoof temperature was monitored using thermistors attached to data-logging devices as previously described (van Eps and Pollitt 2004), except that the thermistors were attached to the hoof surface rather than being embedded in the hoof wall.

Two separate experiments were conducted, differing only with regards to the endpoint. In the first experiment (n = 8) the horses were subjected to euthanasia and tissues collected 24 h after the OF bolus dose. In the second experiment (n = 6) the horses were subjected to euthanasia immediately upon recognition of Obel Grade 1 lameness (OG1) (Obel 1948). Recognition of the onset of OG1 lameness in both experiments involved careful visual monitoring for a subjective increase in the frequency of weight shifting between the forelimbs. Additionally, after removal of the ice boot and prior to euthanasia, all horses were further evaluated for lameness: each horse was walked toward and away from the observer and circled to the right and left. If mild or no lameness was detected at the walk, the horses were also trotted toward and away from the observer. The horses were graded for lameness using the system described by Obel (1948). Although subjective observation was the primary means used to detect OG1 lameness (and thus the endpoint in Experiment 2), the horses in Experiment 2 were also fitted with human pedometer devices (Yamax digiwalker sw700)2 taped onto the antebrachium of both forelimbs to provide additional data on frequency of weight shifting. The total pedometer readings were recorded every 2 h and the pedometers then reset. The pedometers were assessed in the OG1 group of horses to determine if they could be used as a more objective, quantitative measurement of the primary feature of OG1 lameness, the frequent, incessant shifting of weight between limbs. In addition to the lameness monitoring, each horse also had its appetite, demeanour, oral mucous membrane capillary refill time, faecal output, heart rate and pulse quality monitored and recorded every 2 h as indicators of clinical status (data are not included in the results). At the determined endpoint for the 2 groups, each horse was anaesthetised with a combination of xylazine (1 mg/kg bwt i.v.), ketamine (2.2 mg/kg bwt i.v.) and thiopental sodium (2 mg/kg bwt i.v.) then placed in lateral recumbency. A deep surgical plane of anaesthesia was maintained with further doses of thiopental sodium (1 mg/kg bwt i.v.) as required. The forelimbs were removed rapidly by disarticulation of the metacarpophalangeal joint after placement of a tourniquet and 1.5 cm thick sagittal sections of the digit were cut with a band saw. The dorsal lamellae were rapidly dissected from the hoof and third phalanx and sections were snap frozen immediately in liquid nitrogen. Each horse was subjected to euthanasia with pentobarbital sodium (20 mg/kg bwt i.v.) once the samples were collected.

Control lamellar tissue (CON) from a previous study (Leise et al. 2011) was used for comparison with the NON-RX and CRYO feet in the present study. This CON tissue had been harvested in an identical fashion as described for the present study, and was sourced from 7 clinically normal horses that underwent sham treatment (water by nasogastric tube) as part of the other study (Leise et al. 2011).

RNA isolation and cDNA synthesis

Total RNA was extracted from 3 separate sections of forelimb lamellar tissue from the CRYO and NON-RX forelimbs of each horse from the current study and from archived control forelimb lamellar tissue using a kit (Absolutely RNA Miniprep)3 which includes a DNase treatment to remove genomic DNA contamination. The PolyA mRNA was then isolated (mRNA extraction kit)4 and used to make complementary DNA (cDNA) for each horse via reverse transcription (Retroscript)5 using a total of 400 ng of mRNA. The cDNA was frozen at -20°C until used for real-time quantitative polymerase chain reaction (RT-qPCR) analysis.

RT-qPCR

Real-time qPCR was performed using a thermocycler (LightCyler)4 and quantification with external standards was performed with the fluorescent format for SYBR Green I dye as previously described (Waguespack et al. 2004a,b). Equine specific primers for CXCL-1 (Faleiros et al. 2009a), CXCL-6 (Faleiros et al. 2011), cyclooxygenase-2 (COX-2) (Waguespack et al. 2004a), E-selectin, intercellular adhesion molecule-1 (ICAM-1), IL-1β, IL-6, IL-10, CXCL-8, monocyte chemotactic protein-1 (MCP-1) and MCP-2 (Leise et al. 2011; Faleiros et al. 2011) and 3 housekeeping genes (β-actin, β-2 microglobulin, glyceraldehyde-3-phosphate dehydrogenase) (Waguespack et al. 2004a) were used as previously described. All primers were screened using gel electrophoresis and melt curve analysis (LightCyler)4 to confirm amplification of a single cDNA fragment of the correct melting temperature and size (Waguespack et al. 2004b). Amplified cDNA fragments of each gene were ligated into a vector (TOPO 010 E. coli)6 and the vectors linearised with Hind III restriction enzyme6 for the use of templates to generate a standard curve for the RT-qPCR reaction (Waguespack et al. 2004a,b). Amplified cDNA fragments were sequenced after cloning to confirm correct DNA sequence for the products of each primer (Waguespack et al. 2004b). All PCR reactions were performed in glass capillaries in 20 µl volumes (5 µl sample DNA and 15 µl PCR master mixture). The master mix included 1 unit Taq polymerase6, 0.2 units uracil-N-glycosylase4, 1:10,000 dilution of SYBR Green stock solution, forward and reverse primers, PCR Nucleotide plus4 and PCR buffer. The PCR buffer (20 mmol/l Tris-HCl) contained 0.05% each of Tween 20 and a nonionic detergent. Amplification occurred for 40–45 cycles with the annealing temperature set at 1–5°C below melting temperature for each specific set of primers; extension was set at 72°C for 5 s and fluorescence acquisition for 10 s in the SYBR Green format. Single fluorescence acquisition in each cycle was specified at 76–82°C, depending on the melting temperature of the cDNA product of interest as previously described (Waguespack et al. 2004a,b). After cycling, melting curves of PCR products were acquired by stepwise increase of the temperature from 65 to 95°C. Standard curves were performed for each gene product of interest and water was included as a negative control. Standards and target samples were prepared in separate capillaries but always amplified during the same PCR run. In order to document that the OF protocol had resulted in increased lamellar inflammatory gene expression, RT-qPCR reactions were first performed on CON, NON-RX DEV and NON-RX OG1 cDNA samples (all in the same PCR run) for all mediators of interest. In order to compare the effect of digital hypothermia on lamellar inflammatory gene expression in OF-treated animals to normal lamellar gene expression, RT-qPCR reactions were also performed on 3 key inflammatory mediators (IL-6, IL-8, and COX-2) using CON, DEV and OG1 cDNA samples. Finally, to assess the effect of digital hypothermia on inflammatory gene expression in the OF model, RT-qPCR reactions were performed on NON-RX and CRYO cDNA samples from both DEV and OG1 groups.

All amplification reactions were performed in duplicate from the individual lamellar cDNA samples from each horse (i.e. lamellar cDNA samples from the individual horses were not pooled).

The data for the 3 housekeeping genes were assessed by use of geNorm7 to determine which 2 genes received the best acceptable score to then be used to make a normalisation factor for each sample as previously described (Blikslager et al. 2006). The corrected copy number value was determined for each sample by dividing the amplification data obtained by RT-qPCR for the different genes by the normalisation factor of the selected housekeeping genes in the same sample. Fold-changes (increase or decrease) in cytokine expression were determined by comparing normalised values between CRYO and NON-RX groups, and the NON-RX and archived control groups.

Data analysis

The horses were divided into 2 groups for analysis: those that developed Obel Grade 1 lameness prior to euthanasia (OG1 group, n = 7) and those that were not lame at euthanasia (DEV group, n = 7). The normalised RT-qPCR results of NON-RX limbs from both these groups were compared with each other, as well as the archived control samples, using Kruskal-Wallis analysis and Dunn's post tests. Normalised RT-qPCR results were then compared between the treated (CRYO) and untreated (NON-RX) limbs within the DEV and OG1 groups using Wilcoxon signed ranks tests. Pedometer counts were compared over time using Friedman analysis with Dunn's post tests. Statistics were performed using Graphpad Prism8. Significance was set at P<0.05.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Authors' declaration of interests
  8. Source of funding
  9. Acknowledgements
  10. Manufacturers' addresses
  11. References

Clinical and hoof temperature data

All horses developed diarrhoea, depression, tachycardia and tachypnoea. The onset of diarrhoea was more rapid after OF dosing in horses from Experiment 2 (mean ± s.e. 10.3 ± 0.2 h) compared with those from Experiment 1 (13.8 ± 0.9 h). Persistent tachycardia (mean heart rate >45 beats/min) developed after 8 h in both experiments, and the heart rates at 20 h were similar (68 ± 5.6 beats/min in Experiment 1; 70 ± 7.4 in Experiment 2). All horses, except one (from Experiment 1), developed pyrexia during the study period. Pyrexia (rectal temperature >38.5°C) developed earlier in the horses from Experiment 2 (14 h) than those from Experiment 1 (18 h); however, the mean rectal temperatures at 20 h were similar (39.1 ± 0.4°C in Experiment 1; 39.2 ± 0.2°C in Experiment 2). The clinical signs indicated that the majority of the horses in the DEV group responded to the OF administration (in contrast to ‘nonresponders’ in another CHO model which did not become febrile) (Leise et al. 2011).

Seven horses (one horse from Experiment 1 and all 6 horses from Experiment 2) developed OG1 lameness between 20 and 28 h after OF dosing, and were included in the OG1 group. The remaining 7 horses from Experiment 1 were sound at the time of euthanasia (24 h) and were included in the DEV group. Limb movement data from the pedometers (Experiment 2 only) documented a steady increase in the pedometer count frequency for the NON-RX limbs from 16 h onward (Fig 1). Compared with the 2 h time point, count frequency was increased in the NON-RX limbs at 18 and 20 h (P<0.05). In all individual cases, the pedometer data demonstrated an increase in NON-RX count frequency 2–4 h prior to visual recognition of shifting behaviour.

image

Figure 1. Pedometer count frequency of the untreated limbs (NON-RX) compared with the cryotherapy treated limbs (CRYO) in Experiment 2. The horses (n = 6) were recognised subjectively to develop OG1 lameness between 20 and 28 h; however, a review of the pedometer data revealed an earlier increase in weight shifting frequency in the NON-RX limbs after 16 h that was significant (compared with the 2 h time point) at 18 and 20 h (P<0.05)*. Although the pedometer data was not used to determine OG1 onset (and therefore the endpoint for horses in Experiment 2) in the current study, these results indicate that the pedometers provide an objective, quantitative indication of forelimb weight shifting that may be superior to visual observation. This may prove useful in future studies. ^denotes visual recognition of OG1 and removal of a single horse from the experiment at the corresponding time point.

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Hoof wall surface temperature during the experiment was 4.2 ± 0.52°C (mean ± s.e.) for the CRYO feet and 23.1 ± 1.4°C for the NON-RX feet.

Lamellar mRNA concentrations of inflammatory molecules: NON-RX vs. archived control

Lamellar mRNA concentrations of the majority of inflammatory molecules were increased (P<0.05) in the NON-RX feet of the OG1 group compared with archived control tissue (Table 1). CXCL8 was the only inflammatory molecule with increased (P<0.05) lamellar mRNA concentration in the NON-RX feet at the DEV time point.

Table 1. Lamellar tissue inflammatory mediator mRNA expression data: control (CON) compared with untreated (NON-RX) limbs after laminitis induction in the developmental (DEV) and Obel Grade 1 laminitis (OG1) groups
Inflammatory mediatorCONDEV NON-RXOG1 NON-RX
  1. The mRNA data is expressed as cDNA copies per normalisation factor (interquartile range); median fold increase over CON. *denotes significant difference from CON (P<0.05);^denotes significant difference in OG1 NON-RX compared with DEV NON-RX (P<0.05).

E-selectin
 mRNA11,393 (7774–19,261)19,009 (5287–34,871); 1.584,094 (70,876–187,450);6.7*
ICAM-1   
 mRNA16,783 (12,659–27,774)17,071 (13,961–28,771); 1.1161,696 (74,998–526,902); 8.4*^
CXCL-1   
 mRNA2919 (2347–5704)41,545 (16,438–78,688); 9.2592,566 (47,652–150,100); 20.6*
CXCL-6   
 mRNA377 (319–624)1031 (629–302,217); 2.116,326 (1762–38,044); 33.7*
CXCL-8 (IL-8)   
 mRNA250 (158–555)26,005 (16,439–44,682); 64.4*132,674 (14,205–153,722); 328.4*
MCP-1   
 mRNA1957 (997–2726)1966 (0–2423); 13035 (0–18,842); 1.6
MCP-2   
 mRNA223,662 (148,241–24,454)307,926 (185,172–522,226); 1.5432,761 (293,362–965,389); 2.1
IL-1β   
 mRNA3608 (3115–9633)14,025 (11,097–27,913); 2.117,428 (9872–45,273); 2.6
IL-6   
 mRNA209 (134–547)1257 (1056–6198); 3.9124,708 (6506–574,128); 385*
IL-10   
 mRNA2681 (1494–5756)6703 (2375–9958); 1.752873 (2030–4788); 0.75
COX-2   
 mRNA148 (105–526)817 (568–986); 2.513,522 (249–24,314); 40.7*

Lamellar mRNA concentrations of inflammatory molecules: CRYO vs. archived control

No differences were present (P>0.05) between CRYO samples at either the DEV or OG1 time points when compared to CON samples for any of the 3 mediators assessed (IL-6, IL-8, COX-2).

Effect of digital hypothermia on lamellar endothelial adhesion molecule mRNA concentration

Digital hypothermia (CRYO) was associated with a decrease (P<0.05) in lamellar mRNA concentrations of both adhesion molecules (ICAM-1 and E-selectin) in the OG1 group and ICAM-1 in the DEV group, when compared to the NON-RX feet (Fig 2).

image

Figure 2. Lamellar tissue mRNA expression data for the adhesion molecules E-selectin (a) and ICAM-1 (b). Digital hypothermia was associated with significantly (P<0.05) decreased expression of both adhesion molecules in the OG1 group and significantly decreased ICAM-1 in the DEV group. Fold change is comparison of sample data to mean CRYO value.

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Effect of digital hypothermia on lamellar chemokine mRNA concentration

Digital hypothermia was associated with a reduction (P<0.05) in lamellar mRNA concentrations of the neutrophil chemokines CXCL1 and CXCL8 in the CRYO feet compared to the NON-RX feet in both the DEV and OG1 groups (Fig 3), with the reduction of lamellar CXCL8 in the OG1 group (52.4-fold) being most marked. Hypothermia was associated with a decrease (P<0.05) in CXCL6 mRNA concentration in the OG1 group only. For the monocyte chemokines (MCP-1/2), hypothermia resulted in a small decrease (P<0.05) in MCP-2 at both the DEV and OG1 time points (data not shown).

image

Figure 3. Lamellar tissue mRNA expression data for the neutrophil chemokines CXCL-1 (a), CXCL-6 (b) and IL-8/CXCL8 (c). Digital hypothermia was associated with significantly (P<0.05) decreased expression of all the chemokines in the OG1 group and CXCL-1 and CXCL-8 in the DEV group. Fold change is comparison of sample data to mean CRYO value.

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Effect of digital hypothermia on lamellar proinflammatory cytokine and COX-2 mRNA concentration

Digital hypothermia resulted in a mild reduction in expression of the proinflammatory cytokines IL-1β and IL-6 in the DEV group, and a marked reduction of the same cytokines in the OG1 group (P<0.05; Fig 4). IL-10 mRNA concentration was mildly increased (1.8-fold) in the CRYO limbs of the DEV group (P<0.05), but not different in the OG1 group (data not shown). There was a marked reduction P<0.05) in COX-2 mRNA concentration (104-fold) in the CRYO limbs of the OG1 group (Fig 4).

image

Figure 4. Lamellar tissue mRNA expression data for inflammatory cytokines IL-1β (a) and IL-6 (b), as well as COX-2 (d). Digital hypothermia was associated with significantly (P<0.05) decreased expression of IL-1β, IL-6 and COX-2 in the OG1 group and IL-1β in the DEV group. Note the open triangle and circle in the OG1 IL-6 graph, indicating the lamellar concentrations from the NON-RX and CRYO feet of the individual horse exhibiting the greatest IL-6 response.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Authors' declaration of interests
  8. Source of funding
  9. Acknowledgements
  10. Manufacturers' addresses
  11. References

In this study, we assessed mRNA concentrations of proinflammatory cytokines, chemokines, endothelial adhesion molecules and COX-2 in order to study the effect of local hypothermia on these diverse molecules associated with inflammatory injury in human diseases and in equine diseases including laminitis. The molecules were studied at the mRNA level due to the facts that: 1) the data can be compared to numerous previous laminitis studies that have reported mRNA concentrations of these mediators (Waguespack et al. 2004a,b; Belknap et al. 2007; Loftus et al. 2007; Leise et al. 2011); 2) we have optimised techniques to obtain accurate data for the molecules of interest at the mRNA concentration even though equine-specific reagents are not available for accurate assessment of all of these molecules at the protein level and 3) expression of these molecules is primarily controlled at either the transcriptional level or by mRNA stability (Collins et al. 1995; Fan et al. 2005; Anderson 2008; Harper and Tyson-Capper 2008), both of which are accurately represented by mRNA concentrations.

We assessed the effect of constant hypothermia on lamellar inflammatory signalling in OF-treated horses that were either not yet exhibiting signs of lameness 24 h post OF administration (Developmental [DEV] group, n = 7), or exhibiting early clinical signs of laminitis (Obel grade 1 laminitis [OG1] group, n = 7). The data revealed the presence of a marked inflammatory process within the lamellar tissue during laminitis induction with OF that, similar to a recent report in the traditional CHO model (corn starch/wood flour mix) (Leise et al. 2011), was more profound at the onset of lameness (OG1 group) than during the late developmental period (DEV group). Although it is impossible to determine if the animals in the DEV group in the present study would have progressed to clinical laminitis, these animals demonstrated clinical signs that typically precede laminitis development in the OF model (van Eps and Pollitt 2006). The majority of DEV horses also developed fever and increased lamellar expression of chemokines (i.e. CXCL8/IL-8), 2 events recently reported as being indicative of an appropriate response in the traditional CHO laminitis model (Leise et al. 2010; Faleiros et al. 2011). The considerable heterogeneity between horses in the lamellar mRNA concentrations for the inflammatory mediators of interest in the NON-RX samples (lamellar samples from feet kept at ambient temperature post OF administration) is also similar to that reported in the traditional CHO and BWE models (Waguespack et al. 2004b; Leise et al. 2011); this heterogeneity may reflect the temporal variability between animals in the onset of events inherent to the CHO models (compared with BWE), a genetic heterogeneity in this outbred population that may make some animals more responsive to the CHO model than others, or both. In the OF model, we (authors A.V.E. and C.P.) have also noted temporal variability in the onset of clinical signs of laminitis between groups of horses in different OF studies; the only differences between these groups is likely to be subtle differences in the 4 week preparation period (including differences in feed quality) prior to OF administration. These temporal differences were noted in the present study in terms of key clinical events. The horses in Experiment 2 developed diarrhoea, pyrexia and lameness earlier than those in Experiment 1; 4 of 6 horses from Experiment 2 (compared with only one of 8 from Experiment 1) had developed OG1 laminitis by 24 h. The separate preparation and feeding of these groups (for logistical reasons) probably resulted in the temporal differences seen.

Interestingly, in the face of the heterogenous response in mediator expression to the CHO models, the exposure of the lamellae to hypothermia resulted in a much more consistent pattern of gene expression across animals, especially at the OG1 time point. This consistency was most remarkable in the animals that exhibited the most dramatic responses in the untreated feet (NON-RX), but maintained similar low mRNA concentrations in the corresponding treated (CRYO) feet to that of the other animals. An example of this can be seen in Figure 4, where a >800-fold increase in IL-6 mRNA in the NON-RX foot of one OG1 horse is paired with a low concentration in the corresponding CRYO foot from the same horse that approximates the other CRYO samples within the group. When laminar mRNA concentrations of 3 key mediators (IL-6, CXCL8/IL-8, COX-2) were compared between the CRYO laminar samples and the archived CON samples, there were no significant differences, indicating that hypothermia reduced inflammatory gene expression back to control/normal levels.

Digital hypothermia markedly reduced the expression of key inflammatory mediators associated with many of the events reported to contribute to organ injury in human sepsis and also in different experimental models of laminitis. One of the hallmarks of endothelial activation in the microcirculation leading to adhesion and extravasation of circulating leucocytes into the target tissue is the expression of adhesion molecules and chemokines. Endothelial adhesion molecules E-selectin and ICAM-1 are both reported to undergo a substantial increase in expression in both the BWE and starch CHO models (Loftus et al. 2007; Leise et al. 2011) and were both increased in the current study. As has been reported in models of human disease (Kira et al. 2005), hypothermia induced a decrease in both ICAM-1 and E-selectin gene expression, which reportedly results in decreased leucocyte emigration into affected tissues (Hildebrand et al. 2005). The marked downregulation of lamellar chemokine mRNA concentration induced by hypopthermia (over 50-fold decrease for IL-8 in OG1 animals) is also likely to blunt any adhesion and extravasation of leucocytes into the lamellar tissue. Lamellar leucocyte emigration was not assessed in the current study.

Hypothermia resulted in a consistent downregulation of lamellar proinflammatory cytokine gene expression, similar to that previously reported in other tissues in models of inflammatory disease (Westermann et al. 1999; Lim et al. 2003; Webster et al. 2009). Interestingly, as reported in the lung in a rodent sepsis model (Lim et al. 2003), IL-10, considered largely an anti-inflammatory cytokine that may play a protective role in the early stages of inflammatory disease, underwent a mild but significant increase in the hypothermic lamellae in the DEV group of the current study.

Although there is intense interest in the suppressive effects of therapeutic hypothermia on adhesion molecule and cytokine expression, there is limited information in the scientific literature regarding the effect of hypothermia on COX-2 gene expression in models of inflammatory disease. COX-2 is of great importance in inflammatory diseases in both man and horses, not only due to the central role that prostanoids play in the regulation of vascular and inflammatory events, but also due to the fact that it is a target for NSAIDs, the most commonly administered anti-inflammatory medications. Lamellar COX-2 gene expression is reported to be greatly increased in several models of laminitis (Waguespack et al. 2004a; Loftus et al. 2007; Leise et al. 2011). Studies using cell culture models in vitro to assess the effect of hypothermia on cell signalling have reported conflicting results including either an induction or a decrease in COX-2 expression upon exposure to hypothermia (Gibbons et al. 2003; Diestel et al. 2008). In our study, hypothermia caused a dramatic decrease in COX-2 gene expression, which, considering the broad ranging effects of the different prostanoids produced downstream of COX-2 (including vasomotor, platelet aggregation and proinflammatory effects), may be as important in the pathogenesis of laminitis as the inhibition of cytokines.

Inhibition of expression of inflammatory molecules such as cytokines and COX-2 may not be the only protective effect of digital hypothermia; the previously described inhibition of matrix metalloprotease (MMP)-2 gene expression in the same model indicates that hypothermia-induced inhibition of degradative enzymes may also decrease the incidence of lamellar injury (van Eps and Pollitt 2004). MMP expression is induced both by proinflammatory cytokines and by many of the upstream signalling molecules responsible for cytokine expression (i.e. NF-B) (Fanjul-Fernandez et al. 2010), thus hypothermia may inhibit MMP expression through inhibition of cytokine expression or by inhibition of upstream signalling molecules capable of induction of a diverse array of inflammatory molecules including cytokines and MMPs. Finally, hypothermia may also inhibit the activity of proteases already present in the affected lamellae (van Eps and Pollitt 2004).

The consistent inhibition of inflammatory signalling in the present study is one of the few examples of an efficacious preventative therapy for laminitis, tested in a controlled laboratory environment. The work underscores the importance of this treatment modality in the horse at risk of laminitis. The limiting factors regarding the use of cryotherapy in the clinical setting are the labour-intensive nature of its application with currently available equipment and the lack of information regarding its efficacy when initiated in the horse with acute disease (after the onset of clinical signs). Further investigations are warranted not only on the efficacy of cryotherapy when initiated in the horse already exhibiting signs of laminitis, but also on central cellular signalling pathways occurring further upstream of the broad array of inflammatory events documented to be blocked by cryotherapy. Mimicking the actions of hypothermia, a future prophylactic/therapeutic regimen could inhibit the myriad of inflammatory mediators reported to be involved in laminitis by inhibiting a single, upstream therapeutic target. Candidates for such inhibition include the NFκB and MAPK signalling pathways, both of which are therapeutic targets for human disease and have been shown to be inhibited by hypothermia in recent studies related to human disease (Schmitt et al. 2007; Diestel et al. 2008). If a central signalling mechanism is identified, it may be possible to administer a targeted therapy locally (via regional limb perfusion) to address the events in the digit while avoiding systemic complications (including immunosuppression) associated with blocking these central signalling pathways.

In conclusion, the data here further support the use of digital hypothermia as a prophylactic therapy in the presented horse at risk of laminitis. Further investigation of cell signalling mechanisms upstream of the investigated inflammatory molecules as future pharmacological targets, and investigation of the efficacy of cryotherapy initiated at the onset of clinical signs of laminitis are warranted.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Authors' declaration of interests
  8. Source of funding
  9. Acknowledgements
  10. Manufacturers' addresses
  11. References

Presented in part at the American College of Veterinary Internal Medicine Conference, Anaheim, 2010.

Manufacturers' addresses

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Authors' declaration of interests
  8. Source of funding
  9. Acknowledgements
  10. Manufacturers' addresses
  11. References

1 Bigfoot Ice Boots, Esk, Queensland, Australia.

2 Yamasa Tokei Keiki, Tokyo, Japan.

3 Stratagene; LaJolla, California, USA.

4 Roche, Indianapolis, Indiana, USA.

5 Ambion Inc, Austin, Texas, USA.

6 Invitrogen, Carlsbad, California, USA.

7 Ghent University, Ghent, Belgium.

8 GraphPad Software, San Diego, California, USA.

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  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Authors' declaration of interests
  8. Source of funding
  9. Acknowledgements
  10. Manufacturers' addresses
  11. References
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