Reisinger N, Schaumberger S, Nagl V, Hessenberger S, Schatzmayr G
Milk Thistle Extract and Silymarin Inhibit Lipopolysaccharide Induced Lamellar Separation of Hoof Explants in Vitro
Toxins (Basel). 2014 Oct; 6(10): 2962–2974. Published online 2014 Oct 6
Milk Thistle Extract and Silymarin Inhibit Lipopolysaccharide Induced Lamellar Separation of Hoof Explants in Vitro
Toxins (Basel). 2014 Oct; 6(10): 2962–2974. Published online 2014 Oct 6
Introduction
The paper states that "bacterial endotoxins have been suggested to contribute to the pathophysiology of laminitis", and in support cites:
Bailey SR, Adair HS, Reinemeyer CR, Morgan SJ, Brooks AC, Longhofer SL, Elliott J
Plasma concentrations of endotoxin and platelet activation in the developmental stage of oligofructose-induced laminitis
Vet Immunol Immunopathol. 2009 Jun 15;129(3-4):167-73. Epub 2008 Nov 7
Bailey's paper states "endotoxin (lipopolysaccharide; LPS)"..."does not cause laminitis when administered alone". At the most, it seems "endotoxin may contribute in the initiation of the early inflammatory changes observed in experimental models of acute laminitis".
But more recent research has found that LPS administration does not cause laminitis:
Tadros EM, Frank N
Effects of continuous or intermittent lipopolysaccharide administration for 48 hours on the systemic inflammatory response in horses
Am J Vet Res. 2012 Sep;73(9):1394-402
"Horses (given LPS for 48 hours) developed LPS tolerance within approximately 24 hours after administration was started, and the method of LPS administration did not affect the magnitude or duration of systemic inflammation. Laminitis was not induced in horses".
Tadros EM, Frank N, Newkirk KM, Donnell RL, Horohov DW
Effects of a "two-hit" model of organ damage on the systemic inflammatory response and development of laminitis in horses
Vet Immunol Immunopathol. 2012 Nov 15;150(1-2):90-100 Epub 2012 Sep 7
Horses were treated with LPS or saline, and then given oligofructose. Pre-treatment with LPS made no difference to whether horses developed laminitis or not.
Tóth F, Frank N, Chameroy KA, Bostont RC
Effects of endotoxaemia and carbohydrate overload on glucose and insulin dynamics and the development of laminitis in horses
Equine Vet J. 2009 Dec;41(9):852-8
Horses treated with LPS had signs of systemic inflammation (which would be expected - LPS stimulates an inflammatory response), but none developed clinical laminitis.
For more examples see Lipopolysaccharides.
The paper states that "bacterial endotoxins have been suggested to contribute to the pathophysiology of laminitis", and in support cites:
Bailey SR, Adair HS, Reinemeyer CR, Morgan SJ, Brooks AC, Longhofer SL, Elliott J
Plasma concentrations of endotoxin and platelet activation in the developmental stage of oligofructose-induced laminitis
Vet Immunol Immunopathol. 2009 Jun 15;129(3-4):167-73. Epub 2008 Nov 7
Bailey's paper states "endotoxin (lipopolysaccharide; LPS)"..."does not cause laminitis when administered alone". At the most, it seems "endotoxin may contribute in the initiation of the early inflammatory changes observed in experimental models of acute laminitis".
But more recent research has found that LPS administration does not cause laminitis:
Tadros EM, Frank N
Effects of continuous or intermittent lipopolysaccharide administration for 48 hours on the systemic inflammatory response in horses
Am J Vet Res. 2012 Sep;73(9):1394-402
"Horses (given LPS for 48 hours) developed LPS tolerance within approximately 24 hours after administration was started, and the method of LPS administration did not affect the magnitude or duration of systemic inflammation. Laminitis was not induced in horses".
Tadros EM, Frank N, Newkirk KM, Donnell RL, Horohov DW
Effects of a "two-hit" model of organ damage on the systemic inflammatory response and development of laminitis in horses
Vet Immunol Immunopathol. 2012 Nov 15;150(1-2):90-100 Epub 2012 Sep 7
Horses were treated with LPS or saline, and then given oligofructose. Pre-treatment with LPS made no difference to whether horses developed laminitis or not.
Tóth F, Frank N, Chameroy KA, Bostont RC
Effects of endotoxaemia and carbohydrate overload on glucose and insulin dynamics and the development of laminitis in horses
Equine Vet J. 2009 Dec;41(9):852-8
Horses treated with LPS had signs of systemic inflammation (which would be expected - LPS stimulates an inflammatory response), but none developed clinical laminitis.
For more examples see Lipopolysaccharides.
The paper then suggests that perfusing cut-off legs with LPS caused changes to the lamellae, citing:
Patan-Zugaj B, Gauff FC, Licka TF
Effects of the addition of endotoxin during perfusion of isolated forelimbs of equine cadavers
Am J Vet Res. 2012 Sep;73(9):1462-8
in which forelimbs from slaughtered horses were perfused with 80 ng LPS/l for 10 hours, and increased width and other changes were seen in the laminae. However, does it follow that something that happens under unnatural circumstances in a dead leg would happen in a live horse with naturally occurring LPS levels (particularly given that Tadros and Frank 2012 above found that when horses were given LPS for 48 hours, they developed tolerance to LPS within 24 hours)?
The paper states "laminitis is an inflammation of the lamellar tissue". This is only in SIRS laminitis - which is likely to make up no more than 10% of all cases of laminitis. By far the majority of laminitis cases - thought to be around 90% - are endocrine, i.e. caused by EMS or PPID, and recent research has suggested that there is little inflammation involved in endocrine laminitis - see Inflammation.
It is suggested that "phytogenic substances with anti-inflammatory properties might have the potential to neutralize LPS in the gut". But the research looks at hoof tissue, not the gut....
Patan-Zugaj B, Gauff FC, Licka TF
Effects of the addition of endotoxin during perfusion of isolated forelimbs of equine cadavers
Am J Vet Res. 2012 Sep;73(9):1462-8
in which forelimbs from slaughtered horses were perfused with 80 ng LPS/l for 10 hours, and increased width and other changes were seen in the laminae. However, does it follow that something that happens under unnatural circumstances in a dead leg would happen in a live horse with naturally occurring LPS levels (particularly given that Tadros and Frank 2012 above found that when horses were given LPS for 48 hours, they developed tolerance to LPS within 24 hours)?
The paper states "laminitis is an inflammation of the lamellar tissue". This is only in SIRS laminitis - which is likely to make up no more than 10% of all cases of laminitis. By far the majority of laminitis cases - thought to be around 90% - are endocrine, i.e. caused by EMS or PPID, and recent research has suggested that there is little inflammation involved in endocrine laminitis - see Inflammation.
It is suggested that "phytogenic substances with anti-inflammatory properties might have the potential to neutralize LPS in the gut". But the research looks at hoof tissue, not the gut....
Results
Substances were incubated with LPS then treated with Polymyxin B (an antibiotic with known activity against LPS) - 97% effective at reducing LPS; milk thistle - 64% effective; silymarin (75% effective).
Hoof explants cultivated with LPS showed more tissue separation (dermal-epidermal) than controls when pulled apart with forceps.
Hoof explants cultivated with the antibiotic PMB and LPS had significantly less separated tissue than explants without the antibiotic when pulled apart by forceps.
Hoof explants cultivated with larger concentrations of milk thistle or silymarin had significantly less separated tissue than explants without milk thistle or silymarin when pulled apart by forceps.
Substances were incubated with LPS then treated with Polymyxin B (an antibiotic with known activity against LPS) - 97% effective at reducing LPS; milk thistle - 64% effective; silymarin (75% effective).
Hoof explants cultivated with LPS showed more tissue separation (dermal-epidermal) than controls when pulled apart with forceps.
Hoof explants cultivated with the antibiotic PMB and LPS had significantly less separated tissue than explants without the antibiotic when pulled apart by forceps.
Hoof explants cultivated with larger concentrations of milk thistle or silymarin had significantly less separated tissue than explants without milk thistle or silymarin when pulled apart by forceps.
Discussion
Increased plasma endotoxin levels have been measured in horses following oligofructose administration:
Bailey et al. 2009 horses given 10 g/kg BW (so 5 kg for a 500 kg horse!) oligofructose (presumably by nasogastric tube) had peak plasma LPS levels of 2.4 +/- 1.0 pg/ml 8 hours after OF administration, and 5/6 horses showed signs of clinical laminitis and histopathological changes in their lamellae. "These data indicate that small quantities of endotoxin may move into the circulation from the large intestine after the sharp decrease in pH that occurs as a result of carbohydrate fermentation".
Sprouse RF, Garner HE, Green EM
Plasma endotoxin levels in horses subjected to carbohydrate induced laminitis
Equine Vet J. 1987 Jan;19(1):25-8
13/20 horses developed Obel grade 3 laminitis after carbohydrate overload, and of these, 11 had increases in plasma endotoxin from control levels of less than 0.1 ng/litre to values ranging from 2.4 to 81.53 ng/litre.
Pretreatment with LPS increased the incidence of laminitis when horses were given oligofructose:
Toth et al. 2009 - 0/8 horses given LPS only developed laminitis; 2/8 horses given 5 g/kg BW (so 2.5 kg for a 500 kg horsre) oligofructose by nasogatric tube developed laminitis; 5/8 horses given LPS then oligofructose developed laminitis.
LPS administration alone has failed to induce laminitis -
but administration of LPS has affected behaviour, haematologic and coagulation values, increased heart reat and body temperature and caused decreased digital blood flow and laminar perfusion (and appeared to cause clinical signs of laminitis!):
Duncan SG, Meyers KM, Reed SM, Grant B
Alterations in coagulation and hemograms of horses given endotoxins for 24 hours via hepatic portal infusions
Am J Vet Res. 1985 Jun;46(6):1287-93
Horses given infusions of 1 µg/kg BW LPS into the hepatic portal vein per hour for 24 hours (12 mg LPS/500 kg horse) collapsed in the first hour (without an accompanying hypotension), had decreased neutrophils and increased circulating fibrinogen, and a shortening of the recalcification tests, 1-stage prothrombin time, and activated partial thromboplastin time. All horses showed signs of hoof discomfort and "either stood with the 4 feet together beneath the body or continually shifted their weight from one front foot to the other. Hoof temperature decreased approximately 3 degrees (C) during this time and remained decreased for the duration of the experiment".
Ingle-Fehr JE, Baxter GM
Evaluation of digital and laminar blood flow in horses given a low dose of endotoxin
Am J Vet Res. 1998 Feb;59(2):192-6
6 healthy horses were given IV 0.1 µg/kg BW endotoxin (50 µg/500 kg horse). Compared to controls, endotoxin treatment caused a significant decrease in digital blood flow, increases in heart rate and body temperature, and decreased laminar perfusion.
It is hypothesised that LPS is not able to induce laminitis on its own, but plays an important role in the development of laminitis.
Endotoxins (ex-vivo, i.e. infused into a dead leg) have a negative effect on the lamellar tissue.
There is some suggestion that after 24 hours, cells start to repair the damage induced by LPS.
Polymyxin B (PMB) is known to bind and neutralize endotoxins, and has been used on horses:
Morresey PR, Mackay RJ
Endotoxin-neutralizing activity of polymyxin B in blood after IV administration in horses
Am J Vet Res. 2006 Apr;67(4):642-7
5 healthy horses were given:
1. 1 mg of polymyxin B (PMB)/kg BW was given IV.
2. 1 mg of PMB/kg BW was given IV every 8 hours for 5 treatments.
Conclusions: PMB given as multiple infusions to healthy horses did not accumulate in the vascular compartment and appeared safe. Results support repeated IV use of 1 mg of polymyxin B/kg at 8-hour intervals as treatment for endotoxemia.
Barton MH, Parviainen A, Norton N
Polymyxin B protects horses against induced endotoxaemia in vivo
Equine Vet J. 2004 Jul;36(5):397-401
4 groups of 6 healthy horses received 20 ng endotoxin/kg BW IV plus one of:
5000 u PMB/kg BW IV 30 mins before endotoxin infusion;
5000 u PMB/kg BW IV 30 mins after endotoxin infusion;
1000 u PMB/kg BW IV 30 mins prior to endotoxin infusion;
saline (controls).
Neutrophil count, serum tumour necrosis factor (TNF) activity, plasma thromboxane B2 concentration and urine GGT:creatinine ratio were measured.
PMB treatment before or after endotoxin infusion significantly reduced fever, tachycardia and serum TNS, compared to controls. Horses that received 5000 u PMB/kg BW IV 30 mins before endotoxin infusion had the best response.
Conclusion: "PMB may be a safe and effective treatment of endotoxaemia, even when administered after onset. Although nephrotoxicity was not demonstrated with this model, caution should be exercised when using PMB in azotaemic patients".
Mild adverse side effects have been seen with PBD treatment:
MacKay et al 1999 - transient tachypnea, sweating and increased plasma thromboxane B2 concentration, but prior treatment with ketoprofen eliminated all PBD-induced signs.
Schwarz et al. 2013 - 6/18 horses being treated with 5000 iu polymyxin/kg BW IV every 8 hours for endotoxaemia showed a self-limiting weak ataxic gait between 24 and 36 hours after starting treatment. Concurrent use of neostigmine may have potentiated PBD side effects.
Increased plasma endotoxin levels have been measured in horses following oligofructose administration:
Bailey et al. 2009 horses given 10 g/kg BW (so 5 kg for a 500 kg horse!) oligofructose (presumably by nasogastric tube) had peak plasma LPS levels of 2.4 +/- 1.0 pg/ml 8 hours after OF administration, and 5/6 horses showed signs of clinical laminitis and histopathological changes in their lamellae. "These data indicate that small quantities of endotoxin may move into the circulation from the large intestine after the sharp decrease in pH that occurs as a result of carbohydrate fermentation".
Sprouse RF, Garner HE, Green EM
Plasma endotoxin levels in horses subjected to carbohydrate induced laminitis
Equine Vet J. 1987 Jan;19(1):25-8
13/20 horses developed Obel grade 3 laminitis after carbohydrate overload, and of these, 11 had increases in plasma endotoxin from control levels of less than 0.1 ng/litre to values ranging from 2.4 to 81.53 ng/litre.
Pretreatment with LPS increased the incidence of laminitis when horses were given oligofructose:
Toth et al. 2009 - 0/8 horses given LPS only developed laminitis; 2/8 horses given 5 g/kg BW (so 2.5 kg for a 500 kg horsre) oligofructose by nasogatric tube developed laminitis; 5/8 horses given LPS then oligofructose developed laminitis.
LPS administration alone has failed to induce laminitis -
but administration of LPS has affected behaviour, haematologic and coagulation values, increased heart reat and body temperature and caused decreased digital blood flow and laminar perfusion (and appeared to cause clinical signs of laminitis!):
Duncan SG, Meyers KM, Reed SM, Grant B
Alterations in coagulation and hemograms of horses given endotoxins for 24 hours via hepatic portal infusions
Am J Vet Res. 1985 Jun;46(6):1287-93
Horses given infusions of 1 µg/kg BW LPS into the hepatic portal vein per hour for 24 hours (12 mg LPS/500 kg horse) collapsed in the first hour (without an accompanying hypotension), had decreased neutrophils and increased circulating fibrinogen, and a shortening of the recalcification tests, 1-stage prothrombin time, and activated partial thromboplastin time. All horses showed signs of hoof discomfort and "either stood with the 4 feet together beneath the body or continually shifted their weight from one front foot to the other. Hoof temperature decreased approximately 3 degrees (C) during this time and remained decreased for the duration of the experiment".
Ingle-Fehr JE, Baxter GM
Evaluation of digital and laminar blood flow in horses given a low dose of endotoxin
Am J Vet Res. 1998 Feb;59(2):192-6
6 healthy horses were given IV 0.1 µg/kg BW endotoxin (50 µg/500 kg horse). Compared to controls, endotoxin treatment caused a significant decrease in digital blood flow, increases in heart rate and body temperature, and decreased laminar perfusion.
It is hypothesised that LPS is not able to induce laminitis on its own, but plays an important role in the development of laminitis.
Endotoxins (ex-vivo, i.e. infused into a dead leg) have a negative effect on the lamellar tissue.
There is some suggestion that after 24 hours, cells start to repair the damage induced by LPS.
Polymyxin B (PMB) is known to bind and neutralize endotoxins, and has been used on horses:
Morresey PR, Mackay RJ
Endotoxin-neutralizing activity of polymyxin B in blood after IV administration in horses
Am J Vet Res. 2006 Apr;67(4):642-7
5 healthy horses were given:
1. 1 mg of polymyxin B (PMB)/kg BW was given IV.
2. 1 mg of PMB/kg BW was given IV every 8 hours for 5 treatments.
Conclusions: PMB given as multiple infusions to healthy horses did not accumulate in the vascular compartment and appeared safe. Results support repeated IV use of 1 mg of polymyxin B/kg at 8-hour intervals as treatment for endotoxemia.
Barton MH, Parviainen A, Norton N
Polymyxin B protects horses against induced endotoxaemia in vivo
Equine Vet J. 2004 Jul;36(5):397-401
4 groups of 6 healthy horses received 20 ng endotoxin/kg BW IV plus one of:
5000 u PMB/kg BW IV 30 mins before endotoxin infusion;
5000 u PMB/kg BW IV 30 mins after endotoxin infusion;
1000 u PMB/kg BW IV 30 mins prior to endotoxin infusion;
saline (controls).
Neutrophil count, serum tumour necrosis factor (TNF) activity, plasma thromboxane B2 concentration and urine GGT:creatinine ratio were measured.
PMB treatment before or after endotoxin infusion significantly reduced fever, tachycardia and serum TNS, compared to controls. Horses that received 5000 u PMB/kg BW IV 30 mins before endotoxin infusion had the best response.
Conclusion: "PMB may be a safe and effective treatment of endotoxaemia, even when administered after onset. Although nephrotoxicity was not demonstrated with this model, caution should be exercised when using PMB in azotaemic patients".
Mild adverse side effects have been seen with PBD treatment:
MacKay et al 1999 - transient tachypnea, sweating and increased plasma thromboxane B2 concentration, but prior treatment with ketoprofen eliminated all PBD-induced signs.
Schwarz et al. 2013 - 6/18 horses being treated with 5000 iu polymyxin/kg BW IV every 8 hours for endotoxaemia showed a self-limiting weak ataxic gait between 24 and 36 hours after starting treatment. Concurrent use of neostigmine may have potentiated PBD side effects.
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