TREATMENT OF EXPERIMENTAL EQUINE LAMINITIS WITH DOXYCYCLINE

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: ANIMAL HEALTH
• Reporting Frequency: Annual
• Accession No.: 0216724
• Project Start Date: Apr 1, 2008
• Project End Date: Dec 31, 2010
• Program Code: [(N/A)]- (N/A)

Recipient Organization
LOUISIANA STATE UNIVERSITY
202 HIMES HALL
BATON ROUGE,LA 70803-0100

Performing Department
VETERINARY CLINICAL SCIENCES

Non Technical Summary
Acute laminitis is a severely debilitating disease that affects the soft tissues of horses' feet. Although it has been extensively researched, the exact mechanism of action of this disease is still unknown. It is thought that during times of systemic illness, substances are released from the gut that cause a cascade of inflammatory reactions resulting in the release of mediators in the foot. Some of these mediators are enzymes, or MMPs, that break down the soft tissues and allow rotation of the bone within the hoof. This project will help determine if doxycycline, a commonly used antibiotic and known MMP inhibitor, is effective in inhibiting MMP synthesis and release in the horse. It will also determine if doxycycline can prevent or treat lameness associated with experimentally-induced laminitis. The results of this study could provide an affordable new alternative for the treatment of equine laminitis.

Animal Health Component

25%

Research Effort Categories

Basic

50%

Applied

25%

Developmental

25%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
315	3810	1020	50%
315	3810	1160	50%

Knowledge Area
315 - Animal Welfare/Well-Being and Protection;

Subject Of Investigation
3810 - Horses, ponies, and mules;

Field Of Science
1160 - Pathology; 1020 - Physiology;

Keywords

laminitis

equine

matrix metalloproteinase

Goals / Objectives
The overall hypothesis of this project is that doxycyline will prevent the lameness and laminar structural disturbances associated with experimentally-induced laminitis through MMP inhibition. Specifically, the aims of this study are to: 1) determine if doxycycline can prevent lameness in CHO laminitis; 2) determine if doxycycline can prevent laminar structural damage associated with CHO laminitis; 3) determine if doxycycline acts through transcriptional inhibition of MMP-2 and MMP-9 and if this can be localized to the digital laminae; 4) determine if transcriptional inhibition of MMPs by doxycycline can be localized to circulating leukocytes; and 5) confirm that doxycycline decreases MMP-2 and MMP-9 activities in plasma of horses with CHO.

Project Methods
This will be accomplished by experimentally-inducing laminitis and then treating the horses with doxycycline via nasogastric intubation. During the course of the experiment (~60 hours), blood samples will be collected for MMP activity and gene expression analysis. At the conclusion of the study, the horses will be euthanized and laminar tissues collected for microscopic structural analysis, MMP gene analysis, and achival of tissues for future studies.

Progress 04/01/08 to 12/31/10

Outputs
OUTPUTS: The purpose of this study was to determine the effectiveness of pentoxifylline (PTX), oxytetracycline, and doxycycline in the treatment of equine laminitis using a corn starch overload (CSO) model. A corn starch gruel was administered to horses via nasogastric tube. Clinical parameters and digital venous samples were collected for 60 hours following CSO administration or upon reaching Obel grade 3 lameness, whichever occurred first. Horses in the treatment group received PTX, doxycycline, or oxytetracycline immediately prior to CSO administration, then every 12 hours thereafter until the completion of the study. Lameness was evaluated and clinical parameters recorded at designated times throughout the study. Plasma MMP-2 and MMP-9 activities were assessed using zymography. Carbohydrate gruel (CSO; 17.6 g/kg) administered to 20 THB geldings, ages 3-7 yrs. Control horses (n=7)received no other treatment and the treated horses received either pentoxifylline (PTX; n=7, 8.5 mg/kg IV in 1 L saline over 30 min), oxytetracycline (n=5; 10 mg/lg IV), or doxycycline (n=1; 10 mg/kg, PO) administered immediately prior to CSO, then q 12 hrs until end of study. Obel grade, TEMP, HR, and RR were recorded q 4 hrs for 36 hrs, then q 2 hrs until end of study. Digital venous blood was collected q 4 hrs, then q 2 hrs until end of study. Horses were euthanized at 60 hrs post CSO administration or upon reaching Obel grade 3 lameness, whichever occurred first. Plasma samples were analyzed for MMP-2 and MMP-9 activities using gelatin zymography. Obel grade data was analyzed using Wilcoxon signed-rank test. Clinical parameters and MMP data were anlayzed using Student's T-test. PARTICIPANTS: Susan C. Eades is a faculty memeber and the principal investigator on this project. Lee Ann Fugler is a postdoctoral fellow and performed work on horses, assays, and data analysis. Mike Keowen is a research assistant that assisted with the horse work. TARGET AUDIENCES: Target audience for this work includes equine researchers and veterinary practitioners. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Six of the seven control horses developed Obel grade 3 (median = 3) while 5/7 PTX treated horses were Obel grade 2 or less (median = 1). Four out of five horses treated with oxytetracycline or doxycycline developed Obel grade 3 lameness similar to the control horses. Pentoxifylline significantly reduced lameness in horses with laminitis induced by corn starch. All horses developed fever and tachycardia. Control horses developed tachypnea following CSO. RR did not significantly increase in the PTX group. PTX significantly decreased the mean temperature 36 - 40 hrs post-CSO compared with control horses. MMP-2 activity slightly decreased in control horses, but remained unchanged in PTX horses compared with baseline (T=0) values. MMP-9 activity mildly decreased in both Control and PTX horses compared with baseline (T=0) values. PTX decreased MMP-9 at 24 hrs compared with Control horses.

Publications

• No publications reported this period

Progress 01/01/09 to 12/31/09

Outputs
OUTPUTS: Data has been collected from 10 Thoroughbred geldings (n=5 control, n=5 PTX) administered a carbohydrate gruel via nasogastric tube. Clinical parameters and digital venous samples were collected for 60 hours following CHO administration or upon reaching Obel grade 3 lameness whichever occurred first. Horses in the treatment group received 8.5 mg/kg PTX in 1 L saline IV over 30 minutes beginning 12 hours prior to CHO administration, then every 12 hours thereafter until the completion of the study. Matrix metalloproteinase was measured in digital blood throughout the study. PARTICIPANTS: Susan C. Eades, Principal Investigator, performed animal care and in vivo experiments. Lee Ann Fugler, Post Doctoral Fellow, performed animal care, in vivo and in vitro experiments. Mike Keowen, Technician, participated in animal care. TARGET AUDIENCES: Veterinarians, Horse Owners, Trainers, Racing Industry PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
All control horses (5/5) developed Obel grade 3 lameness, four of them within 32 hours post-CHO. However, only one PTX horse reached Obel grade 3. Of the remaining four PTX horses, two never became lame, one reached Obel grade 2 and then returned to almost complete soundness by 60 hours, and one fluctuated between Obel grades 1 and 2 during the entire study. MMP analysis is being performed at this time. Pentoxifylline is a treatment option to help prevent and control equine laminitis. The optimum dosage regimen must be devised.

Publications

• Lee Ann Fugler, DVM, PhD; Susan C. Eades, DVM, PhD; Catherine E. Koch, MA; Mike L. Keowen. Clinical and matrix metalloproteinase inhibitory effects of pentoxifylline on carbohydrate overload laminitis. Journal of Equine Veterinary Science _ Vol -, No - (2010), in press.