Study On Establishment Of Acute Laminitis Model Of Dairy Cow Induced By Oligofructose And Related Pathogenesis

Dairy cow laminitis is a diffuse,serous and aseptic inflammation in the claws,and is recognized as one of the four major diseases.This disease can cause solar yellow staining/hemorrhage/ulcers,white line disease and deformed hoof in dairy cows.It can cause hoof pain,lameness,and decrease in milk production,weight,feed rate of return and reproductive performance.A series of adverse effects not only seriously affect the level of animal welfare,but also cause huge economic losses to the breeding enterprises(households)and hinder the healthy development of the dairy industry.In clinical practices,dairy cow laminitis is often secondary to local or systemic inflammatory diseases such as rumen acidosis,rumenitis,metritis,mastitis,and pleuropneumonia.Combining most of domestic and foreign dairy cow farms are in order to increase milk production,they frequently feed excessive high-energy diets so that leads to the occurrence of common metabolic diseases such as rumen acidosis,then the incidence of laminitis is also increasing.Moreover,since the onset of laminitis in dairy cows is inside the hoo f,it is diffculty to observe and detect directly,and dairy cows are relatively slow to painf ul stimulation,always show lamed symptoms when disease is serious.This has made the timely detection and accurate diagnosis and treatment of this disease great difficulty.According to literatures,scientists generally agree that most of dairy cow laminitis originates from(sub-)acute rumen acidosis caused by high-energy diet feeding.By simulating this clinical conditions,scientists built an acute laminitis model of dairy cow induced by oligofructose overload,and confirmed that this model has similar clinical manifestations and characteristic histopathological changes with acute laminitis cases.However,in the past 20 years,dairy cow lamin itis just remained in simple phenotypic research level,and the related etiology and pathogenesis are still unclear.Its scientific research progress is seriously lagging behind that of equine laminitis,making the development of its diagnosis and treatment technology more difficult.Therefore,this study based on the successful induction of laminitis in dairy cows with oligofructose,for the first time to perform related hematology and histological research.The inflammatory response of laminitis(inflamma tory molecules,inflammatory cells and related signal pathways),metalloproteinase degradation,vascular dysfunction and visceral dysfunction are explored,aiming to explain the changes before and after the occurrence of laminitis providing theoretical basis for follow-ups.In this study,12 healthy Chinese Holstein dairy cows were selected,and they were randomly divided into treatment group and control group,with 6 in each g roup.The cows in the treatment group were perfused with 17 g/kg of oligofructose(dissolved in 2 L/100 kg of deionized water)via a rumen tube at 0 h,and the control cows were perfused with 2 L/100 kg of deionized water.Before perfusion(-72 h),and after perfusion 0 h,6 h,12 h,18 h,24 h,36 h,48 h,60 h and 72 h,the treatments and controls were monitored by clinical indicators and related indicators score items,and collected 20 m L jugular vein blood.After 72 hours of oligofructose infusion,the treatments had a locomotion score ? 2 and a continuous pain response in the same hoof claw,which met the criteria for determining acute laminitis of dairy cow,indicating successfully establish acute laminitis model of dairy cow by oligofructose overload.Then,the treatments and controls would be euthanized,and laminar tissues would be collected to prepare pathological sections to observe the characteristic histological changes of laminitis.In hematology,continuous monitoring of changes in laminitic dairy cow blood routine indicators,serum inflammatory factors(cytokines,chemokines,inflammatory mediators and inducers),acute phase proteins,vasoactive substances,internal organs(liver,kidney and heart)function and electrolyte balance indicators.In histology,ultrastructural observations of laminar tissue,the gene expression of metalloproteinases and their inhibitors,the gene expression of inflammatory factors(cytokines,chemokines and inflammatory mediators),and infiltration and activation of inflammatory cells,and detection of key protein expression in NF-?B and MAPK inflammatory signaling pathways.The experimental results found:(1)Clinical manifestations of laminitic dairy cows: depression,lack of eating and drinking,severe watery diarrhea,weight transfering occured,obvious hoof band swelling,hyperactive pulsation of digital artery,increased pain in hoof claws,increased locomotion score,heart rate increased,respiratory rate decreased,rectal temperature rised,diastolic blood pressure rised,the temperature of plantar skin and capsule temperature rised,rumen contraction rate slowed down,the p H value of rumen liquid dropped.(2)The pathological characteristics of laminar tissue from laminitic dairy cows: diffuse hemorrhage plaques in the dermal lamellae were visible by naked eye.The HE sections showed elongated epidermal lamellae,sharp tips;most epidermal basal cells edema,have multiple layers,and their nucleus were lightly stained,the chromatin reticulum was rough;hyperaemia,hemorrhage,and inflammatory infiltration of monocytes and granulocytes were common;apoptotic cells and microthrombi could be seen in blood vessels.The PAS sections showed a fuzzy basement membrane,and it(partially)separated from the epidermal basal cells.(3)Dynamic monitoring of blood routine indicators and serum inflammatory factors in laminitic dairy cows: the total number of white blood cells,neutrophil ratio,hematocrit and platelet count increased;cytokines(IL-1,IL-6,IL-8 and IL-10),chemokines(CXCL-1,CXCL-6,MCP-1 and MCP-2),inflammatory mediators(E-selectin,ICAM-1,COX-2,i NOS and PAI-1)and inducing factors(LPS,HIS and LA)levels were increased.(4)Dynamic monitoring of serum acute-phase proteins and vasoactive substances in laminitic dairy cows: the contents of CRP,SAA and PCT increased,and the contents of Ia Ip decreased;the contents of PG1-2,5-HT,TXB-2 and ET-1 increased.(5)Dynamic monitoring of visceral function and electrolyte balance indicators in laminitic dairy cows: liver function(AST and ALT activity increased,TBILI content increased);kidney function(Crea and BUN content increased);the heart function(CK and LDH activity increased);electrolyte balance indicators(Ca,Cl and P content increased).(6)Ultrastructural observation of laminar tissue from laminitic dairy cows: the lamina densa was separated from the epidermal basal cell membrane;the number of hemidesmosomes on the cell membrane were reduced;the basal cytoskeleton tension filaments were deeply stained and aggregated;the cell nucleus moved to the basement membrane area and their boundary were not clear,the chromatin was marginalized.(7)The gene expression of metalloproteinases and their inhi bitors in the laminar tissues from laminitic dairy cows: the gene expression of MMPs family(MMP-2 and MMP-9)and ADANTSs family(ADAMTS-4 and ADAMTS-5)increased,while TIMPs family(TIMP-2)gene expression decreased.(8)Gene expression of inflammatory factors in the laminar tissue from laminitic dairy cows:the gene expression of cytokines(IL-1?,IL-6 and IL-8),chemokines(CXCL-1 and MCP-2),adhesion molecules(ICAM-1 and E-selectin)and inflammatory mediators(COX-2,i NOS and PAI-1)increased.(9)Infiltration and activation of inflammatory cells in the laminar tissues from laminitic dairy cows: the number of positive cells in MAC387 and CD163 increased around the deep blood vessels of the dermal lamella and at the junction of epidermal lamella and dermal lamellae.Meanwhile,the degree of increase around the deep blood vessels of dermal lamellae was more obvious.(10)Detection of key proteins of NF-?B and MAPK inflammatory signaling pathway in laminar tissue from laminitic dairy cows: the protein expression of the NF-?B signaling pathway(P-I?B?,P-I?B?/T-I?B?,P-P50,P-P50/T-P50,P-P65,T-P65 and P-P65/T-P65)increased;protein expression of the MAPK signal pathway(TLR4,My D88,P-P38,P-P38/T-P38,P-ERK,P-ERK/TERK,P-JNK,T-JNK and P-JNK/T-JNK)increased.Above all,conclusions:(1)The acute laminitis model of dairy cows is successfully built via the administration of oligofructose overload,and the treatments had typical clinical manifestations and laminar histopathological characteristics of acute laminitis.(2)During the oligofructose-induced acute laminitis of dairy cows,the increased total number of white blood cells,and the increased serum content of cytokines,chemokines,inflammatory mediators,inducing factors and acute phase proteins suggest the occurrence of systemic inflammation;abnormal levels of vasoactive substances suggest vasomotor dysfunction;liver/kidney/heart function as well electrolyte inbalance indicators suggest electrolyte balance disorders.(3)In the laminar tissue from laminitic dairy cows induced by oligofructose,increased gene expression of MMP-2,MMP-9,ADAMTS-4 and ADAMTS-5,and decreased gene expression of TIMP-2 cause the decreased number of hemidesmosomes and the increased distance from the lamina dense to basal cells,leading to the separation of basement membrane and epidermal basal cells.(4)In laminar tissues from laminitic dairy cows induced by oligofructos,infiltrated monocytes and granulocytes as well as cytokines,chemokines and inflammatory mediators produced from activated NF-?B and MAPK inflammatory signaling pathways togrther lead to the occurrence of inflammation in laminar tissues.(5)This study confirmed that acute laminitis of dairy cows is the local manifestation of systemic diseases in the hoof,and inflammation response,metalloproteinase degradation and vascular dysfunction play important roles in the happen and development of laminitis,this provides a scientific basis for further research and formulation of prevention and treatment strategies for the disease.