Making and Observing an Animal Model of Alcohol-induced Osteonecrosis of Femoral Head of Cock in the Sensitized Condition with Horse-serum

Background and Purpose:A bulk and long-term drinking can cause osteonecrosis of the femoral head(ONFH),and alcoholism has been confirmed are a definite cause of ONFH. Making an ideal animal model can provide a good carrier for the in-depth study of pathogenesis and preventive measures of alcohol-induced ONFH.Since Yi-Sheng Wang reported for the first time that they successfully made an animal model of alcohol-induced ONFH by pouring into stomach a high dose of strong wine to rabbits in1998,several scholars carried on the related experiment research,they proved that the method of pouring into stomach imitate the drinking of humanity,and the histology variety of the animal model made by this method was in accordance with the early pathologic characteristic of ONFH in humanity,so the method of making an animal model by pouring alcohol into the stomach is better than the other methods. But there are still some deficiencies in the method,for example,it is diferent bettwen humanity and limbs of animals such as rabbits and rats in weight-beating characteristics,and the cycle is too long.In order to solve these problems,we successfully make an animal model of alcohol-induced ONFH of cock by the method of pouring the stong alcohol into stomach in the sensitized condition with horse-serum, there are some advantages in the method,for example,the cycle of make animal model is short,the success rate is high,and more important is that it can imitate the both lower extremities weight as humanity.Materials and Methods:64 healthy adult cocks(offered by the experimental animal center of Henan province) were randomly divided into 4 groups:Group A were injected intravenously horse serum(offered by GIBICO company,USA) 10 ml/(kg·d) two times at two weeks intervals,and were given strong wine,45%alcohol content,in 10 ml/(kg·d) by pouring into stomach since two weeks after the last time horse serum injection;Group B were simple given strong wine by same method,at the same time with Group A; Group C were simple injected horse serum by same method,at the same time with Group A.Group D is no wine or horse serum treated;Group C and Group D were both given N·S,in 10 ml/(kg·d) by same method,at the same time with Group A.All animals were sacrificed in batches at the end of 4th、8th week after the first pouting into the stomach.The pathological changes of livers and femoral heads were studied under the optical microscop and scanning electron microscope(SEM).Results:1.General conditions:there was no animal death in the course of experiment. After horse serum injection,the animals no significant changes in the general condition.At the end of the 4th week after the first pouring into stomach,the animals of Group A and Group B occurred the following changes,such as,poor appetite, weight loss,hair gloss poor and mental state a little less.At the end of the 8th week after the first pouting into stomach,the animals of Group A and Group B occurred the following changes,such as,poor appetite,weight loss obviously,hair gloss disappear and mental state less.But the general conditions of the animals in Group C and Group D is good,there is no significant change compared to the general conditions before the experiment.2.Histological changes in liver:At the end of the 4th week after the first pouting into stomach,there were no significant histological changes in livers of the four groups.At the end of the 8th week after the first pouting into stomach,there were some obvious changes in the livers of animals in Group A,such as,the volume of livers increased,the texture of the livers became crisp,the appearance of the livers was primrose yellow,the cut noodle of the livers had some soapy feeling.Observed under the optical microscope,there were some significant changes in the livers of animals in Group A,such as,there were many fat vacuoles presented in the liver cell, the changs were more significant in hepatic lobule central area.there were many orange fat particles bestrew in the hepatic lobule central area in fat stain;There were some similar changes in the livers of animals in Group B,but the degree is much less than that in Group A.The livers of the animals in Group C and Group D were dark red and soft,and there was no any histological change under the optical microscope.3.Histological changes in femoral head:General observation:At the end of the 4th week after the first pouring into stomach,there was no significant change in general morphology of the femoral head of animals in each group.At the end of the 8th week after the first pouring into stomach,the femoral heads of animals in Group A were crisp,easy to chisel and cut compared to that in the other groups.Observed under the optical microscope:At the end of the 4th week after the first pouring into stomach,there were no significant histological changes in femoral heads of the animals in four groups,there were only a little changes took part in the femoral head of anmals in Group A,such as the reduction of intramedullary hematopoietic tissue; At the end of 8th week after the first pouring into stomach,the pathological changes is significant,mainly in the femoral head cartilage zone,in group A,intramedullary hematopoietic tissue can be seen significantly reduced,subchondral bone osteoblasts were filled with orange lipid in fat stain.Fet cell become proliferation and hypertrophy,adipose tissue accumulated significantly..The trabecular bone became thinning,sparse significantly,and there are multiple fracture in trabecular bone.The structural of trabecular bone was disordered.There were two animals in Group B we can find the femoral head subchondral bone marrow hematopoietic tissue reduce and adipose tissue increase,one of the two animals in Group B,we can find some trabecular bone become thinning,sparse,broken and empty bone lacunae increase. There were two animals in Group C we can find the femoral head subchondral bone marrow hematopoietic tissue reduced and adipose tissue increased,there was no such abnormal changes in the femoral heads of the animals in Group D.The fraction of trabecular bone area(32.2±2.58)%was lower,the empty bone lacuna(21.60±2.12) %was significantly increased,the average diameter of the largest fat cells (47.22±2.79)μm significantly increased,there were statistically significant differences in Group A compared to the other three groups,P＜0.01.4.The incidence of osteonecrosis:At the end of the 4rth week after the first pouting into stomach,all of the incidence of osteonecrosis in the four groups were 0. At the end of the 8th week after the first pouting into stomach,there were seven cocks occurred ONFH in Group A(7/8,five cocks occurred bilateral ONFH,two cocks occurred unilateral ONFH,the proportion of grade 1 and grade 2 is 3/9),the incidence of osteonecrosis of Group A is 87.5%;there were two cocks occurred ONFH in Group B(2/8,both two cocks occurred unilateral ONFH,the proportion of grade 1 and grade 2 is 1/1),the incidence of osteonecrosis of Group B is 25%;there were two cocks occurred ONFH in Group C(2/8,both two cocks occurred unilateral ONFH,the proportion of grade 1 and grade 2 is 1/0),the incidence of osteonecrosis of Group C is 25%;there was no cock occurred ONFH in Group D.There were statistically significant differences in Group A compared to the other three groups,P＜0.01.5.Observation through Scanning Electron Microscopy(SEM):At the end of the 8th week after the first pouting into stomach,the femoral heads were examined through scanning electron microscopy(SEM).In Group A,we can find the indentation of the femoral heads surface,accompanied by a number of cracks,and the bone trabecula loss its inhere modality,became thinner,sparse,with several fracture,and the empty bone lacuna was significantly increased,but there were no such obvious changes in Group B,C or D.Conclusions:1.In the sensitized condition with horse-serum,we successfully make an animal model of alcohol-induced ONFH of cock by the method of pouting the stong alcohol into stomach,the cycle of making animal model can be shorten,and the success rate also can be enhanced.2.Cocks can be chosen to make animal model of alcohol-induced osteonecrosis of femoral head,and they have the advantage of imitate the both lower extremities weight as humanity;