A Study On The Mechanism Of The Model Of The Gubi Mixture Slowing Down The Rats’ Cervical Disc Degeneration Induced By Upright Standing Because Of Removed Double Forelimbs

Objective: The study is aimed to establish a model of rats’ cervical disc degenerationinduced by upright standing because of removed double forelimbs, similar to theprocess of human cervical disease caused by cervical disc degeneration, and based onthe success of this model, observe the effect of hospital-made Gubi mixture on rats’cervical disc degeneration induced by upright standingMethods: Experimental animals and grouping: Select SD rats with15day’s pregnancyand feed them in separate cages; select208-day-old young rats and randomly dividethem into two groups, namely the normal group and the double-forelimbs-removedmodel group. Modeling method: Conduct intramuscular anesthesia on the ratsaccording to the proportion of0.1g/kg body weight, remove the double forelimbs ofthe young rats, shave their fir and clean them, and then disinfect their upper limb skinwith iodine, transversely incise the skin at the position1/3of the length of theforelimb to the tip of the forelimbs, strip the skin and fascia to expose theneurovascular bundle under the deltoid and ligate with silk thread, snip with bonerongeur the humerus at the far-end of the ligated part and cut off the skin, muscle,vessels and nerves, finishing the amputation of the upper limbs. Disinfect the rats withgentamicin and then suture the muscles, fascia, skin one after another, and finallycoat the sutured parts with chlortetracycline eye ointment to prevent infection.Feedthe rats first in ordinary breeding cages after the operation, and kill the normal groupand the model group6months thereafter. Take their whole cervical spines asspecimens, and, through fixation, decalcification, dehydration, embedding, slicing,flattening, toasting, HE staining, toluidine blue staining, safranine fast green stainingand CollagenⅩ, Collagen Ⅰimmunofluorescence test, evaluate if the model issuccessful.Based on the success of the model, select12SD rats with15days’pregnancy, feed them separately. Select eighty10-day-old rats and randomly divide them into two groups: Group A (to be raised for6months) and Group B (to be raisedfor9months), each group has40rats. Cut off all their double forelimbs (using thesimilar modeling and feeding methods with the above ones). When the rats come to be5months old, randomly further divide Group A into4groups of10rats, namelyGroup C (model group), Group D (saline group), Group E (Gubi mixture group) andGroup F (positive control group). Administer drug intervention to each group, exceptthe model group, for one month: administer saline by gavage by1ml/100g weight toGroup D; administer Gubi mixture by gavage by1ml/100g weight to Group E;administer Glucosomine And Indomethacin erterucoated Tablets by gavage by1ml/100g weight to Group F. Administer these drugs two times a day, one in themorning and the other in the afternoon, for4successive weeks.When the four groupsof rats come to be6months old, kill them and take their cervical spines andsurrounding muscles as specimens. Then, conduct fresh frozen section HE stainingobservation, and deal with the cervical specimens through fixation, decalcification,dehydration, embedding, slicing, flattening, toasting, HE staining, toluidine bluestaining, safranine fast green staining and Collagen, CollagenⅠ immunofluorescencetest. When rats of Group B rats come to be8months old, randomly divide them into4groups (grouping method is the same with Group A), and administer drugs to themthrough the same method with Group A, for4successive weeks. When the rats are9months old, kill them, and make and observe their specimens and do tests with thesame method with Group A.Results:The structure of intervertebral discs of the normal control group is normal:the nucleus pulposus is in the center, the anulus fibrosus surround the nucleuspulposus and are neatly arranged in a clear hierarchy and manifest neat and intactlamellar structure, the cartilage endplate is intact; there appear a dividing layerbetween the calcified and non-calcified layer of the cartilage endplate; growthcartilage is in columnar arrangement, and closely clings to vertebrae. In the modelgroups, the shape of intervertebral disc changes: cracks and shrinkage appear in thenucleus pulposus, some of the fiber inside the nucleus pulposus are distorted anddisarranged, with blurred boundaries with the nucleus pulposus; the vertebral structureis partially damaged; the intervertebral disc has undergone some morphologicalchanges, for example, degeneration and necrosis happen to its chondrocytes, and thecartilage endplate is calcified and fractured; the height of intervertebral disc is reduced.Collagen Ⅰimmunofluorescence shows: the expression proportion ofCollagen Ⅰ positive cells of the model groups is higher than that of the normal controlgroup. Collagen Ⅹimmunofluorescence shows: the expression proportion ofCollagen Ⅹpositive sells of the model groups is higher than that of the normal controlgroup. Compared with other three groups, although the anulus fibrosus ofintervertebral disc of Group E (Gubi mixture group) is distorted, its structure is stillintact, and the layers are still clear. The degree of shrinkage of nucleus pulposus islow; the calcified cartilage layer, and the dividing layer between the calcified andnon-calcified layer have advanced toward the surface of the cartilage endplate; thestaining of some chondrocytes is uneven, and there appear a dividing layer betweenthe calcified and non-calcified layer of the cartilage endplate. Collagen ⅠImmunofluorescence shows: the expression proportion of positive cells of Gubimixture group is lower than that of the other three groups. Collagen Ⅹimmunofluorescence also shows: the expression proportion of positive cells of Gubimixture group is lower than that of the other three groups.Conclusion:The study has successfully established a model of rats’ cervical discdegeneration induced by upright standing because of removed double forelimbs. Thismodel is simple to operate and duplicate, similar to the process of human cervicaldisease caused by cervical disc degeneration. Gubi mixture can slow down the processof rats’ cervical disc degeneration induced by upright standing because of removeddouble forelimbs, by reducing the Collagen Ⅰa nd Collagen Ⅹexpression ofextracellular matrix.