Established A Model Of The Rats’ Lumbar Disc Degeneration Induced By Upright Standing Because Of Removed Double Forelimbs And Study The Mechanism Of The Gubi Mixture Intervention

Objective: The study is aimed to establish a model of rats’ lumbar disc degenerationinduced by upright standing because of removed double forelimbs, similar to theprocess of human lumbar intervertebral disc protrusion caused by lumbar discdegeneration, and based on the success of this model, observe the effect ofhospital-made Gubi mixture on rats’ lumbar disc degeneration induced by uprightstanding.Methods: Experimental animals and grouping: Select SD rats with15day’spregnancy and feed them in separate cages; select208-day-old young rats andrandomly divide them into two groups, namely the normal group and thedouble-forelimbs-removed model group. Modeling method: Conduct intramuscularanesthesia on the rats according to the proportion of0.1g/kg body weight, removethe double forelimbs of the young rats, shave their fir and clean them, and thendisinfect their upper limb skin with iodine, transversely incise the skin at the position1/3of the length of the forelimb to the tip of the forelimbs, strip the skin and fasciato expose the neurovascular bundle under the deltoid and ligate with silk thread, snipwith bone rongeur 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. Disinfectthe rats with gentamicin and then suture the muscles, fascia, skin one after another, and finally coat the sutured parts with chlortetracycline eye ointment to preventinfection. Feed the rats first in ordinary breeding cages after the operation, and killthe normal group and the model group6months thereafter. Take their whole lumbarspines as specimens, and, by fixation, decalcification, dehydration, embedding,slicing, flattening, toasting, HE+Al new blue-staining, toluidine blue staining,safranine fast green staining and CollagenⅠ, Collagen Ⅹ, TNF-αimmunofluorescence test, evaluate if the model is successful. Based on the successof the model, select12SD rats with15days’ pregnancy, feed them separately. Selecteighty10-day-old rats and randomly divide them into two groups: Group A (to beraised for6months) and Group B (to be raised for9months), each group has40rats.Cut off all their double forelimbs (using the similar modeling and feeding methodswith the above ones). When the rats come to be5months old, randomly furtherdivide Group A into4groups of10rats, namely Group C (model group), Group D(saline group), Group E (Gubi mixture group) and Group F (positive control group).Administer drug intervention to each group, except the model group, for one month:administer saline by gavage by1ml/100g weight to Group D; administer Gubimixture by gavage by1ml/100g weight to Group E; administer Glucosomine AndIndomethacin erterucoated Tablets by gavage by1ml/100g weight to Group F.Administer these drugs two times a day, one in the morning and the other in theafternoon, for4successive weeks. When the four groups of rats come to be6monthsold, kill them and take their lumbar spines as specimens. Then deal with the lumbarspecimens through fixation, decalcification, dehydration, embedding, slicing,flattening, toasting, HE+Al new blue-staining, toluidine blue staining, safranine fastgreen staining and CollagenⅠ, CollagenⅩ,TNF-α immunofluorescence test. Whenrats of Group B rats come to be8months old, randomly divide them into4groups(grouping method is the same with Group A), and administer drugs to them throughthe same method with Group A, for4successive weeks. When the rats are9monthsold, kill them, and make and observe their specimens and do tests with the samemethod 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 vertebralstructure is partially damaged; the intervertebral disc has undergone somemorphological changes, for example, degeneration and necrosis happen to itschondrocytes, and the cartilage endplate is calcified and fractured; the height ofintervertebral disc is reduced. Immunofluorescence shows: the expression proportionof CollagenⅠ, CollagenⅩ,TNF-α positive sells of the model groups is higher thanthat of the normal control group. After drug intervention,compared with other threegroups, although the anulus fibrosus of intervertebral disc of Group E (Gubi mixturegroup) is distorted, its structure is still intact, and the layers are still clear. The degreeof shrinkage of nucleus pulposus is low; the calcified cartilage layer, and thedividing layer between the calcified and non-calcified layer have advanced towardthe surface of the cartilage endplate; the staining of some chondrocytes is uneven,and there appear a dividing layer between the calcified and non-calcified layer of thecartilage endplate. CollagenⅠImmunofluorescence shows: the expression proportionof positive cells of Gubi mixture group is lower than that of the other three groups.CollagenⅩ Immunofluorescence shows: the expression proportion of positive cellsof Gubi mixture group is lower than that of the other three groups. TNF-αimmunofluorescence 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’ lumbar discdegeneration induced by upright standing because of removed double forelimbs.This model is simple to operate and duplicate, similar to the process of humanlumbar intervertebral disc protrusion caused by lumbar disc degeneration. Gubi mixture can slow down the process of rats’ lumbar disc degeneration induced byupright standing because of removed double forelimbs, by reducing CollagenⅠ,CollagenⅩ and TNF-α expression of extracellular matrix.