Effect Of Elevated Cerebrospinal Fluid Leptin Level On Osteoporotic Rabbits Model

Objective: To explore effect of increasing cerebrospinal fluid(CSF)leptin level by administration from the foramen magnum to the cerebellum medulla oblongata pool in osteoporosis in rabbit model established by ovariectomy(OVX)and methylprednisolones hemisuccinate(MPH)with observing the changes of femora bone mineral density and testing the bone metabolism.Method:(1)New Zealand white rabbits were used for experiment.36 animals were divided into an OP model group(n=30)and a Sham group(n=6)with a random number table.The bilateral OVX combined with MPH(1mg/kg/d)intramuscular injection for 8 weeks after the operation was used to establish the OP rabbit model.In Sham group animals,only bilateral ovarian dissociation was performed,and the abdominal cavity was closed.Then,sham animals were intramuscularly injected daily for 8 weeks after the operation with a volume of NS equivalent with the OP group injection volume.Blood(2 ml)was collected at before and 2,4 and 8 weeks.Serum E2 was measured with an ACA,and serum Ca was measured with an ABA.The femora BMD were measured with DEXA before and 8 weeks after the operation.(2)30 OP rabbits,which were established in Experiment(1),were equally divided into a Control group(Ctr),a Normal Saline group(NS),a Low dose leptin group(Low),a Middle dose leptin group(Middle)and a High dose leptin group(High)(n=6).Animals were puncture to the cerebellum medulla oblongata pool.Control animals were only punctured without injection of the drug or NS on days 0,3,and 7 after establishing the OP rabbit model.For NS animals,0.05 ml NS was injected into the cerebellomedullary cistern(ICV).For the Low,Middle and High animals,recombinant rabbit leptin at concentrations of 0.01μg/μl,0.1μg/μl and 1.0μg/μl,respectively,was similarly administered.CSF(0.5 ml)and blood(2 ml)collected before and on days 3,7,14,28,42 and 56.CSF leptin and serum GH,IGF-1,and OC concentration were measured by ELISA kit.Serum Ca,P,and ALP were measured with ABA.The femora BMD were measured with DEXA before and 28 and 56 days.Rabbits were sacrificed by perfusing the heart with 4% paraformaldehyde in PBS on the 56 th day.The OB-R m RNAexpression in thalamus tissue,and BMP-2,Col-Ⅰ m RNA expression in femur was conducted by real-time PCR.The OC and BMP-2 expression in thalamus tissue and femora was conducted by IHC.Result:(1)Serum estradiol(E2)levels were significantly reduced in the OP group compared to Sham animals at weeks 2,4 and 8 after surgery.In contrast,serum Ca was significantly elevated in OP model animals at weeks 4 and 8 after surgery.DEXA showed that OP rabbits had decreased femur BMD at the 8th week.(2)CSF leptin was significantly elevated in Low,Middle and High on days 3,7,14,28,42 and 56 and the peak value presented on the 7th day.Femora BMD were significantly elevated in Low,Middle and High on the 28 th and 56 th day.Elevated femur BMD and the dose of administration were found to be positively correlated.Serum GH was significantly elevated in Low,Middle and High on days 7,14,28,42 and 56,serum IGF-1 was significantly elevated on days 14,28,42 and 56,and serum OC was significantly elevated on days 28,42 and 56.Serum Ca and ALP were significantly reduced in the Low,Middle and High groups on days 14,28,42 and 56.However,serum P was reduced slightly in all groups.Expression production of femur BMP-2 and COL-I m RNA was significantly levated in Low,Middle,High.The levels of OC and BMP-2 expression in fibroblasts(femur)were found to be higher in the Low,Middle and High than Ctr and NS.However,there was no obvious OC and BMP-2 expression in nerve cells(brain)in all groups.Conclusion: The bilateral OVX combined with methylprednisolone hemisuccinate(MPH,1 mg/kg/d)intramuscular injection for 8 weeks on adult female rabbits could establish the rabbit osteoporosis model effectively.Elevated cerebrospinal fluid leptin level on osteoporotic rabbits model could promote bone metabolism,reduce bone loss,reverse the reducing of bone mineral density in osteoporosis,treatment of osteoporosis caused by bilateral ovariectomy combined with methylprednisolone,and it was positively correlated the dose of administration.