Effect Of 6-hydroxydopamine-induced Sympathectomy On Bone Metabolism In Rats

Bone is continuously remodeled and regulated by various systemic and local factors. Neural regulation of bone metabolism mediated by osteoblastic and osteoclastic cells has been demonstrated in histochemical and pharmacological studies. These recent findings suggest that the extension of axons of sympathetic and peripheral sensory nerves to osteoblastic and osteoclastic cells is required for the dynamic neural regulation of local bone metabolism. Bone envelopes are innervated by both myelinated and unmyelinated fibers belonging to the sensory nervous systems, releasing sensory calcitonin gene-related peptide (CGRP), and substance P(SP). CGRP and substance P, have been shown to be involved in the direct and/or indirect regulation of osteoblasts and osteoclasts.[1-7] The effect of sympathetic nervous system on bone metabolism was not well documented. 6-Hydroxydopamine, or 6-OHDA, is a neurotoxin used by neurobiologists to selectively kill dopaminergic and noradrenergic neurons. 6-OHDA enters the neurons via the dopamine and noradrenaline reuptake transporters. 6-OHDA is often used in conjunction with a selective noradrenaline reuptake inhibitor to selectively kill dopaminergic neurons only. As a neurotoxin, 6-OHDA can selectively destroy sympathetic nerve terminals. Meanwhile, the peripheral application without blood-brain barrier and axonal damage of peripheral sympathetic fibers, is a recognized chemical removal of sympathetic agents.[8-9]In this study, three different dose of systemic 6-OHDA treatment was used to destroy the sympathetic nervous system in adult rats. Analysis of mRNA levels by RT-PCR,μCT, bone histomorphometry, biomechanical testing and serum chemistry were employed to evaluate the effect of Sympatholytic by 6-OHDA induced on bone metabolism and whether or not this effect is dose-dependent.Methods:Thirty two eight-week-old male Wistar rats weighing 250±30g were divided to four groups(control, 6-OHDA 100mg/kg, 6-OHDA 200mg/kg and 6-OHDA 300mg/kg group). 6-OHDA and vehicle (saline) were intraperitoneal injection 5 days. Four weeks later, all the rats were sacrificed humanely and the femur, tibiae and plasma samples were harvested.In vitro imaging of the right proximal tibia was performed with aμCT(Locus SP)scanner, relative bone volume (BV/TV), trabecular number (Tb.N), thickness (Tb.Th), and separation (Tb.Sp) were calculated.The left femur and the sixth lumbar_L6 were used for 3-point bending test and for compression test by a servohydraulic materials testing system. Both displacement and load were recorded for biomechanical property analysis.mRNA was extracted from the right proximal femur by use of QIAzol Lysis reagent, total mRNA was treated with DNase I and cDNA was synthesized by using random primers and Moloney murine leukaemia virus reverse transcriptase. The cDNA was amplified by PCR using specific primers for RANKL, OPG and Bactin. PCR amplification was performed by using a GeneAmp PCR System. PCR products were electrophoresed on a 3% NuSive GTG agarose gel, stained with ethidium bromide, and detected on a fluoroimage analyzer. The blood sample was centrifuged and the serum was taken.The osteocalcin and TRAP 5b content in the plasma was tested by ELISA. The left proximal tibia were fixed in 4% paraformaldehyde and decalcified in 20% EDTA_{PH 7.4}. The specimens were dehydrated in ethanol and embedded in paraffin, sectioned longitudinally at 4μm thick, stained for tartrate-resistant acid phosphatase (TRAP). Histomorphometry was conducted to quantify the number of osteoclasts (Oc.N/BS) and osteoclast surface (Oc.S/BS).Results:μCT image showed the trabecular bone volume increased and separation decreased in the proximal tibia in 6-OHDA 300mg/kg group, BV/TV was increased by 18.9% and Tb.Sp was reduced by 13%.Lumber_L6 compression test showed the bone mechanical property parameter strength, ductility and toughness was increased by 19.7%_P<0.01, 28.7%_P<0.05 and 40.4%_P<0.05 respectively in the 6-OHDA 300mg/kg group in contrast to the control group. In femur midshaft 3-point bending test, all of the mechanical properties had no significant difference between the 6-OHDA treatment groups and control group.Expressions of mRNA for OPG increased a little in the 6-OHDA treated groups but no significant difference in contrast to the control group. RANKL expression was decreased significantly by 27% in the high-dose 6-OHDA 300mg/kg group vs. control group.The content of osteocalcin in plasma had no significant difference between the c6-OHDA treatment groups and the control group but TRAP5b was decreased significantly by 50.3% in the 6-OHDA 300mg/kg group vs. the control group.The number of osteoclasts (Oc.N/BS) and osteoclast surface (Oc.S/BS) were decreased significantly in the 6-OHDA 200mg/kg group and 6-OHDA 300mg/kg group by 45% and 25%.Conclusion:In this study, we found the skeletal structure of adult rat was protected by high-dose_300mg/kg 6-OHDA treatment. Trabecular volume of the proximal tibia was increased and trabecular separation was decreased, the biomechanical property such as strength, ductility and toughness was also increased in the trunk of lumber 6, which indicated high-dose 6-OHDA treatment restrained trabecular resorption and perforation and bone mechanical quality enhanced. Histomorphometric evidence of decreased osteoclast number and surface under 6-OHDA treatment supported the results above.