Comparative analysis of vertebral transcriptome in Japanese seabass (Lateolabrax japonicus) fed diets with varying phosphorus/calcium levels.

Aquaculture jeopardizes the aquatic environment by discharge of the most dietary phosphorus (P) into the water. Reducing the dietary P level is a common approach for decreasing the P discharge but it may result in increased risk of P deficiency leading to vertebral deformities. However, the molecular mechanism of vertebral deformities is poorly understood. We assessed vertebral transcriptome and compared the genes associated with bone metabolism in Japanese seabass (Lateolabrax japonicus) fed three diets containing different P and Ca levels including: diet I (0.4% P, 0.3% Ca), diet II (0.8% P, 0.3% Ca) and diet III (0.8% P, 3% Ca). The results showed that P deficiency reduces the ossification of vertebrae and induces visible vertebral deformities. Moreover, 256 gens were up-regulated and 125 genes were down-regulated in fish fed P deficient diets. Furthermore, administration of the diet with adequate P and Ca excess (diet III) resulted in the significant enhancement in expression of 19 genes and reduced expression of 93 genes. Comparing group II with group III, expression of 109 genes was up-regulated and expression of 1369 genes was down-regulated. Gene ontology enrichment analysis revealed significant alterations in biological functions by P deficiency. In summary, these findings indicated that both dietary P shortage and Ca excess lead to reduced differentiation and proliferation of osteoblast and induce a higher activity of osteoclastogenesis, which could subsequently impair vertebral mineralization and cause skeletal deformities.

Pulsed electromagnetic fields prevented the decrease of bone formation in hindlimb-suspended rats by activating sAC/cAMP/PKA/CREB signaling pathway.

Microgravity is one of the main threats to the health of astronauts. Pulsed electromagnetic fields (PEMFs) have been considered as one of the potential countermeasures for bone loss induced by space flight. However, the optimal therapeutic parameters of PEMFs have not been obtained and the action mechanism is still largely unknown. In this study, a set of optimal therapeutic parameters for PEMFs (50 Hz, 0.6 mT 50% duty cycle and 90 min/day) selected based on high-throughput screening with cultured osteoblasts was used to prevent bone loss in rats induced by hindlimb suspension, a commonly accepted animal model to simulate the space environment. It was found that hindlimb suspension for 4 weeks led to significant decreases in femoral and vertebral bone mineral density (BMD) and their maximal loads, severe deterioration in bone micro-structure, and decreases in levels of bone formation markers and increases in bone resorption markers. PEMF treatment prevented about 50% of the decreased BMD and maximal loads, preserved the microstructure of cancellous bone and thickness of cortical bone, and inhibited decreases in bone formation markers. Histological analyses revealed that PEMFs significantly alleviated the reduction in osteoblast number and inhibited the increase in adipocyte number in the bone marrow. PEMFs also blocked decreases in serum levels of parathyroid hormone and its downstream signal molecule cAMP, and maintained the phosphorylation levels of protein kinase A (PKA) and cAMP response element-binding protein (CREB). The expression level of soluble adenylyl cyclases (sAC) was also maintained. It therefore can be concluded that PEMFs partially prevented the bone loss induced by weightless environment by maintaining bone formation through signaling of the sAC/cAMP/PKA/CREB pathway. Bioelectromagnetics. 39:569-584, 2018. © 2018 Wiley Periodicals, Inc.

Ginsenoside Rd attenuates mitochondrial permeability transition and cytochrome C release in isolated spinal cord mitochondria: involvement of kinase-mediated pathways.

Ginsenoside Rd (Rd), one of the main active ingredients in Panax ginseng, has multifunctional activity via different mechanisms and neuroprotective effects that are exerted probably via its antioxidant or free radical scavenger action. However, the effects of Rd on spinal cord mitochondrial dysfunction and underlying mechanisms are still obscure. In this study, we sought to investigate the in vitro effects of Rd on mitochondrial integrity and redox balance in isolated spinal cord mitochondria. We verified that Ca2+ dissipated the membrane potential, provoked mitochondrial swelling and decreased NAD(P)H matrix content, which were all attenuated by Rd pretreatment in a dose-dependent manner. In contrast, Rd was not able to inhibit Ca2+ induced mitochondrial hydrogen peroxide generation. The results of Western blot showed that Rd significantly increased the expression of p-Akt and p-ERK, but had no effects on phosphorylation of PKC and p38. In addition, Rd treatment significantly attenuated Ca2+ induced cytochrome c release, which was partly reversed by antagonists of Akt and ERK, but not p-38 inhibitor. The effects of bisindolylmaleimide, a PKC inhibitor, on Rd-induced inhibition of cytochrome c release seem to be at the level of its own detrimental activity on mitochondrial function. Furthermore, we also found that pretreatment with Rd in vivo (10 and 50 mg/kg) protected spinal cord mitochondria against Ca2+ induced mitochondrial membrane potential dissipation and cytochrome c release. It is concluded that Rd regulate mitochondrial permeability transition pore formation and cytochrome c release through protein kinases dependent mechanism involving activation of intramitochondrial Akt and ERK pathways.

Spinal involvement and muscle cramps in electrically elicited muscle contractions.

Electrical stimulation of innervated muscles has been investigated for many decades with alternations of high and low clinical interest in the fields of rehabilitation medicine and sports sciences. Early work demonstrated that afferent fibers have lower thresholds and are usually activated first (therefore eliciting an H-reflex). In the case of nerve trunk stimulation, the order of recruitment is mostly conditioned by the axonal dimension and excitability threshold. In the case of muscle motor point stimulation, the spatial distribution of nerve branches plays a predominant role. Sustained stimulation produces a progressive increase of force that is often maintained in subsequent voluntary activation by stroke patients. This observation suggested a facilitation mechanism at the spinal and/or supraspinal level. Such facilitation has been observed in healthy subjects as well, and may explain the generation of cramps elicited during stimulation and sustained for dozens of seconds after the stimulation has been interrupted. The most recent interpretations of facilitation resulting from peripheral stimulation focused on presynaptic (potentiation of neurotransmitter release from afferent fibers) or postsynaptic (generation of "persistent inward currents" in spinal motor neurons or interneurons) mechanisms. The renewed attention to these phenomena is once more increasing the interest toward electrical stimulation of the neuromuscular system. This is an opportunity for a structured investigation of the field aimed to resolving elements of confusion and controversy that still plague this area of electrophysiology.

Dietary fatty acids and inflammation in the vertebral column of Atlantic salmon, Salmo salar L., smolts: a possible link to spinal deformities.

Vegetable oils (Vo) are an alternative to fish oil (Fo) in aquaculture feeds. This study aimed to evaluate the effect of dietary soybean oil (Vo diet), rich in linoleic acid, and of dietary fish oil (Fo diet) on the development of spinal deformities under bacterial lipopolysaccharide (LPS)-induced chronic inflammation conditions in Atlantic salmon, Salmo salar L. Fish [25 g body weight (BW)] were fed the experimental diets for 99 days. On day 47 of feeding (40 g BW), fish were subjected to four experimental regimes: (i) intramuscular injections with LPS, (ii) sham-injected phosphate-buffered saline (PBS), (iii) intraperitoneally injected commercial oil adjuvant vaccine, or (iv) no treatment. The fish continued under a common feeding regime in sea water for 165 more days. Body weight was temporarily higher in the Vo group than in the Fo group prior to immunization and was also affected by the type of immunization. At the end of the trial, no differences were seen between the dietary groups. The overall prevalence of spinal deformities was approximately 14% at the end of the experiment. The Vo diet affected vertebral shape but did not induce spinal deformities. In groups injected with LPS and PBS, spinal deformities ranged between 21% and 38%, diet independent. Deformed vertebrae were located at or in proximity to the injection point. Assessment of inflammatory markers revealed high levels of plasma prostaglandin E2 (PGE2) in the Vo-fed and LPS-injected groups, suggesting an inflammatory response to LPS. Cyclooxigenase 2 (COX-2) mRNA expression in bone was higher in fish fed Fo compared to Vo-fed fish. Gene expression of immunoglobulin M (IgM) was up-regulated in bone of all LPS-injected groups irrespective of dietary oil. In conclusion, the study suggests that Vo is not a risk factor for the development of inflammation-related spinal deformities. At the same time, we found evidence that localized injection-related processes could trigger the development of vertebral body malformations.

Proliferation and osteoblastic differentiation of bone marrow stem cells: comparison of vertebral body and iliac crest.

Bone marrow stem cells (BMSCs) can be obtained from the vertebral body (VB) and iliac crest (IC) for augmenting spinal arthrodesis. However, it is still not evaluated, which of the two sites would have a better BMSCs potential on Proliferation and osteoblastic differentiation is still not evaluated. Fourteen patients (10 men and 4 women) undergoing posterolateral lumbar arthrodesis and pedicle screw instrumentation were involved. The mean age was 54.7 years (range 31-75 years). Bone marrow aspirates were obtained from the vertebral body through the bilateral pedicle and were quantified relative to matched, bilateral aspirates from the iliac crest that were obtained from the same patient and at the same time. The mononuclear cell count and concentration of BMSCs were calculated and compared. Proliferation and osteoblastic differentiation of each of the BMSCs were characterized using biochemical and molecular biology techniques. Concentration (cells/mL) of BMSCs from VB and IC were 3.73 × 10(3) and 3.19 × 10(3), respectively (P > 0.05). VB and IC exhibited similar proliferation pattern at 3, 5 and 7 days, but BMSCs from the VB exhibited an increased mineralization staining with Alizarin Red S at 14 days. BMSCs from both anatomic sites expressed comparable levels of CD29, CD34, CD44, CD90 and CD105. VB and IC displayed similar levels of expression of ALP, type I collagen and osterix, but VB expressed higher level of osteocalcin and Runx-2, especially at 14 and 21 days. Our studies show that BMSCs from VB have osteogenic differentiation potential similar to IC. Based on these findings, we suggest that BMSCs from VB would be comparable candidates for osseous graft supplementation especially in spinal fusion procedures.

Magnetic stimulation of the spine: the role of tissues and their modelling.

Numerical modelling of magnetic stimulation in the spine is a scarce subject in the literature, although it has been gaining clinical acceptance. In the present work we present the results from a simplified computational model of the spine. The results indicate that it is necessary to use a numerical technique for solving the problem, which takes into account tissue dispersion and both dielectric properties (conductivity and permittivity), since a difference of 14% in the induced electric fields was found when displacement currents were included. With respect to the role of tissues in stimulation efficiency, it was confirmed that water-rich tissues lead to a shielding effect of the spinal cord. However, this effect becomes smaller at the height of the intervertebral discs, resulting in an increase of the field inside the spine.

Pain mechanisms: labeled lines versus convergence in central processing.

The issue of whether pain is represented by specific neural elements or by patterned activity within a convergent somatosensory subsystem has been debated for over a century. The gate control theory introduced in 1965 denied central specificity, and since then most authors have endorsed convergent wide-dynamic-range neurons. Recent functional and anatomical findings provide compelling support for a new perspective that views pain in humans as a homeostatic emotion that integrates both specific labeled lines and convergent somatic activity.

Successful transpedicular lumbar interbody fusion by means of a composite of osteogenic protein-1 (rhBMP-7) and hydroxyapatite carrier: a comparison with autograft and hydroxyapatite in the sheep spine.

STUDY DESIGN: Transpedicular lumbar interbody fusion (TLIF) was performed in a sheep model comparing three treatment groups: a composite of osteogenic protein (OP)-1 and hydroxyapatite carrier (HA), HA without OP-1, and autograft. OBJECTIVE: To evaluate the efficacy of the composite of OP-1 and HA (HA-OP-1) in achieving reliable TLIF. SUMMARY OF BACKGROUND DATA: Anterior fusion techniques directly address disc-related problems and achieve primary axial stability. However, they are characterized by high morbidity. Alternatively, the theoretically advantageous posterior TLIF technique using autograft fails clinically because it lacks compressive stability. METHODS: In 36 sheep, lumbar vertebrae L4 to L6 were instrumented posteriorly. Endoscopically assisted TLIF of L4 to L5 was performed. In 12 sheep, the defect was filled with injectable HA-OP-1. Another 12 sheep were treated with HA and another 12 with autograft. Animals were killed at 8 weeks and evaluated by radiologic, histologic, and histomorphometric analysis and by fluorochrome labeling. RESULTS: Only 10 autograft sheep were available for evaluation. Radiologically and histologically, TLIF with HA-OP-1 led to a fusion rate of 10 in 12 compared with autograft (one in 10 fused) and HA (two in 12 fused) ( = 0.0016). Semiquantitative radiologic and histologic scoring also revealed significant differences with superiority of HA-OP-1 ( = 0.0011). Compared with HA, HA-OP-1 presented significantly more ossification at the bone-cement interface ( = 0.0003) and less cement resorption ( = 0.0209). In four of 12 HA sheep, excessive resorption was responsible for local aseptic inflammation. CONCLUSIONS: Biointegration of the osteoconductive HA does not occur, because shear forces cause early HA fracture, subsequent fragmentation, and gross resorption (initiating severe inflammation in four of 12 sheep). In contrast, osteoinductive effects of HA-OP-1 enable bio-integration, resulting in full osseous composite sheathing and solid fusion. By use of this composite, TLIF is successfully applied in sheep. Harvesting autograft and the anterior approach are avoided.

Direct current electrical stimulation increases the fusion rate of spinal fusion cages.

STUDY DESIGN: A randomized experimental evaluation of direct current stimulation in a validated animal model with an experimental control group, using blinded radiographic, biomechanical, histologic, and statistical measures. OBJECTIVES: To evaluate the efficacy of the adjunctive use of direct current stimulation on the fusion rate and speed of healing of titanium interbody fusion cages packed with autograft in a sheep lumbar interbody fusion model. SUMMARY OF BACKGROUND DATA: Titanium lumbar interbody spinal fusion cages have been reported to be 90% effective for single-level lumbar interbody fusion. However, fusion rates are reported to be between 70% and 80% in patients with multilevel fusions or with risk factors such as obesity, tobacco use, or metabolic disorders. The authors hypothesized that direct current stimulation would increase the fusion rate of titanium interbody fusion cages packed with autograft in a sheep lumbar interbody fusion model. METHODS: Twenty-two sheep underwent lumbar discectomy and fusion at L4-L5 with an 11- x 20-mm Bagby and Kuslich (BAK) cage packed with autograft. Seven sheep received a BAK cage and no current. Seven sheep had a cage and a 40-microA current applied with a direct current stimulator. Eight sheep had a BAK cage and a 100-microA current applied. All sheep were killed 4 months after surgery. The efficacy of electrical stimulation in promoting interbody fusion was assessed by performing radiographic, biomechanical, and histologic analyses in a blinded fashion. RESULTS: The histologic fusion rate increased as the direct current dose increased from 0 microA to 40 microA to 100 microA (P < 0.009). Histologically, all animals in the 100-microA group had fusions in both the right and left sides of the cage. Direct current stimulation had a significant effect on increasing the stiffness of the treated motion segment in right lateral bending (P < 0.120), left lateral bending (P < 0.017), right axial rotation (P < 0.004), left axial rotation (P < 0.073), extension (P < 0.078), and flexion (P < 0.029) over nonstimulated levels. CONCLUSION: Direct current stimulation increased the histologic and biomechanical fusion rate and the speed of healing of lumbar interbody spinal fusion cages in an ovine model at 4 months.

Control of lamprey locomotor neurons by colocalized monoamine transmitters.

Neurons in the central nervous system (CNS) often store more than one neurotransmitter, but as yet the functional significance of this type of coexistence is poorly understood. 5-Hydroxytryptamine (5-HT) modulates calcium-dependent K+ channels (KCa) responsible for the postspike afterhyperpolarization in different regions of the CNS. In lamprey, 5-HT neurons control apamine-sensitive KCa channels in spinal locomotor network interneurons, thereby in addition regulating the duration of locomotor bursts. We report here that these spinal 5-HT neurons also contain dopamine. Like 5-HT, dopamine causes a reduction of the afterhyperpolarization, but in this case it is due to a reduction of calcium entry during the action potential, which results in a reduced activation of KCa. 5-HT and dopamine are both released from these midline neurons, and both reduce the afterhyperpolarization through two distinctly different, but complementary cellular mechanisms. The net effect of dopamine (10-100 microM) on the locomotor network is similar to that of 5-HT, and the effects of dopamine and 5-HT are additive at the network level.

Characterization of endosteal osteoblastic cells isolated from mouse caudal vertebrae.

We have developed a reliable procedure for isolating endosteal osteoblasts from mouse trabecular bone. Endosteal osteoblasts were obtained by migration and proliferation of the cells from the metaphyseal bone surface of caudal vertebrae onto nylon meshes. The isolated cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. The cell population consisted of 95% alkaline-phosphatase-positive cells. The cell level of alkaline phosphatase was elevated (1.19 +/- 0.26 (SD) mumol PNP/mn/mg protein) and the enzyme activity was heat-inhibitable, indicating its skeletal origin. Light and electron microscopic observation revealed that cells have morphologic and ultrastructural appearance of typical osteoblasts with high protein synthesis activity. Osteoblasts grown in multilayers in the presence of 50 micrograms/ml ascorbic acid produced within 4 days an abundant fibrous intercellular collagenous matrix forming nodules in which osteocyte-like cells were embedded. Immunolabeling revealed synthesis of type I collagen but no detectable type III collagen. In presence of 7 mM beta-glycerophosphate the matrix became mineralized after 14-21 days of culture. Mineralization could not be induced by mouse skin fibroblasts cultured under similar conditions. The mineral deposits were closely associated with the collagen matrix, consisted of EDTA-removable, Von Kossa and alizarin red S stainable material and were composed of hydroxyapatite crystals identified by X-ray electron probe microanalysis. The isolated endosteal osteoblasts also displayed an intense (+457%) increase in intracellular cAMP production in response to human (1-34) PTH (2 x 10(-8) M) stimulation. The confluent cells responded to 20 nM 1,25(OH)2D3 by a significant 45% reduction in heat labile alkaline phosphatase activity. This procedure allowed us to isolate from trabecular bone a cell population that differentiates into osteoblasts in vitro, respond to calcitropic hormones and that retains its capacity to form a calcified bone tissue in culture. This method provided us a culture system for investigating the differentiation and metabolism of endosteal osteoblastic bone forming cells.