Rat Lumbar Research Articles

Validation and the role of PDK4 relevant to ferroptosis in degenerative lumbar disc disease

BackgroundFerroptosis was involved in the pathogenesis of intervertebral disc degeneration (IVDD). However, the exact mechanism of IVDD associated with ferroptosis still required deeper studies.MethodThe differentially expressed genes (DEGs) in rat lumbar disc tissue between the control and IVDD group treated with IL-1β were detected by RNA sequencing (RNA-seq). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were performed on DEGs. We further screened the differential expressed ferroptosis-related genes (DEFRGs). Besides, a protein-protein interaction (PPI) network of DEFRGs was constructed by STRING database. The Cytoscape database identified significant modules and the hub genes. The loss function of PDK4 by siRNA inference was investigated in NPCs by CCK8 assay, ELISA assay, and the analysis of ferroptosis indicators.ResultDEGs were identified using RNA-seq. KEGG pathway analysis showed that these genes were mainly involved in Parkinson’s disease, oxytocin signaling pathway, calcium ion signaling pathway, AMPK signaling pathway, and glucagon signaling pathway. Eight hub genes (including LDHA, PKM, EP300, EGFR, EGLN1, SCD, PDK4, and FABP4) were found by the PPI network and Cytoscape on a total of 25 ferroptosis-related genes that were identified in rat lumbar disc tissue after IVDD treatment. PDK4 silencing promoted NPCS proliferation, decreased the levels of the proinflammatory factors, and suppressed ferroptosis.ConclusionThe study suggested the potential roles of ferroptosis-related genes in IVDD and further revealed the role of PDK4 in the progression of IVDD.

Selenium nanoparticles ameliorate lumbar disc degeneration by restoring GPX1-mediated redox homeostasis and mitochondrial function of nucleus pulposus cells

Intervertebral disc degeneration (IVDD) is a prevalent musculoskeletal disorder that involves the excessive accumulation of reactive oxygen species (ROS), resulting in mitochondrial dysfunction and matrix metabolism imbalance in nucleus pulposus cells (NPCs). Selenium, an indispensable trace element, plays a crucial role in maintaining mitochondrial redox homeostasis by being incorporated into antioxidant selenoproteins as selenocysteine. In this study, we employed a straightforward synthesis method to produce selenium nanoparticles (SeNPs) with consistent size and distribution, and evaluated their potential protective effects in ameliorating IVDD. In a simulated inflammatory environment induced by interleukin-1beta (IL-1β) in vitro, SeNPs demonstrated a protective effect on the matrix synthesis capacity of NPCs through the up-regulation of aggrecan and type II collagen, while concurrently suppressing the expression of matrix degradation enzymes including MMP13 and ADAMTS5. Additionally, SeNPs preserved mitochondrial integrity and restored impaired mitochondrial energy metabolism by activating glutathione peroxidase1 (GPX1) to rebalance redox homeostasis. In a rat lumbar disc model induced by puncture, the local administration of SeNPs preserved the hydration of nucleus pulposus tissue, promoted matrix deposition, and effectively mitigated the progression of IVDD. Our results indicate that the enhancement of GPX1 by SeNPs may offer a promising therapeutic approach for IVDD by restoring mitochondrial function and redox homeostasis.

The regulatory mechanism of cyclic GMP-AMP synthase on inflammatory senescence of nucleus pulposus cell

BackgroundCellular senescence features irreversible growth arrest and secretion of multiple proinflammatory cytokines. Cyclic GMP-AMP synthase (cGAS) detects DNA damage and activates the DNA-sensing pathway, resulting in the upregulation of inflammatory genes and induction of cellular senescence. This study aimed to investigate the effect of cGAS in regulating senescence of nucleus pulposus (NP) cells under inflammatory microenvironment.MethodsThe expression of cGAS was evaluated by immunohistochemical staining in rat intervertebral disc (IVD) degeneration model induced by annulus stabbing. NP cells were harvested from rat lumbar IVD and cultured with 10ng/ml IL-1β for 48 h to induce premature senescence. cGAS was silenced by cGAS specific siRNA in NP cells and cultured with IL-1β. Cellular senescence was evaluated by senescence-associated beta-galactosidase (SA-β-gal) staining and flow cytometry. The expression of senescence-associated secretory phenotype including IL-6, IL-8, and TNF-a was evaluated by ELISA and western blotting.ResultscGAS was detected in rat NP cells in cytoplasm and the expression was significantly increased in degenerated IVD. Culturing in 10ng/ml IL-1β for 48 h induced cellular senescence in NP cells with attenuation of G1-S phase transition. In senescent NP cells the expression of cGAS, p53, p16, NF-kB, IL-6, IL-8, TNF-α was significantly increased while aggrecan and collagen type II was reduced than in normal NP cells. In NP cells with silenced cGAS, the expression of p53, p16, NF-kB, IL-6, IL-8, and TNF-α was reduced in inflammatory culturing with IL-1β.ConclusioncGAS was increased by NP cells in degenerated IVD promoting cellular senescence and senescent inflammatory phenotypes. Targeting cGAS may alleviate IVD degeneration by reducing NP cell senescence.

Delivering cells via ECM mimetic hydrogel microspheres for promoting the reconstruction of rat lumbar nucleus pulposus

Nucleus pulposus (NP) discectomy is a clinical procedure for the treatment of late-stage intervertebral disc (IVD) degeneration; however, it is associated with the potential for serious complications. The reconstruction of the NP tissue presents challenges due to its avascular structure and prolonged compression within the IVD. In this study, we have developed a novel biomimetic hydrogel microsphere composed of gelatin methacryloyl (GelMA) and fucoidan, a polysaccharide derived from marine brown seaweed, to mimic the extracellular matrix (ECM) of the native NP. The GelMA and fucoidan (GelMA@FD) hydrogel microspheres demonstrate favorable cytocompatibility and significantly enhance the synthesis of matrix components in nucleus pulposus cells (NPCs), including aggrecan and type II collagen. Transcriptome sequencing analysis demonstrates a correlation between the augmentation of matrix synthesis and the enrichment of genes and pathways related to mitochondrial function. Subsequent molecular investigations provide evidence that NPCs cultured on GelMA@FD exhibit enhanced mitochondrial functions, including elevated adenosine triphosphate production and respiratory chain factor expression, alongside a reduction in mitochondrial reactive oxygen species. When implantated into rat lumbar IVDs that have undergone discectomy, the incorporation of NPCs within GelMA@FD hydrogel microspheres facilitates the regeneration of the NP and reinstates its structural and biomechanical properties. This is substantiated by the observed augmentation in IVD disc height and water content within the NP compartment. Collectively, these results demonstrate the potential of employing cell-laden GelMA@FD hydrogel microspheres as a promising strategy for NP regeneration in degenerated IVDs.

Inducing Angiogenesis in the Nucleus Pulposus.

Bone morphogenetic protein (BMP) gene delivery to Lewis rat lumbar intervertebral discs (IVDs) drives bone formation anterior and external to the IVD, suggesting the IVD is inhospitable to osteogenesis. This study was designed to determine if IVD destruction with a proteoglycanase, and/or generating an IVD blood supply by gene delivery of an angiogenic growth factor, could render the IVD permissive to intra-discal BMP-driven osteogenesis and fusion. Surgical intra-discal delivery of naïve or gene-programmed cells (BMP2/BMP7 co-expressing or VEGF165 expressing) +/- purified chondroitinase-ABC (chABC) in all permutations was performed between lumbar 4/5 and L5/6 vertebrae, and radiographic, histology, and biomechanics endpoints were collected. Follow-up anti-sFlt Western blotting was performed. BMP and VEGF/BMP treatments had the highest stiffness, bone production and fusion. Bone was induced anterior to the IVD, and was not intra-discal from any treatment. chABC impaired BMP-driven osteogenesis, decreased histological staining for IVD proteoglycans, and made the IVD permissive to angiogenesis. A soluble fragment of VEGF Receptor-1 (sFlt) was liberated from the IVD matrix by incubation with chABC, suggesting dysregulation of the sFlt matrix attachment is a possible mechanism for the chABC-mediated IVD angiogenesis we observed. Based on these results, the IVD can be manipulated to foster vascular invasion, and by extension, possibly osteogenesis.

Characterization of the L4/L5 rat facet capsular ligament macromechanical and microstructural responses to tensile failure loading

Low back pain is a prevalent condition that affects the global population. The lumbar facet capsular ligament is a source of pain since the collagenous tissue of the ligament is innervated with sensory neurons that deform with the capsule’s stretch. Regional differences in the microstructural and macrostructural anatomy of the spinal facets affect its capsule’s mechanical behavior. Although there are many studies of the cervical facet in human and rodent models, the lumbar capsular ligament’s multiscale behavior is less well-defined. This study characterizes the macroscale and fiber-scale changes of the rat lumbar facet capsule during tensile failure loading. An integrated polarized light imaging setup captured local fiber alignment during 0.08 mm/s distraction of 7 lumbar facets. Force, displacement, strain, and circular variance were measured at several points along the failure curve: the first instance when the local collagen fiber network realigns differentially (anomalous realignment), yield, the first peak in force corresponding to the capsule’s first failure, and peak force, defined as ultimate rupture. Those outcomes were compared across events. While each of force, displacement, and average maximum principal strain increased with applied tension, so did the circular variance of the collagen, suggesting that the fibers were becoming more disorganized. From the fiber alignment maps collected at each mechanical event, the number of anomalous realignment events were counted and found to increase dramatically with loading. The increased collagen disorganization and increasing regions of such disorganization in the facet capsule during loading can provide insights about how loading to the ligament afferent nerves may be activated and thereby produce pain.

Brushite-coated Mg–Nd–Zn–Zr alloy promotes the osteogenesis of vertebral laminae through IGF2/PI3K/AKT signaling pathway

Biodegradable magnesium (Mg) alloys have been extensively investigated in orthopedic implants due to their suitable mechanical strength and high biocompatibility. However, no studies have reported whether Mg alloys can be used to repair lamina defects, and the biological mechanisms regulating osteogenesis are not fully understood. The present study developed a lamina reconstruction device using our patented biodegradable Mg-Nd-Zn-Zr alloy (JDBM), and brushite (CaHPO4·2H2O, Dicalcium phosphate dihydrate, DCPD) coating was developed on the implant. Through in vitro and in vivo experiments, we evaluated the degradation behavior and biocompatibility of DCPD-JDBM. In addition, we explored the potential molecular mechanisms by which it regulates osteogenesis. In vitro, ion release and cytotoxicity tests revealed that DCPD-JDBM had better corrosion resistance and biocompatibility. We found that DCPD-JDBM extracts could promote MC3T3-E1 osteogenic differentiation via the IGF2/PI3K/AKT pathway. The lamina reconstruction device was implanted on a rat lumbar lamina defect model. Radiographic and histological analysis showed that DCPD-JDBM accelerated the repair of rat lamina defects and exhibited lower degradation rate compared to uncoated JDBM. Immunohistochemical and qRT-PCR results showed that DCPD-JDBM promoted osteogenesis in rat laminae via IGF2/PI3K/AKT pathway. This study shows that DCPD-JDBM is a promising biodegradable Mg-based material with great potential for clinical applications.

Lumbar endplate microfracture injury induces Modic-like changes, intervertebral disc degeneration and spinal cord sensitization – an in vivo rat model

BACKGROUND CONTEXTEndplate (EP) injury plays critical roles in painful IVD degeneration since Modic changes (MCs) are highly associated with pain. Models of EP microfracture that progress to painful conditions are needed to better understand pathophysiological mechanisms and screen therapeutics. PURPOSEEstablish in vivo rat lumbar EP microfracture model and assess crosstalk between IVD, vertebra and spinal cord. STUDY DESIGN/SETTINGIn vivo rat EP microfracture injury model with characterization of IVD degeneration, vertebral remodeling, spinal cord substance P (SubP), and pain-related behaviors. METHODSEP-injury was induced in 5 month-old male Sprague-Dawley rats L4–5 and L5–6 IVDs by puncturing through the cephalad vertebral body and EP into the NP of the IVDs followed by intradiscal injections of TNFα (n=7) or PBS (n=6), compared with Sham (surgery without EP-injury, n=6). The EP-injury model was assessed for IVD height, histological degeneration, pain-like behaviors (hindpaw von Frey and forepaw grip test), lumbar spine MRI and μCT, and spinal cord SubP. RESULTSSurgically-induced EP microfracture with PBS and TNFα injection induced IVD degeneration with decreased IVD height and MRI T2 signal, vertebral remodeling, and secondary damage to cartilage EP adjacent to the injury. Both EP injury groups showed MC-like changes around defects with hypointensity on T1-weighted and hyperintensity on T2-weighted MRI, suggestive of MC type 1. EP injuries caused significantly decreased paw withdrawal threshold, reduced axial grip, and increased spinal cord SubP, suggesting axial spinal discomfort and mechanical hypersensitivity and with spinal cord sensitization. CONCLUSIONSSurgically-induced EP microfracture can cause crosstalk between IVD, vertebra, and spinal cord with chronic pain-like conditions. CLINICAL SIGNIFICANCEThis rat EP microfracture model was validated to induce broad spinal degenerative changes that may be useful to improve understanding of MC-like changes and for therapeutic screening.

Effects of the administration of Shinbaro 2 in a rat lumbar disk herniation model.

The current standard for the pharmacological management of lumbar disk herniation (LDH), involving non-steroidal anti-inflammatory drugs, muscle relaxants, and opioid analgesics, often carries a risk of adverse events. The search for alternative therapeutic options remains a vital objective, given the high prevalence of LDH and the critical impact on the quality of life. Shinbaro 2 is a clinically effective herbal acupuncture against inflammation and various musculoskeletal disorders. Therefore, we explored whether Shinbaro 2 exerts protective effects in an LDH rat model. The results showed that Shinbaro 2 suppressed pro-inflammatory cytokines, interleukin-1 beta, tumor necrosis factor-alpha, disk degeneration-related factors, matrix metalloproteinase-1,-3,-9, and ADAMTS-5 in LDH rats. Shinbaro 2 administration reinstated a behavioral activity to a normal level in the windmill test. The results indicated that Shinbaro 2 administration restored spinal cord morphology and functions in the LDH model. Therefore, Shinbaro 2 exerted a protective effect in LDH via actions on inflammatory responses and disk degeneration, indicating that future research is warranted to assess the action mechanisms further and validate its effects.

Relationship between mechanical and microstructural parameters of rat lumbar spine in different ages

Exploring the relationships between microstructure and mechanical properties of bones may provide effective suggestions for increasing bone strength and reducing osteoporotic fracture. In this research, the tissue-level mechanical parameters, microstructure parameters of cancellous bone, and apparent mechanical parameters of L6 vertebral body were calculated in female SD rats aged 1-, 3-, 5-, 7-, 9-, 11-, 13-, 15-, 16-, and 17-month-old. Data were processed with Kruskal–Wallis test, linear regression and Spearman’s rank correlation analysis. Appropriately increasing the plate Tb.N could enhance mechanical properties of bone. Tb.Th and Tb.N were two key factors in determining the tissue-level mechanical properties of cancellous bone. The microstructure could significantly predict mechanical parameters. Our findings may help to further understand the mechanism of osteoporotic fractures.

Transient receptor potential melastatin-7 in the rat dorsal root ganglion

AimsTransient receptor potential melastatin-7 (TRPM7) is a selective cation permeable channel which plays important roles in cellular and developmental biology such as cell proliferation, survival, differentiation and migration. This channel is also known to be necessary for transmitter release in the peripheral nervous system. In this study, immunohistochemistry for TRPM7 was conducted in the rat lumbar dorsal root ganglion (DRG). MethodsTriple immunofluorescence methods were used to demonstrate distribution of TRPM7 and its relationship to other TRP channels in the DRG. Retrograde tracing and double immunofluorescence methods were also performed to know peripheral targets of DRG neurons containing TRPM7 and TRP vanilloid 1 (TRPV1). In addition, transection of the sciatic nerve was conducted to demonstrate an effect of the nerve injury on TRPM7expression in the DRG. ResultsTRPM7-immunoreactivity was expressed by 53.9% of sensory neurons in the 4th lumbar DRG. TRPM7-immunoreactive (-IR) DRG neurons mostly had small (<600 µm²) and medium-sized (600–1200 µm²) cell bodies. By triple and double immunofluorescence methods, approximately 70% of TRPM7-IR DRG neurons contained TRPV1-immunoreactivity. Although the number of DRG neurons co-expressing TRPM7 and TRPM8 was small in the DRG, almost all of TRPM8-IR DRG neurons co-expressed TRPM7-immunoreactivity. By combination of retrograde tracing method and immunohistochemistry, TRPM7 was expressed by half of DRG neurons innervating the plantar skin (61.9%) and gastrocnemius muscle (51.2%), and 79.6% of DRG neurons innervating the periosteum. Co-expression of TRPM7 and TRPV1 among periosteum DRG neurons (75.7%) was more abundant than among cutaneous (53.2%) and muscular (40.4%) DRG neurons. DRG neurons which co-expressed these ion channels in the periosteum had smaller cell bodies compared to the skin and muscle. In addition, the sciatic nerve transection decreased the number of TRPM7-IR neurons in the DRG (approximately 60% reduction). The RT-qPCR analysis also demonstrated reduction of TRPM7 mRNA in the injured DRG. ConclusionThe present study suggests that TRPM7 is mainly located in small nociceptors in the DRG. The content of TRPM7 in DRG neurons is probably different among their peripheral targets. TRPM7 in DRG neurons may be able to respond to noxious stimulation from their peripheral tissues. The nerve injury can decrease the level of TRPM7 mRNA and protein in DRG neurons.

Advanced oxidation protein products induce annulus fibrosus cell senescence through a NOX4-dependent, MAPK-mediated pathway and accelerate intervertebral disc degeneration.

BackgroundIntervertebral disc degeneration (IVDD) is closely associated with senescence. Annulus fibrosus (AF) cell senescence is a crucial driver of AF tissue tearing and fissures, thereby exacerbating IVDD. Increased advanced oxidative protein products (AOPPs) were found in human degenerative discs and aged rat discs and may be involved in IVDD. This study aimed to explore the mechanism of AOPPs-induced senescence in AF cells.MethodsThe pathological effects of AOPPs in vivo were investigated using a rat lumbar disc persistent degeneration model and a rat caudal disc puncture model. Rat primary AF cells were selected as in vitro models, and AOPPs were used as direct stimulation to observe their pathological effects. Setanaxb (NOX1/4 inhibitor), apocynin (NADPH oxidase inhibitor) and adenovirus (ADV) packed NADPH oxidase 4 (NOX4) specific shRNAs were used for pathway inhibition, respectively. Finally, adeno-associated viruses (AAVs) packed with NOX4-specific blocking sequences were used to inhibit the in vivo pathway.ResultsAOPPs accumulated in the rat lumbar and caudal degenerative discs. Intra-discal loading of AOPPs up-regulated the expression of NOX4, p53, p21, p16, IL-1β, and TNF-α, ultimately accelerating IVDD. Exposure of AOPPs to AF primary cells up-regulated NOX4 expression, induced phosphorylation of mitogen-activated protein kinases (MAPK), triggered senescence and increased IL-1β and TNF-α. Apocynin, setanaxib, and ADV pre-cultured AF cells abrogated AOPPs-induced senescence. AAV-mediated inhibition of NOX4 expression in vivo reduced the expression of p53, p21, p16, IL-1β and TNF-α in vivo and delayed IVDD.ConclusionsAOPPs induced AF cell senescence through a NOX4-dependent and MAPK-mediated pathway.

Function of KCNQ2 channels at nodes of Ranvier of lumbar spinal ventral nerves of rats

Previous immunohistochemical studies have shown the expression of KCNQ2 channels at nodes of Ranvier (NRs) of myelinated nerves. However, functions of these channels at NRs remain elusive. In the present study, we addressed this issue by directly applying whole-cell patch-clamp recordings at NRs of rat lumbar spinal ventral nerves in ex vivo preparations. We show that depolarizing voltages evoke large non-inactivating outward currents at NRs, which are partially inhibited by KCNQ channel blocker linopirdine and potentiated by KCNQ channel activator retigabine. Furthermore, linopirdine significantly alters intrinsic electrophysiological properties of NRs to depolarize resting membrane potential, increase input resistance, prolong AP width, reduce AP threshold, and decrease AP amplitude. On the other hand, retigabine significantly decreases input resistance and increases AP rheobase at NRs. Moreover, linopirdine increases excitability at NRs by converting single AP firing into multiple AP firing at many NRs. Saltatory conduction velocity is significantly reduced by retigabine, and AP success rate at high stimulation frequency is significantly increased by linopirdine. Collectively, KCNQ2 channels play a significant role in regulating intrinsic electrophysiological properties and saltatory conduction at NRs of motor nerve fibers of rats. These findings may provide insights into how the loss-of-function mutation in KCNQ2 channels can lead to neuromuscular disorders in human patients.

Role of Voltage-Gated K+ Channels and K2P Channels in Intrinsic Electrophysiological Properties and Saltatory Conduction at Nodes of Ranvier of Rat Lumbar Spinal Ventral Nerves.

Ion channels at the nodes of Ranvier (NRs) are believed to play essential roles in intrinsic electrophysiological properties and saltatory conduction of action potentials (AP) at the NRs of myelinated nerves. While we have recently shown that two-pore domain potassium (K2P) channels play a key role at the NRs of Aβ-afferent nerves, K+ channels and their functions at the NRs of mammalian motor nerves remain elusive. Here we addressed this issue by using ex vivo preparations of lumbar spinal ventral nerves from both male and female rats and the pressure-patch-clamp recordings at their NRs. We found that depolarizing voltages evoked large noninactivating outward currents at NRs. The outward currents could be partially inhibited by voltage-gated K+ channel blockers, largely inhibited by K2P blockers and cooling temperatures. Inhibition of the outward currents by voltage-gated K+ channel blockers, K2P blockers, or cooling temperatures significantly altered electrophysiological properties measured at the NRs, including resting membrane potential, input resistance, AP width, AP amplitude, AP threshold, and AP rheobase. Furthermore, K2P blockers and cooling temperatures significantly reduced saltatory conduction velocity and success rates of APs in response to high-frequency stimulation. Voltage-gated K+ channel blockers reduced AP success rates at high-frequency stimulation without significantly affecting saltatory conduction velocity. Collectively, both K2P and voltage-gated K+ channels play significant roles in intrinsic electrophysiological properties and saltatory conduction at NRs of motor nerve fibers of rats. The effects of cooling temperatures on saltatory conduction are at least partially mediated by K2P channels at the NRs.SIGNIFICANCE STATEMENT Ion channels localized at the NRs are believed to be key determinants of saltatory conduction on myelinated nerves. However, ion channels and their functions at the NRs have not been fully studied in different types of mammalian myelinated nerves. Here we use the pressure-patch-clamp recordings to show that both K2P and voltage-gated K+ channels play significant roles in intrinsic electrophysiological properties and saltatory conduction at NRs of lumbar spinal ventral nerves of rats. Furthermore, cooling temperatures exert effects on saltatory conduction via inhibition of ion channels at the NRs. Our results provide new insights into saltatory conduction on myelinated nerves and may have physiological as well as pathologic implications.

Associations of Lumber Disc Degeneration With Paraspinal Muscles Myosteatosis in Discogenic Low Back Pain.

Accompanied with intervertebral disc (IVD) degeneration, increasing fat infiltration of paraspinal muscles may be related to discogenic low back pain (DLBP), but their relationship is still unclear and the classical animal models are not completely applicable. The purpose of this study was to assess the paraspinal muscle fat infiltration in patients with DLBP by quantitative MRI, and to develop a novel DLBP rat model to explore the potential relationship between DLBP paraspinal muscle fat infiltration and TNF-α levels. We measured the proton density fat fraction (PDFF) of the multifidus and erector spinae muscles of 70 DLBP patients and 36 healthy volunteers by using quantitative MRI IDEAL-IQ. In addition, we developed a DLBP experimental rat model by puncturing the L4/5 and L5/6 IVDs under the guidance of X-ray fluoroscopy. Then various behavioral experiments, MRI and pathological examination of IVDs were used to evaluate the performance of the DLBP animal model. The gait analysis, hot plate test, acetone test, grasping test and tail suspension test were used to evaluate the pain and muscle dysfunction in rats. Through quantitative MRI and histological examination, the degeneration of IVDs and fat infiltration in the muscles were observed in vivo and ex vivo. Enzyme linked immunosorbent assay detects the level of TNF-α in rat IVDs and paraspinal muscles. In the human study, compared with healthy volunteers, the PDFF of multifidus and erector muscles of DLBP patients increased significantly at L4/5 and L5/S1 levels (p<0.05). In the rat experiment, compared with control group and sham group, DLBP group had reduced gait score, shortened response time to cold and heat stimuli, prolonged bending time, and shortened struggling time. Rat lumbar MRI T2WI showed that the signal intensity of L4/5 and L5/6 IVDs were progressively decreased. Histological examination revealed that IVDs had increased collagen fibers, reduced nucleus pulposus, thickened annulus fibrosus, and distorted shape. The PDFF of multifidus muscle at L4/5 and L5/6 level in the DLBP group were more than that in other groups (p<0.05), and HE staining and oil red O staining of paraspinal muscles showed that the muscle bundle space of the DLBP group muscles increased, and the muscle tissues Increased lipid droplets. Finally, the expression of TNF-α in IVDs and paraspinal muscles in the DLBP group were significantly higher than that in the control group (p<0.05). It is reliable and feasible to establish a DLBP rat model by puncturing the lumbar IVDs under the guidance of X-ray fluoroscopy. The degeneration of lumbar IVDs with DLBP leads to the occurrence of fat infiltration of paraspinal muscles, which is related to the expression of TNF-α.

Ex vivo biomechanical evaluation of Acute lumbar endplate injury and comparison to annulus fibrosus injury in a rat model

Back pain is often associated with intervertebral disc (IVD) degeneration, and IVD degeneration phenotypes are commonly characterized by annulus fibrosus (AF)-driven and endplate (EP)-driven phenotypes. Few studies of EP injury exist in animal models, even though clinical studies show EP lesions are strongly associated with IVD pathology and pain. This project established an ex-vivo rat lumbar EP injury model and characterized effects of EP injury on motion segment biomechanical properties, as compared to AF injury, a common way of inducing IVD degeneration. Lumbar motion segments (39 total vertebra-IVD-vertebra sections) assigned to Intact (L1/L2), AF injury and EP injury (L3/L4 and L5/L6 randomly selected), and biomechanically tested in axial tension-compression, stress-relaxation and torsional testing in pre-injury and post-injury conditions using a repeated-measures design. EP injury involved superior vertebra endplate puncture transcorporeally and obliquely. AF injury involved mid-line punctures anterior and bilaterally. Axial ROM, tensile stiffness, hysteresis, and neutral zone stiffness were significantly affected by EP injury but not AF injury. Torque range, torsional stiffness and torsional neutral zone stiffness were significantly affected by AF injury but not EP injury. Stress-relaxation fast time constant was decreased for EP injury. EP and AF injuries induced distinct biomechanical changes in lumbar motion segments with EP injury having the largest impact on axial biomechanical properties and AF injury most prominently affecting torsional properties. This study deepens the understanding of biomechanical mechanism of EP-driven low back pain and provides methods and biomechanical characterization for future in vivo studies.

Manual therapy regulates oxidative stress in aging rat lumbar intervertebral discs through the SIRT1/FOXO1 pathway.

With the increasing burden of a globally aging population, low back pain has become one of the most common musculoskeletal disorders, caused mainly by intervertebral disc (IVD) degeneration. There are currently several clinical methods to alleviate back pain, but there is scarce attention paid as to whether they can improve age-related IVD degeneration. It is therefore difficult to conduct an in-depth evaluation of these methods. A large number of clinical studies have shown that manual therapy (MT), a widely used comprehensive alternative method, has effects on pain, the mechanisms of which require further study. In this study, MT was performed on aging rats for 6 months, and their behaviors were compared with those of a non-intervention group of aging and young rats. After the intervention, all rats were examined by X-ray to observe lumbar spine degeneration, and the IVD tissues were dissected for detection, including pathological staining, immunofluorescence, Western bolt, etc. This study demonstrated the possibility that MT intervention delay the lumbar IVD degeneration in aging rats, specifically improving the motor function and regulating senescence-associated β-galactosidase, p53, p21, p16, and telomerase activity to retard the senescence of cells in IVDs. Moreover, MT intervention can modify oxidative stress, increase the expression of SIRT1 and FOXO1 in IVDs and decrease ac-FOXO1 expression, suggesting that MT can reduce oxidative stress through the SIRT1/FOXO1 pathway, thereby playing a role in delaying the aging of IVDs. This study shows that drug-free, non-invasive mechanical interventions could be of major significance in improving the physical function of the elderly.

κ-opioid receptor is involved in electroacupuncture analgesia via inhibition of spinal microglial Toll-like receptor 4 in neuropathic pain rats

To observe the effect of electroacupuncture (EA) on the expression of lumbar spinal κ-opioid receptor (KOR) and Toll-like receptor 4(TLR4) in microglia in neuropathic pain rats, so as to explore the role of cross-talk between KOR and TLK4 in EA-induced alleviation of chronic neuropathic pain. Wistar male rats were randomized into control, model, EA and EA plus KOR inhibitor (EA+inhibitor) groups (n=18 in each group). The neuropathic pain model was established in rats by ligature of the right sciatic nerve. EA was applied at bilateral "Zusanli"(ST36) and "Yanglingquan"(GB34) for 30 min, once daily for 5 days. JDTic dihydrochloride (a KOR inhibitor) was administrated by intraperitoneal injection before EA intervention. The difference value of paw withdrawal thermal latency (PWLD) of the bilateral hind-limbs was used as the thermal pain reaction level. At the end of experiments, the rat's lumbar spinal cord (L2-L4) was taken for detecting the expression of CD68 mRNA (a marker of the activated microglia) and Iba-1 (a marker for the activated and resting microglia) immunoactivity, and dynorphin content, and KOR mRNA and TLR4 protein (in immunomagnetic microbead method separated microglia) by using fluorescence quantitative PCR, immunofluorescence, radioimmunoassay and Western blot, separately. Compared with the control group, a strong thermal hyperalgesia was induced, the expression levels of Iba-1 and CD68 mRNA in the spinal cord, TLR4 protein of the spinal microglia were significantly increased(P<0.01) in the model group. The microglia were characterized by somatic hypertrophy and thickened branches in the model group. After EA intervention, the PWLD, the expression of Iba-1, CD68 mRNA and TLR4 protein of the microglia were significantly decreased(P<0.05), while the content of spinal dynorphin and the expression of KOR mRNA of the microglia increased in the EA group relative to the model group(P<0.05). The hypertrophic microglia shrinked slightly in the EA group. After injection of KOR inhibitor, the PWLD and expression levels of Iba-1, CD68 mRNA and TLR4 protein were significantly increased(P<0.05), and the expression of KOR mRNA was significantly decreased(P<0.05) in the EA+inhibitor group in comparison with the EA group. The analgesia effect of EA may partly mediated by spinal microglial KOR and the activation of KOR of microglia may be a target for inhibition of microglial TLR4-induced pro-inflammatory signaling.

Ia EPSPs in rat spinal motoneurons are potentiated after a 5-week whole body vibration.

Whole body vibration (WBV) is often applied as an alternative method for strength training or to prevent muscle force decrease. In this study, we evaluated the influence of WBV on Ia monosynaptic input from muscle spindles because the tonic vibration reflex is responsible for the enhancement of muscle activity observed after WBV. The aim was to investigate whether repeated activation of muscle spindles during WBV may result in altered synaptic excitation of motoneurons. WBV was performed on adult male Wistar rats, 5 days/wk, for 5 wk, and each daily session consisted of four 30-s runs of vibration at 50 Hz. Fast-type medial gastrocnemius motoneurons were investigated intracellularly in deeply anesthetized animals in the experimental (n = 7, 34 motoneurons) and control (n = 7, 32 motoneurons) groups. Monosynaptic Ia excitatory postsynaptic potentials (EPSPs) were evoked by electrical stimulation of afferent fibers from the synergistic lateral gastrocnemius and soleus muscles. Data were analyzed using a mixed linear model. The central latencies of EPSPs were 0.45-0.9 ms with no differences in the mean values between the analyzed groups (P = 0.291). WBV induced an increase of the mean EPSP amplitude by 28% (P = 0.025), correlated with the resting membrane potential and input resistance, and a shortening of the mean EPSP rise time by 11% (P = 0.012). The potentiation of synaptic excitation of motoneurons was not accompanied by changes of passive membrane properties, pointing to synaptic plasticity. This indicates that WBV may support rehabilitation or training processes aimed at increasing muscle strength on the basis of increased motoneuronal drive.NEW & NOTEWORTHY The study provides new information on neuronal plasticity following repeatedly exerted mechanical loading. We demonstrate in electrophysiological experiments on rat lumbar motoneurons that low-volume whole body vibration applied systematically for 5 wk potentiates synaptic excitation from primary muscle afferents. The adaptive changes are expressed by higher amplitudes and shorter rise times of monosynaptic EPSPs evoked in motoneurons of the vibrated group compared with the control.

Early detection of increased marrow adiposity with age in rats using Z-spectral MRI at ultra-high field (7 T).

Nowadays, the drive towards high-field MRI is fueled by the pursuit of higher signal-to-noise ratio, spatial resolution, and imaging speed. However, high field strength is associated with field inhomogeneity, acceleration of T2 * decay, and increased chemical shift, which may pose challenges to conventional MRI for fat quantification in complex tissues such as bone marrow. With proton MRI spectroscopy (1 H-MRS), on the other hand, it is difficult to produce high resolution. As a novel alternative fat quantification method, high-resolution Z-spectral MRI (ZS-MRI) can achieve fat quantification by acquiring direct saturated images of both fat and water under the same TE , which may be less affected by T2 * decay and field inhomogeneity. To demonstrate ZS-MRI for marrow adipose tissue (MAT) quantification in rat's lumbar spine and the early detection of MAT changes with age. The accuracy of ZS-MRI for fat quantification at ultra-high-field MRI (7 T) was verified with MRS and conventional Dixon MRI in water-oil mixed phantoms with varying fat fraction (FF). Dixon MRI data were processed with iterative decomposition of water and fat with echo asymmetry and least-squares estimation. ZS-MRI was then used to longitudinally monitor the adiposity in the lumbar spine of young healthy rats at 13, 17, and 21 weeks to detect the early changes of FF with age in MAT. Hematoxylin-eosin staining of lumbar spines from separated rat groups was performed for verification. In ex vivo phantom experiments, both Dixon MRI and ZS-MRI were well correlated with 1 H-MRS for the quantification of FF at 7 T (R > 0.99). Compared with Dixon MRI, ZS-MRI showed reduced image artifacts due to field inhomogeneity and presented better agreement with 1 H-MRS for the early detection of increased MAT due to age at 7 T (ZS-MRI R = 0.78 versus Dixon MRI R = 0.34). The increased MAT FF due to age was confirmed by histology. ZS-MRI proves itself as an alternative fat quantification method for bone marrow in rats at 7 T.