Upregulation of Phosphodiesterase 7A Contributes to Concurrent Pain and Depression via Inhibition of cAMP-PKA-CREB-BDNF Signaling and Neuroinflammation in the Hippocampus of Mice.

BACKGROUND: Phosphodiesterases (PDEs) are enzymes that catalyze the hydrolysis of cyclic adenosine monophosphate AMP (cAMP) and/or cyclic guanosine monophosphate (cGMP). PDE inhibitors can mitigate chronic pain and depression when these disorders occur individually; however, there is limited understanding of their role in concurrent chronic pain and depression. We aimed to evaluate the mechanisms of action of PDE using 2 mouse models of concurrent chronic pain and depression. METHODS: C57BL/6J mice were subjected to partial sciatic nerve ligation (PSNL) to induce chronic neuropathic pain or injected with complete Freund's adjuvant (CFA) to induce inflammatory pain, and both animals showed depression-like behavior. First, we determined the change in PDE expression in both animal models. Next, we determined the effect of PDE7 inhibitor BRL50481 or hippocampal PDE7A knockdown on PSNL- or CFA-induced chronic pain and depression-like behavior. We also investigated the role of cAMP-protein kinase A (PKA)-cAMP response element binding protein (CREB)-brain-derived neurotrophic factor (BDNF) signaling and neuroinflammation in the effect of PDE7A inhibition on PSNL- or CFA-induced chronic pain and depression-like behavior. RESULTS: This induction of chronic pain and depression in the 2 animal models upregulated hippocampal PDE7A. Oral administration of PDE7 inhibitor, BRL50481, or hippocampal PDE7A knockdown significantly reduced mechanical hypersensitivity and depression-like behavior. Hippocampal PDE7 inhibition reversed PSNL- or CFA-induced downregulation of cAMP and BDNF and the phosphorylation of PKA, CREB, and p65. cAMP agonist forskolin reversed these changes and caused milder behavioral symptoms of pain and depression. BRL50481 reversed neuroinflammation in the hippocampus in PSNL mice. CONCLUSIONS: Hippocampal PDE7A mediated concurrent chronic pain and depression in both mouse models by inhibiting cAMP-PKA-CREB-BDNF signaling. Inhibiting PDE7A or activating cAMP-PKA-CREB-BDNF signaling are potential strategies to treat concurrent chronic pain and depression.

Comparison of Syn T2-FLAIR and Syn DIR with conventional T2-FLAIR in displaying white matter hyperintensities in migraine patients.

OBJECTIVE: Young migraine patients often present with white matter hyperintensities (WMHs) on magnetic resonance imaging (MRI). This study aimed to analyze whether synthetic (Syn) T2-FLAIR and Syn double inversion recovery (DIR) can reveal WMHs more clearly and sensitively than conventional T2-FLAIR. MATERIALS AND METHODS: Conventional MRI and Syn MRI data from 50 young migraine patients were analyzed prospectively. WMHs in each anatomical region (periventricular, deep white matter, and juxtacortical) were recorded separately. The differences in the clarity of lesion boundaries and the number of lesions displayed in the three sequences in the same anatomical region were analyzed. RESULTS: A total of 80 (periventricular area, 15; deep white matter, 31; juxtacortical area, 34), 163 (17, 50, 96), and 134 (18, 42, 74) lesions were observed with conventional T2-FLAIR, Syn T2-FLAIR, and Syn DIR, respectively. Syn T2-FLAIR and Syn DIR can show lesions more clearly than conventional T2-FLAIR (all P < 0.001). There was no significant difference in the number of lesions observed in the periventricular white matter among the three sequences (P = 0.159, 0.083, 0.322). Syn T2-FLAIR and Syn DIR can detect more lesions in the deep white matter than conventional T2-FLAIR (P < 0.001, P = 0.006). Syn T2-FLAIR revealed more lesions in the juxtacortical white matter than Syn DIR and conventional T2-FLAIR imaging (all P < 0.001), and conventional T2-FLAIR revealed the fewest lesions (P < 0.001). CONCLUSION: Syn T2-FLAIR and Syn DIR sequences can clearly and sensitively detect WMHs, especially in deep and juxtacortical white matter areas.

Steroidal Saponins from Solanum lyratum Thunb.

Four undescribed steroidal compounds along with twenty known compounds were isolated from n-butanol extracted fraction of the whole plants of Solanum lyratum Thunb (SLNF). Their structures were assigned based on analyses of the extensive spectroscopic data (including MS, 1D/2D NMR, and ECD) or comparisons of the NMR data with those reported. Among the knowns, three compounds were isolated from Solanum plants for the first time, while one compound was isolated from S. lyratum for the first time. In addition, the cytotoxicities of these isolates against human colon SW480 and hepatoma Hep3B cells were evaluated by a MTT assay. And, nine of them and SLNF exhibited significant activities against both SW480 and Hep3B cells, while twelve of them significantly inhibited the activities of SW480 cells. This study allows for the exploitation of chemical markers with potential significance in discrimination of Solanum plants, and uncovers the diverse steroidal constituents from S. lyratum dedicated for its future application in cancer treatment.

Xanthones from Gentianella acuta (Michx.) Hulten Ameliorate Colorectal Carcinoma via the PI3K/Akt/mTOR Signaling Pathway.

Colorectal carcinoma (CRC) is a kind of malignant tumor closely related to ulcerative colitis. Xanthone derivatives are one of the most promising therapeutic drugs which have been used in phase I/II clinical trials for cancer therapy. Our previous study indicated that the aerial parts of Gentianella acuta Michx. Hulten (GA) was rich in xanthones and showed a good therapeutic effect on ulcerative colitis in mice, suggesting that GA xanthones might have some therapeutic or ameliorative effects on CRC. However, no relevant study has been reported. This study aims to find the effective substances of GA inhibiting CRC and clarify their mechanism. Solvent extraction, column chromatographic separation, and LC-MS analysis were used to characterize the 70% EtOH extract of GA and track xanthones abundant fraction XF. MTT assay was carried out to clarify the activity of GA fractions; the result showed XF to be the main active fraction. LC-MS analysis was executed to characterize XF, 38 xanthones were identified. Network pharmacology prediction, in vitro activity screening, and molecular docking assay were combined to predict the potential mechanism; the PI3K/Akt/mTOR signaling pathway was found to be most important. Western blot assay on the main active xanthones 1,3,5-trihydroxyxanthone (16), 1,3,5,8-tetrahydroxyxanthone (17), 1,5,8-trihydroxy-3-methoxyxanthone (18), and 1,7-dihydroxy-3,8-dimethoxyxanthone (19) was used to verify the above prediction; these xanthones were found to inhibit the PI3K/Akt/mTOR signaling pathway, and 17 played a significant role among them through Western blot assay using PI3K/AKT/mTOR agonist IGF-1. In conclusion, this study demonstrated that GA xanthones were effective compounds of GA inhibiting CRC by regulating PI3K/Akt/mTOR signaling pathway transduction, at least. Importantly, 1,3,5,8-tetrahydroxyxanthone (17), the most abundant active xanthone in GA, might be a candidate drug for CRC.

Astramalabaricosides A-T, Highly Oxygenated Malabaricane Triterpenoids with Migratory Inhibitory Activity from Astragalus membranaceus var. mongholicus.

Twenty new malabaricane triterpenoids, astramalabaricosides A-T (1-20), were isolated from the roots of Astragalus membranaceus var. mongholicus (Astragali Radix). Their structures were determined by spectroscopic analysis, and the use of the circular dichroism exciton chirality method, quantum chemical calculations, and chemical methods. Malabaricane triterpenoids, an unusual group with the 6-6-5-tricyclic core, are distributed in plants (e.g., Simaroubaceae, Polypodiaceae, and Fabaceae), a marine sponge, and fungi, and their number obtained to date is limited. Compounds 1-20 were characterized as glycosides with a highly oxygenated side chain, and 13-20 were the first cyclic carbonate derivatives among the malabaricane triterpenoids. The stereocluster formed from the continuous hydroxylated chiral carbons in each highly oxygenated side chain and the 6-6-5-tricyclic core system were entirely segregated, and the independent identification of their stereoconfigurations required considerable effort. The migratory inhibitory and antiproliferative activities of 1-20 were evaluated by wound-healing and cell-viability assays, respectively. Most compounds showed significant migratory inhibitory activity, and a preliminary structure-activity relationship was developed. Malabaricane triterpenoids are being reported in the genus Astragalus for the first time.

An innovative adjustable prone positioning frame for treatment of severe kyphosis secondary to ankylosing spondylitis with two-level osteotomy.

PURPOSE: This study aims to introduce an innovative adjustable prone positioning frame (APPF) and explore its feasibility and safety for treatment of severe kyphosis secondary to ankylosing spondylitis (AS) with two-level osteotomy. METHODS: A retrospective, non-controlled study was conducted to illustrate the process where 13 patients diagnosed with severe kyphosis secondary to AS received operations on the APPF. Parameters of chin brow vertical angle (CBVA), global kyphosis (GK), thoracolumbar kyphosis (TLK), lumbar lordosis (LL) and sagittal vertical axis (SVA) were measured. Positioning time, operation time, intraoperative blood loss ahd complications were also determined. The Scoliosis Research Society outcomes instrument (SRS-22) was applied for clinical assessment. RESULTS: All patients were placed on the APPF successfully with the positioning time of 2.92 ± 0.76 min, received operation with 457.00 ± 88.04 min and had blood loss of 2330.77 ± 1423.25 ml. Four cases experienced pain due to tensional skin of the abdomen and one case suffered cerebrospinal fluid leakage postoperatively, but these patients were all cured conservatively. No neurological complications were observed, although sagittal translation occurred in four patients. Significant improvements were detected in CBVA, GK, TLK, LL and SVA postoperatively (P < 0.05), but no significant difference was observed between postoperation and the final follow-up (P > 0.05). The SRS-22 scores at 2 years after operation were significantly higher than those before operation (P < 0.05). CONCLUSION: The innovative APPF provided great convenience to place patients with severe kyphosis secondary to AS in a prone position. Performing two-level osteotomy with the aid of APPF is safe, feasible and effective.

[The Spatiotemporal Expression Patterns of Mechanical-Stress Related Regulatory Proteins in Mouse Molar Development].

OBJECTIVE: In order to better understand the role of mechanical stress in early tooth development, we examined the spatiotemporal expression patterns of mechanical-stress related regulatory protein (actin filament, or F-actin), non-muscle myosin ⅡB (NMⅡB) and vinculin at different stages of tooth development in mice. METHODS: Mouse first mandible molars were used as the research model. Immunofluorescence staining was performed to detect the expression patterns of F-actin, NMⅡB and Vinculin, the key molecules constituting the chemical mechanical system, at bud, cap, early bell and late bell stages of tooth. RESULTS: F-actin, NMⅡB and vinculin were all expressed in the tooth epithelium in an extensive or a limited way at different stages of tooth development, while F-actin was also expressed steadily in the mesenchymal cells. The quantitative analysis of fluorescence intensity showed that F-actin and NMⅡB exhibited significantly increase in the early stage of tooth development, but then dropped to their basal levels at the end of the late bell stage and the early bell stage respectively, with the differences of expression changes between successive developmental stages showing statistically significance ( P<0.05). Vinculin expression, however, remained at a relatively constant level except for the late bell stage when vinculin expression was slightly elevated compared to that of the early bell stage ( P<0.05). CONCLUSIONS: The findings suggest that mechanical stress is involved in early tooth development. F-actin may have an important role in dispersing and transmitting mechanical stress while NMⅡB may participate in tooth epithelial invagination and cusps formation. The findings also suggest that vinculin can respond to the mechanical stimuli and its interaction with cell adhesion molecules may play a role in tooth development. The mechanism of how actomyosin and cell adhesion interact with each other in regulating tooth development still needs further investigation.

How does temperature play a role in the storage of extracellular vesicles?

Extracellular vesicles (EVs) contain specific proteins, lipids, and nucleic acids that can be passed to other cells as signal molecules to alter their function. However, there are many problems and challenges in the conversion and clinical application of EVs. Storage and protection of EVs is one of the issues that need further research. To adapt to potential clinical applications, this type of problem must be solved. This review summarizes the storage practices of EVs in recent years, and explains the impact of temperature on the quality and stability of EVs during storage based on current research, and explains the potential mechanisms involved in this effect as much as possible.

Effects of Benzophenones from Mango Leaves on Lipid Metabolism.

The mango tree (Mangifera indica L.) is a tropical, perennial, woody evergreen plant belonging to the Anacardiaceae. In traditional medicine, dried mango tree leaves were considered useful in treating diabetes and respiratory infections. In this paper, we review the phytochemical research on mango leaves and the mechanisms of benzophenones in lipid metabolism regulation. Thirty-six benzophenones have been isolated from mango leaves; among them, mangiferin is the major compound. Structure-activity relationships of benzophenones in lipid accumulation and the mechanisms of action of mangiferin in lipid metabolism are summarized. After oral administration, mangiferin is partly converted to its active metabolite, northyariol, which contributes to the activation of sirtuin-1 and liver kinase B1 and increases the intracellular AMP level and AMP/adenosine triphosphate ratio, followed by AMP-activated protein kinase phosphorylation, leading to increased phosphorylation of sterol regulatory element-binding protein-1c. Current evidence supports ethnopharmacological uses of mango leaves in diabetes and points toward potential future applications.

Two new naphthalene glycosides from the seeds of Cassia obtusifolia.

A phytochemical study on the seeds of Cassia obtusifolia was carried out, which finally led to obtain two naphthalenes (1 and 2), two naphthopyrans (3 and 4) and twelve anthraquinones (5-16). The structures of all compounds were established mainly by NMR and MS experiments as well as the necessary chemical evidence. Among them, 1 and 2 (obtusinaphthalensides A and B) were identified to be new naphthalene glycosides.

Soluble expression, rapid purification, biological identification of chicken interferon-alpha using a thioredoxin fusion system in E. coli and its antiviral effects to H9N2 avian influenza virus.

In this paper, we report a soluble expression based on Escherichia coli and two-step purification of a novel thioredoxin-tagged chicken interferon-α fusion protein (Trx-rChIFN-α) by using pET32a(+) expression system. The mature ChIFN-α gene was amplified by Reverse transcriptase-polymerase chain reaction (RT-PCR) and subcloned into pET-32a (+) vector prior to transformation into Rosetta (DE3) competent cells. After IPTG induction, the recombinant fusion protein was expressed efficiently in the soluble fraction. The protein purification was performed by nickel affinity chromatography and DEAE anion exchange chromatography. The purified product has a purity of 95% with a yield of 47.3 mg/L of culture. The specific activity of the fusion protein reaches to 2.0 × 107 IU/mg as determined in the CEF/VSV titration system. After excision of the Trx tag by enterokinase, the remaining solo protein was confirmed as rChIFN-α protein by SDS-PAGE, N-terminal sequencing and mass spectrometry. The effects of this Trx-rChIFN-α fusion protein against H9N2 influenza virus infection were also evaluated in ovo. The results showed that the Trx-rChIFN-α protein could significantly reduce the hemagglutination titer of H9N2 virus, and the H9N2 viruses HA gene copy numbers. These findings will enable us to produce large amount and bio-active rChIFN-α protein for future applications.

[N-containing compounds from seeds of Paganum harmala].

To investigate the N-containing compounds in the seeds of Paganum harmala,fourteen compounds were finally isolated from the 95% Et OH extract of P. harmala seeds by using various chromatographic techniques including silica gel,MCI resin,and ODS column chromatography as well as the semi-preparative HPLC. Depending on spectroscopic techniques and comparison with the reported data in the literatures,the structures of all compounds were identified as N-[3-(2-amino-4-methoxyphenyl)-3-oxopropyl]acetamide(1),dehydroharmalacidine(2),harmalacidine(3),harmine N-oxide(4),harmine(5),tetrahydroharmine(6),demethylharmalacidine(7),harmol(8),tetrahydroharmol(9),harmindol ß-D-glucopyranoside(10),tryptophyl ß-D-glucopyranoside(11),pegamineß-D-glucopyranoside(12),vasicol(13) and vasicinone(14). Among them,1 was a new compound,and 2 and 10 were obtained as natural products for the first time.

microRNA-141-3p fosters the growth, invasion, and tumorigenesis of cervical cancer cells by targeting FOXA2.

microRNA (miR)-141-3p has context-dependent effects on tumor progression. In this study, we attempted to explore the expression and function of miR-141-3p in cervical cancer. We found that miR-141-3p expression was significantly increased in cervical cancer specimens relative to normal cervical tissues. Moreover, miR-141-3p levels were associated with tumor size and lymph node metastasis status. Ectopic expression of miR-141-3p significantly increased cervical cancer cell proliferation, colony formation, invasion, and epithelial to mesenchymal transition, whereas depletion of miR-141-3p suppressed cervical cancer cell proliferation and invasion. FOXA2 was identified to be a target of miR-141-3p. Overexpression of miR-141-3p led to a marked inhibition of endogenous FOXA2 in cervical cancer cells. FOXA2 silencing phenocopied the effects of miR-141-3p overexpression on cervical cancer cell proliferation and invasion. Enforced expression of FOXA2 blocked the effects of miR-141-3p on cervical cancer cell proliferation and invasion. miR-141-3p overexpression significantly accelerated the growth of xenograft tumors, which was accompanied by a striking reduction in FOXA2 expression. miR-141-3p acts as an oncogene in cervical cancer largely through repression of FOXA2. Targeting miR-141-3p may represent a potential therapeutic strategy for cervical cancer.

Secoiridoid analogues from the fruits of Ligustrum lucidum and their inhibitory activities against influenza A virus.

A phytochemical study focusing on the secoiridoid components in the fruits of Ligustrum lucidum was carried out, which finally led to the isolation of nine secoiridoid glycosides (1-9) together with two secoiridoids (10, 11). The structures of all compounds were established mainly by NMR and MS experiments as well as the necessary chemical evidence, of which 1, 2, 4 (ligulucisides A-C), 10 and 11 (liguluciridoids A and B) were identified as new secoiridoid analogues. An in vitro antiviral bioassay indicated that 1, 4, 6, and 10 displayed the inhibitory activities against influenza A virus with the IC50 values of 16.5, 12.5, 13.1, and 18.5 µM, respectively, which were better than the positive control Ribavirin (IC50 22.6 µM). .

Respiratory syncytial virus infection up-regulates TLR7 expression by inducing oxidative stress via the Nrf2/ARE pathway in A549 cells.

In order to better understand the early pathways of the pathogenesis of, and immune response to, RSV, herein, we explored the relationship between TLR7 expression and oxidative stress induction following RSV infection in A549 cells. We studied the intervening effects of the Nrf2/ARE pathway agonist butylated hydroxyanisole (BHA) and inhibitor trigonelline (TRI) on TLR7 modulation or oxidative stress induction. For comparison purposes, we set up seven treatment groups in this study, including RSV-treated cells, BHA + RSV-treated cells, TRI + RSV-treated cells, normal cell controls, inactivated RSV controls, BHA controls and TRI controls. We measured changes in TLR7, IL-6, TNF-α mRNA using RT-PCR and IL-6, TNF-α and IL-1ß protein using ELISA as well as TLR7, Nrf2 and HO-1 protein using Western blot in A549 cells from the different treatment groups. We also assessed changes in cell proliferation and measured changes in ·OH and NO in A549 cells from the different treatment groups. The results indicate that TLR7 up-regulation is related to RSV infection and the induction of oxidative stress and that TLR7 expression was mediated by the anti-inflammatory effects of Nrf2/ARE pathway inhibitors or agonists. Our experiments may help elucidate the underlying pathology of RSV infection and suggest potential therapeutic targets for drug development and the prevention of RSV-induced human diseases.

Baicalin Reverses Depressive-Like Behaviours and Regulates Apoptotic Signalling Induced by Olfactory Bulbectomy.

Apoptosis is thought to be involved in neurological disorders including major depression. In this study, we examined whether the polyphenolic compound baicalin could decrease apoptosis in the olfactory bulbectomy (OBX) depression rat model. OBX rats exhibited decreased performance in depression-like behavioural tests and showed evidence of increased oxidative stress, decreased synaptophysin expression, and hippocampal apoptosis. Treatment with baicalin (20 and 40 mg/kg) significantly reversed all of these changes. Baicalin modulated the levels or activity of malondialdehyde, superoxide dismutase, and glutathione peroxidase and prevented apoptotic protease-activating factor-1 expression, effectively suppressing caspase-mediated apoptosis signalling cascades. Our results demonstrate that baicalin has potent antidepressant activity, likely because of its ability to suppress apoptosis.

Baicalin reverse AMPA receptor expression and neuron apoptosis in chronic unpredictable mild stress rats.

Changes of a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor expression could impacts the viability of neurons and brain levels of brain-derived neurotrophic factor (BDNF) expression in the key brain structures in the pathophysiology of depressive disorder. In the present study, chronic unpredictable mild stress (CUMS) degraded performance decreased AMPA receptor expression and increased neuron apoptosis. Treatment with baicalin (20, 40mg/kg) significantly reversed these changes. This study demonstrates that baicalin has potent antidepressant effect, likely through up-regulated the expression of AMPA receptor and suppression neuron apoptosis in CUMS-treated rats.

Enhanced nitrogen removal for low C/N wastewater via preventing futile carbon oxidation and augmenting anammox.

Efficient carbon use is crucial for biological nitrogen removal. Traditional aerobic processes can waste carbon sources, exacerbating carbon deficiency. This study explores an anaerobic/oxic/anoxic system with sludge double recirculation to improve nitrogen removal in low C/N wastewater. This system integrated aerobic nitrification after the carbon intracellular storage, separating carbon and nitrogen by denitrifying glycogen-accumulating organisms (DGAOs) with endogenous partial denitrification and Anammox within the anoxic units. A significant efficiency of 91.02±7.01% chemical oxygen demand (COD) was converted into intracellular carbon in anaerobic units, significantly reducing carbon futile oxidation in the aerobic units by effectively separating COD from ammonia. Intracellular storage of carbon sources and microbial adaptation to carbon scarcity prevent futile oxidation of COD in the aerobic units even with short-term high dissolved oxygen (DO), thereby enhancing nitrogen removal under anoxic conditions with sufficient intracellular carbon source. The microbial analysis identified Candidatus Brocadia as the dominant anammox bacteria, in combination with the activity of DGAOs and other related microbial communities, accounting for 37.0% of the TN removal. Consequently, the system demonstrated remarkable nitrogen removal efficiencies, achieving 81.3±3.3% for total nitrogen (TN) and 98.5±0.9% for ammonia nitrogen while maintaining an effluent COD concentration of 17.2±9.1 mg/L, treating the low C/N of 4.18 in the influent wastewater. The findings in this study provide a sustainable and energy-saving technique for conventional WWTPs to meet strict discharge standards by avoiding futile oxidation of COD and encouraging anammox contributions.

Composite formation of whey protein isolate and OSA starch for fabricating high internal phase emulsion: A comparative study at different pH and their application in biscuits.

The composites formed by whey protein isolate (WPI) and octenyl succinate anhydride (OSA)-modified starch were characterized with a focus on the effect of pH, and their potential in fabricating high internal phase emulsions (HIPEs) as fat substitutes was evaluated. The particles obtained at pH 3.0, 6.0, 7.0, and 8.0 presented a nanosized distribution (122.04 ± 0.84 nm-163.24 ± 4.12 nm) while those prepared at pH 4.0 and 5.0 were remarkably larger. Results from the shielding agent reaction and Fourier transform infrared spectroscopy (FT-IR) showed that the interaction between WPI and OSA starch was mainly hydrophobic at pH 3.0-5.0, while there was a strong electrostatic repulsion at pH 6.0-8.0. A quartz crystal microbalance with dissipation (QCM-D) study showed that remarkably higher ΔD and lower Δf/n were observed at pH 3.0-5.0 after successive deposition of WPI and OSA starch, whereas slight changes were noted for those made at higher pH values. The WPI-OSA starch (W-O) composite-based HIPEs made at pH 3.0 and 6.0-8.0 were physically stable after long-term storage, thermal treatment, or centrifugation. Incorporation of HIPE into the biscuit formula yielded products with a desirable sensory quality.

Effects of tail nerve electrical stimulation on the activation and plasticity of the lumbar locomotor circuits and the prevention of skeletal muscle atrophy after spinal cord transection in rats.

INTRODUCTION: Severe spinal cord injury results in the loss of neurons in the relatively intact spinal cord below the injury area and skeletal muscle atrophy in the paralyzed limbs. These pathological processes are significant obstacles for motor function reconstruction. OBJECTIVE: We performed tail nerve electrical stimulation (TNES) to activate the motor neural circuits below the injury site of the spinal cord to elucidate the regulatory mechanisms of the excitatory afferent neurons in promoting the reconstruction of locomotor function. METHODS: Eight days after T10 spinal cord transection in rats, TNES was performed for 7 weeks. Behavioral scores were assessed weekly. Electrophysiological tests and double retrograde tracings were performed at week 8. RESULTS: After 7 weeks of TNES treatment, there was restoration in innervation, the number of stem cells, and mitochondrial metabolism in the rats' hindlimb muscles. Double retrograde tracings of the tail nerve and sciatic nerve further confirmed the presence of synaptic connections between the tail nerve and central pattern generator (CPG) neurons in the lumbar spinal cord, as well as motor neurons innervating the hindlimb muscles. CONCLUSION: The mechanisms of TNES induced by the stimulation of primary afferent nerve fibers involves efficient activation of the motor neural circuits in the lumbosacral segment, alterations of synaptic plasticity, and the improvement of muscle and nerve regeneration, which provides the structural and functional foundation for the future use of cutting-edge biological treatment strategies to restore voluntary movement of paralyzed hindlimbs.