The efficacy of silver needle therapy for treating low back pain: a protocol for meta-analysis of randomized controlled trials.

Background: As population aging and unhealthy living habits may exacerbate the prevalence and burden of low back pain (LBP), effective treatment and improvement of patient quality of life are particularly critical. Silver needle therapy (SNT), having evolved from traditional acupuncture, involves placing silver needles into muscles, tendons, and fascia for treatment. However, it still lacks robust clinical evidence to substantiate its effectiveness. Therefore, it is necessary to conduct more emphasis on meta-analysis to evaluate the clinical efficacy of SNT for treating LBP. Methods: We will search PubMed, Medline, Cochrane Library, Embase, China National Knowledge Infrastructure (CNKI), and Wanfang Databases up until December 2023 to identify randomized controlled trials of SNT treatment in adult patients with LBP. The primary outcome will be the intensity of pain after pain management. Secondary outcomes will include the Oswestry Disability Index, Japanese Orthopedic Association Back Pain Evaluation Questionnaire, requirement for analgesic drugs, and treatment-related adverse reactions. Two investigators conducted the literature search, selected studies that might meet the inclusion criteria based on the title and abstract, and extracted data from the eligible literature independently and will independently assess the risk of bias using the Revised Cochrane Risk-of-Bias (RoB2) tool. Multivariate analyses (including subgroup analysis, trial sequential analysis (TSA), sensitivity analysis, etc.) will be conducted to improve the quality of evidence. Clinical trial registration: Registration: PROSPERO Registration Number: CRD42023466207, https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42023466207.

High-voltage pulsed radiofrequency improves ultrastructure of DRG and enhances spinal microglial autophagy to ameliorate neuropathic pain induced by SNI.

Neuropathic pain (NeP) is intractable for which many therapies are ineffective. High-voltage pulsed radiofrequency (HVPRF) on dorsal root ganglion (DRG) is considered an effective treatment for NeP. The aim of this study is to explore the therapeutic voltage for the optimal efficacy of PRF and the underlying mechanisms. The radiofrequency electrode was placed close to the L5 DRG of rats with spared nerve injury (SNI) and emitted current by the corresponding voltage in different groups. Four different voltages (45 V, 65 V, 85 V, and 100 V) of PRF on DRG significantly alleviated the SNI-induced NeP, reduced the levels of activating transcription factor 3 (ATF3) in DRG, improved the ultrastructure of DRG, and promoted autophagy in spinal microglia to varying degrees and partially reversed the increased expression of TNF-α and the reduced expression of IL-10 in spinal cord dorsal horn (SCDH). The beneficial effect of 85V-PRF was superior to those of other three PRF treatments. The underlying mechanisms may be related to repairing the DRG damage and improving the DRG ultrastructure while regulating spinal microglial autophagy and thereby alleviating neuroinflammation.

Pulsed radiofrequency on DRG inhibits hippocampal neuroinflammation by regulating spinal GRK2/p38 expression and enhances spinal autophagy to reduce pain and depression in male rats with spared nerve injury.

Evidence indicates that microglial G protein-coupled receptor kinase 2 (GRK2) is a key regulator of the transition from acute to chronic pain mediated by microglial products via the p38 mitogen-activated protein kinase (MAPK) pathway in the spinal cord dorsal horn (SCDH). Increasing studies have shown that autophagic dysfunction in the SCDH and neuroinflammation in the hippocampus underlie NeP. However, whether GRK2/p38MAPK and autophagic flux in the SCDH and hippocampal neuroinflammation are involved in NeP and depression comorbidity has not been determined. Here, we explored the effects of high-voltage pulsed radiofrequency (PRF) (85 V-PRF; HV-PRF) to the dorsal root ganglion (DRG) on pain phenotypes in Wistar male rats with spared nerve injury (SNI) and the underlying mechanisms. The exacerbation of pain phenotypes was markedly relieved by PRF-DRG. The SNI-induced reduction in GRK2 expression, elevation of p-p38 MAPK levels in the SCDH, and increase in IL-1ß and TNF-α levels in the hippocampus were reversed by PRF, which was accompanied by an increase in autophagic flux in spinal microglia. The beneficial effect of 85 V-PRF was superior to that of 45 V-PRF. In addition, the improvements elicited by 85 V-PRF were reversed by intrathecal injection of GRK2 antisense oligonucleotide, and these changes were accompanied by GRK2 downregulation and p-p38 upregulation in the SCDH, increased pro-inflammatory factor levels in the hippocampus, and excessive autophagy in spinal microglia. In conclusion, our data indicate that the application of HV-PRF to the DRG could serve as an excellent therapeutic technique for regulating neuroimmunity and neuroinflammation to relieve pain phenotypes.

Systematic identification of potential key microRNAs and circRNAs in the dorsal root ganglia of mice with sciatic nerve injury.

Background: Neuropathic pain (NeP) is a pathological condition arising from a lesion or disease affecting the somatosensory system. Accumulating evidence has shown that circular RNAs (circRNAs) exert critical functions in neurodegenerative diseases by sponging microRNAs (miRNAs). However, the functions and regulatory mechanisms of circRNAs as competitive endogenous RNAs (ceRNAs) in NeP remain to be determined. Methods: The sequencing dataset GSE96051 was obtained from the public Gene Expression Omnibus (GEO) database. First, we conducted a comparison of gene expression profiles in the L3/L4 dorsal root ganglion (DRG) of sciatic nerve transection (SNT) mice (n = 5) and uninjured mice (Control) (n = 4) to define the differentially expressed genes (DEGs). Then, critical hub genes were screened by exploring protein-protein interaction (PPI) networks with Cytoscape software, and the miRNAs bound to them were predicted and selected and then validated by qRT-PCR. Furthermore, key circRNAs were predicted and filtered, and the network of circRNA-miRNA-mRNA in NeP was constructed. Results: A total of 421 DEGs were identified, including 332 upregulated genes and 89 downregulated genes. Ten hub genes, including IL6, Jun, Cd44, Timp1, and Csf1, were identified. Two miRNAs, mmu-miR-181a-5p and mmu-miR-223-3p, were preliminarily verified as key regulators of NeP development. In addition, circARHGAP5 and circLPHN3 were identified as key circRNAs. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis demonstrated that these differentially expressed mRNAs and targeting miRNAs were involved in signal transduction, positive regulation of receptor-mediated endocytosis and regulation of neuronal synaptic plasticity. These findings have useful implications for the exploration of new mechanisms and therapeutic targets for NeP. Conclusion: These newly identified miRNAs and circRNAs in networks reveal potential diagnostic or therapeutic targets for NeP.

Novel Bipolar High-Voltage Pulsed Radiofrequency Targeting the Cervical Sympathetic Chain for Treating Acute Herpetic Neuralgia.

INTRODUCTION: Postherpetic neuralgia (PHN) in the oral, maxillofacial, neck, and upper limb regions is a refractory neuropathic pain and severely affects the quality of life of patients. Because of the absence of ideal treatments for this condition, relieving pain in the acute stage and preventing the occurrence of PHN are of great clinical significance. However, the optimal intervention for this acute herpetic neuralgia remains obscure. OBJECTIVES: This study aimed to investigate whether bipolar high-voltage pulsed radiofrequency (PRF) targeting the cervical sympathetic chain could effectively treat acute herpetic neuralgia in the oral, maxillofacial, neck, and upper limb regions and reduce the incidence of PHN. MATERIALS AND METHODS: A total of 60 patients with acute herpetic neuralgia in the oral, maxillofacial, neck, and upper limb regions were enrolled. The radiofrequency group (n = 30) received bipolar high-voltage PRF under ultrasound guidance at the level of the transverse processes of C6 and C7 to modulate the cervical sympathetic chain. In the sham group (n = 30), the electrodes were simply placed at the same position as in the radiofrequency group, but no radiofrequency energy was applied. The same treatment was repeated in each group after 72 hours. If patients were reported to have a visual analog scale (VAS) score ≥4, they would receive oral tramadol and gabapentin as rescue analgesics. The VAS score, Pittsburgh Sleep Quality Index (PSQI), 36-Item Short Form Health Survey (SF-36) score, use of tramadol and gabapentin, incidence of PHN, and adverse reactions were recorded to assess the effect and safety of therapy during three months of follow-up. RESULTS: Decreased VAS scores, PSQI scores, and improved SF-36 scores were detected in the two groups at different time points after treatment (all p < 0.05). The VAS scores, PSQI scores, use of tramadol and gabapentin, and incidence of PHN were significantly lower, whereas the SF-36 scores were significantly higher in the radiofrequency group than in the Sham group (all p < 0.05). No serious adverseness related to the treatment was detected in either group. CONCLUSIONS: Bipolar high-voltage PRF treatment targeting the cervical sympathetic chain could effectively relieve acute herpetic neuralgia in the oral, maxillofacial, neck, and upper limb regions and reduce PHN incidence. The efficacy and safety of this novel treatment make it worthy of recommendation for clinical application.

Effects of High-Voltage Pulsed Radiofrequency on the Ultrastructure and Nav1.7 Level of the Dorsal Root Ganglion in Rats With Spared Nerve Injury.

OBJECTIVES: To investigate the analgesic effect of high-voltage pulsed radiofrequency (HV-PRF) on the dorsal root ganglion (DRG) for neuropathic pain induced by spared nerve injury (SNI) in rats, especially the influence of this treatment on the DRG ultrastructure and voltage-gated sodium channel 1.7 (Nav1.7) level in the DRG. MATERIALS AND METHODS: One hundred fifty adult male Sprague-Dawley rats were randomly divided into five groups: Sham, SNI, Free-PRF, standard-voltage PRF (SV-PRF), and HV-PRF. The 45V-PRF and 85V-PRF procedures applied to the left L5 DRG were performed in SV-PRF group and the HV-PRF group, respectively, on day 7 after SNI, whereas no PRF was concurrently delivered in Free-PRF group. The paw mechanical withdrawal threshold (PMWT) was detected before SNI (baseline) and on days 1, 3, 7, 8, 10, 14, and 21. The changes of left L5 DRG ultrastructure were analyzed with transmission electron microscopy on days 14 and 21. The expression levels of Nav1.7 in left L5 DRG were detected by immunofluorescence and Western blot. RESULTS: Compared with the Free-PRF group, PMWT in the SV-PRF group and HV-PRF group were both significantly increased after PRF (all p < 0.05). Meanwhile, the PMWT was significantly higher in the HV-PRF group than that in the SV-PRF group on days 14 and 21 (all p < 0.05). There were statistically significant differences between the SV-PRF and Free-PRF groups (p < 0.05). Similarly, statistically significant difference was found between the HV-PRF and Free-PRF groups (p < 0.05). Especially, comparison of the SV-PRF group and the HV-PRF group revealed statistically significant difference (p < 0.05). The Nav1.7 levels were significantly downregulated in the SV-PRF group and HV-PRF groups compared to that in the Free-PRF group (all p < 0.01). A significantly lower Nav1.7 level was also found in the HV-PRF group compared to that in the SV-PRF group (p < 0.05). CONCLUSIONS: The HV-PRF produces a better analgesic effect than SV-PRF applied to the DRG in SNI rats. The underlying mechanisms may be associated with improving the histopathological prognosis and the downregulation of Nav1.7 levels in the DRG.

Expert consensus on the diagnosis and treatment of myofascial pain syndrome.

Myofascial pain syndrome (MPS) is characterized by myofascial trigger points and fascial constrictions. At present, domestic and foreign scholars have not reached a consensus on the etiology and pathogenesis of MPS. Due to the lack of specific laboratory indicators and imaging evidence, there is no unified diagnostic criteria for MPS, making it easy to confuse with other diseases. The Chinese Association for the Study of Pain organized domestic experts to formulate this Chinese Pain Specialist Consensus on the diagnosis and treatment of MPS. This article reviews relevant domestic and foreign literature on the definition, epidemiology, pathogenesis, clinical manifestation, diagnostic criteria and treatments of MPS. The consensus is intended to normalize the diagnosis and treatment of MPS and be used by first-line doctors, including pain physicians to manage patients with MPS.

Expert consensus of the Chinese Association for the Study of Pain on pain treatment with the transdermal patch.

Chronic pain lasting more than 3 mo, or even several years can lead to disability. Treating chronic pain safely and effectively is a critical challenge faced by clinicians. Because administration of analgesics through oral, intravenous or intramuscular routes is not satisfactory, research toward percutaneous delivery has gained interest. The transdermal patch is one such percutaneous delivery system that can deliver drugs through the skin and capillaries at a certain rate to achieve a systemic or local therapeutic effect in the affected area. It has many advantages including ease of administration and hepatic first pass metabolism avoidance as well as controlling drug delivery, which reduces the dose frequency and side effects. If not required, then the patch can be removed from the skin immediately. The scopolamine patch was the first transdermal patch to be approved for the treatment of motion sickness by the Food and Drug Administration in 1979. From then on, the transdermal patch has been widely used to treat many diseases. To date, no guidelines or consensus are available on the use of analgesic drugs through transdermal delivery. The pain branch of the Chinese Medical Association, after meeting and discussing with experts and based on clinical evidence, developed a consensus for promoting and regulating standard use of transdermal patches containing analgesic drugs.

Microglial BDNF, PI3K, and p-ERK in the Spinal Cord Are Suppressed by Pulsed Radiofrequency on Dorsal Root Ganglion to Ease SNI-Induced Neuropathic Pain in Rats.

Background: Pulsed radiofrequency (PRF) on the dorsal root ganglion (DRG) has been applied to alleviate neuropathic pain effectively, yet the mechanisms underlying pain reduction owing to this treatment are not clarified completely. The activated microglia, brain-derived neurotrophic factor (BDNF), phosphatidylinositol 3-kinase (PI3K), and phosphorylated extracellular signal-regulated kinase (p-ERK) in the spinal cord were demonstrated to be involved in developing neuropathic pain. Also, it has been just known that PRF on DRG inhibits the microglial activation in nerve injury rats. Here, we aim to investigate whether PRF treatment could regulate the levels of BDNF, PI3K, and p-ERK in the spinal cord of rats with spared nerve injury (SNI) via suppressing the spinal microglia activation to ease neuropathic pain. Methods: The rats with SNI were intrathecally treated with minocycline (specific microglia inhibitor) or same volume of dimethyl sulfoxide once daily, beginning from 1 h before nerve transection to 7 days. PRF was applied adjacent to the L4-L5 DRG of rats with SNI at 45 V for 6 min on the seventh postoperative day, whereas the free-PRF rats were treated without PRF. The withdrawal thresholds were studied, and the spinal levels of ionized calcium-binding adapter molecule 1 (Iba1), BDNF, PI3K, and p-ERK were calculated by western blot analysis, reverse transcription-polymerase chain reaction, and immunofluorescence. Results: The paw withdrawal mechanical threshold and paw withdrawal thermal latency decreased in the ipsilateral hind paws after SNI, and the spinal levels of Iba1, BDNF, PI3K, and p-ERK increased on day 21 after SNI compared with baseline (P < 0.01). An intrathecal injection of minocycline led to the reversal of SNI-induced allodynia and increase in levels of Iba1, BDNF, PI3K, and p-ERK. Withdrawal thresholds recovered partially after a single PRF treatment for 14 days, and SNI-induced microglia hyperactivity, BDNF upregulation, and PI3K and ERK phosphorylation in the spinal cord reduced on D14 due to the PRF procedure. Conclusion: Microglial BDNF, PI3K, and p-ERK in the spinal cord are suppressed by the therapy of PRF on DRG to ease SNI-induced neuropathic pain in rats.

Downregulated spinal IRF8 and BDNF in NAC are involved in neuropathic pain-induced depression relief via pulsed radiofrequency on dorsal root ganglion in rat SNI model.

Pulsed radiofrequency (PRF) on the dorsal root ganglion (DRG), which produces remarkable analgesia through high-frequency electromagnetic energy, has become a main therapy for chronic neuropathic pain. The chronic neuropathic pain in patients is frequently accompanied by depression. However, the underlying neurophysiological mechanisms of the treatment of PRF on DRG for the neuropathic pain-induced depression remain unclear. This study was designed to explore the effect of PRF on DRG on the neuropathic pain-induced depression in rats with spared nerve injury (SNI). Here, we found that PRF on DRG or intrathecal injection of the interferon regulatory factor 8 (IRF8) siRNA prevented the increase of mechanical allodynia and depression-like behaviors of rats after receiving SNI. Meanwhile, Western blot, immunohistochemistry, and RT-PCR revealed that PRF on DRG or intrathecal injection of IRF8 siRNA inhibited IRF8 overexpression in the spinal cord and brain-derived neurotrophic factor (BDNF) in NAc. These results suggest that neuropathic pain-induced depression could be attenuated by PRF applied to DRG in SNI rats. The suppressed overexpression of the spinal IRF8 and BDNF in NAc may play an important role and contribute considerably to effectiveness of the therapy by PRF on DRG.

Pulsed Radiofrequency on Dorsal Root Ganglion Relieved Neuropathic Pain Associated with Downregulation of the Spinal Interferon Regulatory Factor 8, Microglia, p38MAPK Expression in a CCI Rat Model.

BACKGROUND: Interferon regulatory factor 8 (IRF8), which is induced by peripheral nerve injury (PNI), plays a key role in activating spinal microglia to release inflammatory cytokines in a p38-dependent way, thereafter results in formation of central sensitization. Pulsed radiofrequency (PRF) on dorsal root ganglion (DRG) alleviates neuropathic pain and inhibits the microglial activation in chronic constriction injury (CCI) rats. However, the consequences of PRF on spinal IRF8 of CCI rats remains unknown. OBJECTIVE: We explore if PRF on DRG of rats with CCI could restrain IRF8, microglia, and p38 hyperactivity in the spinal cord to alleviate neuropathic pain. STUDY DESIGN: A randomized, controlled animal study. SETTING: Department of Pain Management, Fujian Provincial Hospital, Fujian Key Laboratory of Geriatrics, Provincial Clinic College of Fujian Medical University. METHODS: The changes in pain behaviors and the expressions of IRF8, Iba1 and p-p38 in the spinal cord of CCI rats which were administrated with antisense/ mismatch oligodeoxynucleotide of IRF8 were studied. Rats in CCI+AS ODN group, CCI+MM ODN group or CCI+NS group were intrathecally treated with antisense oligodeoxynucleotide of IRF8, mismatch oligodeoxynucleotide of IRF8 or same volume 0.9% NaCl once daily respectively, beginning from the day after nerve transection 12 hours and lasting for 7 days. The effects of PRF on L4-5 DRG of rats with CCI were investigated. PRF was applied adjacent to the L4-5 DRG at an intensity of 45 V for 6 minutes after CCI, whereas the control rats were treated without radiofrequency current. The withdrawal thresholds were studied and the spinal levels of IRF8, ionized calcium-binding adapter molecule 1 (Iba1, microglia characteristic marker) and p-p38 were calculated by ELISA, western blot, RT-PCR, and immunofluorescence. RESULTS: Intrathecal administration of antisense oligodeoxynucleotide of IRF8 led to the reversal of CCI-induced allodynia, lower activation of spinal microglia and p-p38. Withdrawal thresholds were partially recovered after a single PRF treatment for 14 days. CCI-induced IRF8 upregulation, microglia hyperactivity, and p38 phosphorylation in the spinal cord were reduced due to PRF treatment. However, PRF did not alter pain behaviors and pain signals in normal rats. LIMITATIONS: In our study, one time point was selected just to assess the levels of microglia, and p-p38. The changes of IRF8, microglia, p-p38 in the ipsilateral DRG were not investigated. A more detailed study on how PRF on the DRG could further relieve NP is needed. CONCLUSIONS: Restraining IRF8, microglia and p38 hyperactivity in the spinal cord of CCI rats involved in the contribution to the long-lasting analgesia of PRF. KEY WORDS: Neuropathic pain, pulsed radiofrequency, dorsal root ganglion, microglia, p38MAPK, Interferon regulatory factor 8, chronic constriction injury of sciatic nerve.

[Akt involved in diazoxide preconditioning against rat hippocampal neuronal apoptosis induced by anoxia-reoxygenation injury].

OBJECTIVE: Investigate whether preconditioning with diazoxide (DZ), a mitochondrial ATP-sensitive potassium channel opener, could enhance Akt protein to up-regulate Bcl-2/Bax protein ratio against apoptosis. METHODS: Cultured for 9 - 10 d in vitro, the hippocampal neurons of Sprague-Dawley rats were assigned to the following 5 groups randomly: Control (Group A), Anoxia (Group B), Anoxia + DZ100 micromol/L (Group C), Anoxia + DZ100 micromol/L + 5-hydroxydecanoate (Group D), Anoxia + DZ100 micromol/L + LY294002 50 micromol/L (Group E). Prior to oxygen deprivation, the hippocampal neurons were treated with DZ for 1 h per day and this treatment was persisted for 3 d. Each experiment was repeated for three times and each group contained 16 wells or 2 dishes of neurons for each time. Thereafter, neurons were derived of oxygen for 4 h. At 48 h of reoxygenation the neuronal optical density (A) were measured by MTT method, while the apoptotic rates were assayed by annexin V-FITC staining. The expressions of Akt, Bcl-2 and Bax proteins were detected and evaluated by Western blot. RESULTS: Compared with other pretreatment, DZ 100 micromol/L led to the elevation of A, whereas promoted the decrease of apoptotic rate (P < 0.05). Compared with those in other anoxic groups, the expressions of Akt protein and Bcl-2 protein in Group C were increased significantly (P < 0.05), whereas the Bax density were reduced significantly (P < 0.05). Preceding administration of 100 micromol/L DZ took protective effects on the neurons induced by anoxia-reoxygenation. CONCLUSIONS: DZ 100 micromol/L increased Akt protein to up-regulate Bcl-2/Bax protein ratio against apoptosis induced by anoxia-reoxygenation.

Diazoxide preconditioning alleviates apoptosis of hippocampal neurons induced by anoxia-reoxygenation in vitro through up-regulation of Bcl-2/Bax protein ratio.

Activating mitochondrial ATP-sensitive potassium (mitoK(ATP)) channels is a critical event of pharmacological preconditioning, which can enhance neuronal ability against various insults. mitoK(ATP) channels are abundant in neurons and can be selectively opened by diazoxide (DZ). The aim of this study was to determine whether DZ could restrain neuronal apoptosis induced by anoxia-reoxygenation and to reveal the effect of DZ preconditioning on the expressions of Bcl-2 and Bax proteins in cultured hippocampal neurons. Cultured for 9~10 d in vitro, the hippocampal neurons of Sprague-Dawley rats were assigned to the following 5 groups randomly: Control, DZ 0 mumol/L, DZ 30 mumol/L, DZ 100 mumol/L, DZ 100 mumol/L+5-hydroxydecanoate (5-HD, a selective mitoK(ATP) channel blocker) 100 mumol/L. Prior to oxygen deprivation, the hippocampal neurons except those in the control group were treated with DZ or DZ+5-HD for 1 h per day and this treatment persisted for 3 d. Thereafter, neurons were subjected to anoxia for 4 h and followed by reoxygenation. At 24 h of reoxygenation the neuronal survival rates were measured by MTT method, while the apoptotic rates were assayed by annexin V-FITC staining. The expressions of Bcl-2 and Bax proteins were detected with immunocytochemistry and evaluated by Western blot. Anoxia-reoxygenation injury reduced the survival rates and increased apoptotic rates significantly. In comparison with those in other groups, the survival rate in DZ 100 mumol/L group was increased by about 15%, whereas the apoptotic rate was decreased by almost 12% simultaneously. 5-HD could abolish the neuroprotection afforded by 100 mumol/L DZ. Bcl-2 and Bax proteins in the control normoxic neurons were both expressed slightly, while anoxia-reoxygenation led to high expression of Bax protein. The administration of 100 mumol/L DZ enhanced the expression of Bcl-2 protein by nearly 60%, whereas Bax protein was reduced by approximately 30%. Lower concentrations of DZ had no detectable effects on the expressions of Bcl-2 and Bax proteins. However, beneficial effects of DZ on the expressions of Bcl-2 and Bax proteins were reversed after the co-treatment with 5-HD. In conclusion, 100 mumol/L DZ prevented cultured hippocampal neurons from apoptosis induced by anoxia-reoxygenation possibly through up-regulating the expression of Bcl-2 protein and down-regulating the expression of Bax protein.

Diazoxide preconditioning plus subsequent hypothermia increased resistance of rat cultured hippocampal neurons against hypoxia-reoxygenation injury.

BACKGROUND: Cerebral ischemia-reperfusion/hypoxia-reoxygenation insult triggers lots of pathophysiological and biochemical events that separately affect the evolution of cerebral damage. Accordingly, all known effective neuroprotective measures should be taken to get the optimal efficacy of therapy. This study was undertaken to investigate whether diazoxide (DZ) preconditioning combined with the following hypothermia could contribute to synergistic neuroprotection compared with either hypothermia or DZ preconditioning alone. METHODS: Cultured for 9-10 days in vitro, the hippocampal neurons of SD rats were preconditioned with DZ 0 micromol/L or DZ 250 micromol/L for 1 hour per day and this treatment lasted for 3 days. Subsequently, neurons were subjected to deprivation of oxygen for 4 hours at 37 degrees C, 34 degrees C, 30 degrees C and 22 degrees C, respectively. This experiment consisted of 8 groups (4 temperature groups and 4 combination groups) and each group contained 12-well or 2-dish cells. Survival rate, expression of Bcl-2, fluorescence magnitude of intracellular calcium, and concentration of malondialdehyde (MDA) were determined at 24 hours after reoxygenation. RESULTS: The survival rate and expression of Bcl-2 were both increased in individually hypothermic conditions compared with those at 37 degrees C (P < 0.05), whereas intracellular calcium and MDA did the opposite exhibition simultaneously (P < 0.05). 22 degrees C contributed to a higher survival rate and greater expression of Bcl-2 in comparison with other hypothermia (P < 0.05). Preceding administration of 250 micromol/L DZ took the similar effects on the neurons like hypothermia. Moreover, compared with individual hypothermia or DZ preconditioning, the neuronal survival rate and expression of Bcl-2 in the combination group were increased significantly (P < 0.05), whereas the calcium fluorescence density and concentration of MDA were reduced further (P < 0.05). 250 micromol/L DZ preconditioning combined with 22 degrees C provided a maximal neuroprotection. CONCLUSIONS: Compared with either individual hypothermia or DZ preconditioning, the combination of both treatments conferred synergistic protection for cultured hippocampal neurons in vitro against hypoxia-reoxygenation insult.

[Diazoxide preconditioning combined with subsequent hypothermia alleviates anoxia-re-oxygenation injury: experiment with rat hippocampal neurons].

OBJECTIVE: To observe the alleviative effects of preconditioning with diazoxide (DZ), a mitochondrial ATP-sensitive potassium channel opener, combined with subsequent hypothermia on anoxia-re-oxygenation injury. METHODS: Hippocampal neurons of new-born SD rat were cultured. DZ of the concentration of 250 micromol/L was added into the culture fluid. The group of preconditioned cultured neurons were re-divided into 4 subgroups to be treated by hypothermia of different levels: 37 degrees C, 34 degrees C, 30 degrees C, and 22 degrees C, and then were derived of oxygen for 4 hours. The group of neurons without addition of DZ was re-divided into 4 subgroup too to be exposed to different temperatures and then underwent anoxia-re-oxygenation as described above. Thus the experiment was performed on 8 subgroups in total. Twenty-four hours after re-oxygenation the neurons were stained with dimethyl sulfoxide and their levels of absorptivity were measured so as to calculate the survival rate. Modified thiobarbital method was used to detect the concentration of malondialdehyde (MDA) in the culture fluid. Fluorescence probe Fluo-3-AM was added to detect the Ca(2+) concentration in the neurons. RESULTS: The survival rates of the DZ-preconditioned neurons of the 22 degrees C, 30 degrees C, and 34 degrees C subgroups were (94 +/- 8)%, (73 +/- 9)%, (67 +/- 8)% respectively, all significantly higher than that of the 37 degrees C subgroup (57 +/- 6%, all P < 0.01), with significant differences between the 22 degrees C subgroup and the 30 degrees C and 34 degrees C subgroups (both P < 0.01). The survival rates of the non-DZ-preconditioned neurons of the 22 degrees C, 30 degrees C, and 34 degrees C subgroups were 66 +/- 10%, 52 +/- 910%, 48 +/- 9% respectively, all significantly higher than that of the 37 degrees C subgroup (41% +/- 7%, all P < 0.01), with significant differences between the 22 degrees C subgroup and the 30 degrees C and 34 degrees C subgroups (both P < 0.01). The survival rates at different temperatures in the DZ-preconditioned group were all significantly higher than those in the corresponding non-preconditioned subgroups (all P < 0.01). The MDA concentration in the culture fluid of the 22 degrees C subgroup was significantly lower than those of the 30 degrees C, 34 degrees C, and 37 degrees C subgroups in both the DZ-preconditioned and non-preconditioned groups (all P < 0.01). The MDA concentrations of the preconditioned subgroups at different temperatures were all significantly lower than those of the corresponding non-preconditioned subgroups (all P < 0.01). The fluorescent value of the intracellular Ca(2+) of the 22 degrees C subgroup was significantly lower than those of the 30 degrees C, 34 degrees C, and 37 degrees C subgroups in both the DZ-preconditioned and non-preconditioned groups (all P < 0.01). The fluorescent value of the intracellular Ca(2+) of the preconditioned subgroups at different temperatures were all significantly lower than those of the corresponding non-preconditioned subgroups (all P < 0.01). CONCLUSION: Hypothermia alleviates the anoxia-re-oxygenation injury: DZ preconditioning enhances the neuroprotection by hypothermia.