Microglial-induced apoptosis is potentially responsible for hyperalgesia variations during CFA-induced inflammation.

AIMS: Activated microglia is known as a main mediator of inflammatory pain, but the possible mechanisms of its operation are poorly understood. Microglial cells have considered as one of the main sources of pro-inflammatory cytokines in the CNS. PTEN is one of the important targets of pro-inflammatory cytokines and the main mediator of apoptotic cell death. In this study, we investigated the possible effect of microglial activation on PTEN/PI3K/Akt signaling pathway and apoptosis in an inflammatory rat model of Complete Freund's adjuvant (CFA). METHODS: Persistent peripheral inflammation was induced by a subcutaneous injection of CFA into the rats' right hind paw on day 0. Minocycline (a potent selective inhibitor of microglial) was administered intraperitoneally during days 1-21 after CFA injection. Hyperalgesia was assessed on days 0, 7, and 21 using plantar test, then lumbar spinal cord segments were isolated, and the amount of spinal Iba1 (microglial marker), PTEN, P.Akt, and cleaved caspase-3 (a marker of apoptosis activation) were analyzed using Western blot. The spinal TNF-α levels were assayed by ELISA and the microglia numbers were determined using immunohistochemical technique. RESULTS: Results revealed that increased hyperalgesia was concurrent with an increment of Iba1 (P < 0.001), TNF-α (P < 0.001), PTEN (P < 0.01), cleaved caspase-3 (P < 0.001), and a decrement of P.Akt (P < 0.01) during the acute phase of CFA-induced inflammation, while, at the same time as decreasing hyperalgesia during the chronic phase of study, Iba1 and TNF-α expression significantly decreased and PTEN, cleaved caspase-3, and P.Akt restored to baseline on day 0. Minocycline administration reduced the elevation of spinal Iba1 (P < 0.001), TNF-α (0.001), PTEN (P < 0.01), and cleaved caspase-3 (P < 0.001) expression induced by CFA injection, and also restored Akt activity to the baseline on day 0 (P < 0.001). CONCLUSIONS: These results suggest that microglial-mediated pain following CFA injection might be related in part to increased spinal cell apoptosis which probably is mediated by PTEN/PI3K/Akt deregulation.

Synovial interlukin-6 affects apoptosis induction via nuclear factor kappa-B and fractalkine pathway during adjuvant arthritis.

Background: Apoptosis disruptions play substantial roles in pathogenesis of arthritis and its symptoms. Cytokines and their intracellular signaling have pivotal roles in arthritis pathophysiology. This study aimed to investigate the relationship between synovial Interleukin-6 (IL-6), nuclear factor kappa-B (NF-ĸB), and fractalkine (FKN) in the changes of edema and apoptosis during adjuvantinduced knee arthritis. Methods: A total of 240 male Wistar rats were divided into different groups. Arthritis was evoked and the knee edema changes were evaluated by Vernier caliper. Synovial IL-6 was assayed by rat standard ELISA kit. Levels of NF-ĸB, fractalkine, and apoptotic indicators in the synovium were evaluated by Western blot method. Results were expressed as Mean± SEM. To analyze within-group variations, repeated measures ANOVA, followed by post hoc Tukey's test was used (SPSS, 16). Independent samples t test was used to designate significant differences in knee diameter, synovial level of IL-6, apoptotic markers, NF-ĸB, and FKN between groups. Significance level was set at P≤ 0.05. Results: The injection of Complete Freund's Adjuvant (CFA) caused intense knee edema (P< 0.001), which was reduced by implementing anti-IL-6 (P< 0.001), anti-FKN (P< 0.001), Inh-NF-ĸB (P< 0.001), and anti-FKN+ Inh NF-kB (P< 0.001). The results indicated elevated levels of apoptotic markers during the acute phase (P = 0.010), along with an increase in IL-6 (P< 0.001), NF-ĸB (P< 0.001), and FKN (P= 0.030). Although IL-6 (P< 0.001), NF-ĸB (P= 0.001), and FKN (P= 0.007) levels elevation continued during the chronic phase, the apoptosis markers decreased in this phase (P= 0.050). The findings revealed that Anti-IL-6 treatment during different phases of the study could change the synovial NF-ĸB and FKN. Conclusion: It seems that time-dependent variations in apoptotic markers level may be involved in pathogenesis of adjuvant-induced knee arthritis. In conclusion, synovial IL-6 through NF-ĸB- FKN pathway can play an important role in this process.

Long term treatment by mesenchymal stem cells conditioned medium modulates cellular, molecular and behavioral aspects of adjuvant-induced arthritis.

Neuroinflammation plays a crucial role in expression of symptoms of numerous autoimmune and neurodegenerative diseases such as pain during rheumatoid arthritis. Overproduction of pro-inflammatory cytokines and activation of intracellular signaling pathways have been strongly implicated in the generation of pathological pain states, particularly at central nervous system sites and induction of spinal neuroinflammatory symptoms. The wide ranges of research to define new therapeutic approaches, including neuroimmune-modulators like stem cells are in progress. Mesenchymal stem cells conditioned medium (MSC-CM) has anti-inflammatory factors which can regulate the immune responses. The aim of this study was to investigate the effect of administration of MSC-CM on behavioral, cellular and molecular aspects of adjuvant-induced arthritis in male Wistar rats. Complete Freund's adjuvant (CFA)-induced arthritis (AA) was caused by single subcutaneous injection of CFA into the rat's hind paw on day 0. MSC-CM was administered daily (i.p.) and during the 21 days of the study after injection. Hyperalgesia, Edema, Serum TNF-α levels and p38MAPK and NF-κB activities were assessed on days 0,7,14 and 21 of the study. The results of this study indicated the role of MSC-CM in reducing inflammatory symptoms, serum TNF-α levels and activity of intracellular signaling pathway factors during different phases of inflammation caused by CFA. It seems that MSC-CM treatment due to its direct effects on inhibition of intracellular signaling pathways and pro-inflammatory cytokines can alleviate inflammatory symptoms and pain during CFA-induced arthritis.

Inhibition of microglial activity alters spinal wide dynamic range neuron discharge and reduces microglial Toll-like receptor 4 expression in neuropathic rats.

It is believed that neuropathic pain results from aberrant neuronal discharges although some evidence suggests that the activation of glia cells contributes to pain after an injury to the nervous system. This study aimed to evaluate the role of microglial activation on the hyper-responsiveness of wide dynamic range neurons (WDR) and Toll-like receptor 4 (TLR4) expressions in a chronic constriction injury (CCI) model of neuropathic pain in rats. Adult male Wistar rats (230 ± 30 g) underwent surgery for induction of CCI neuropathy. Six days after surgery, administration of minocycline (10, 20, and 40 mg/kg, i.p.) was initiated and continued until day 14. After administration of the last dose of minocycline or saline, a behavioral test was conducted, then animals were sacrificed and lumbar segments of the spinal cord were collected for Western blot analysis of TLR4 expression. The electrophysiological properties of WDR neurons were investigated by single unit recordings in separate groups. The findings showed that after CCI, in parallel with thermal hyperalgesia, the expression of TLR4 in the spinal cord and the evoked response of the WDR neurons to electrical, mechanical, and thermal stimulation significantly increased. Post-injury administration of minocycline effectively decreased thermal hyperalgesia, TLR4 expression, and hyper-responsiveness of WDR neurons in CCI rats. The results of this study indicate that post-injury, repeated administration of minocycline attenuated neuropathic pain by suppressing microglia activation and reducing WDR neuron hyper-responsiveness. This study confirms that post-injury modulation of microglial activity is a new strategy for treating neuropathic pain.

Cellular events during arthritis-induced hyperalgesia are mediated by interleukin-6 and p38 MAPK and their effects on the expression of spinal mu-opioid receptors.

Activation of mitogen-activated protein kinase (MAPK) enzymes in nociceptive plasticity has been extensively studied. P38 MAPK enzyme, which can be activated by cytokines, acts as a crucial intracellular regulator of environmental changes. The aim of this study was to elucidate the cellular events during arthritis-induced hyperalgesia that are mediated by interleukin-6 and p38 MAPK, and their effects on the expression of spinal mu-opioid receptors (MORs), in different stages of arthritis in male Wistar rats. Complete Freund's adjuvant (CFA)-induced arthritis (AA) was caused by subcutaneous injection of CFA into the rats' hindpaw. Anti-IL-6 antibody and p38 MAPK phosphorylation inhibitor were administered during 21 days of study. Spinal MOR, p38, and phosphorylated-p38 (pp38) proteins expressions were detected by Western blotting. Daily treatment with anti-IL-6 antibody and p38 MAPK phosphorylation inhibitor, SB203580, significantly decreased paw edema in AA group. Daily anti-IL-6 and SB203580 administration caused a significant reduction in hyperalgesia in the first week of the study, but increased hyperalgesia in the next 2 weeks in experimental groups compared to the AA control group. Expression of pp38 MAPK protein significantly decreased on the 3, 7, 14, and 21 days in AA+SB203580 and AA+anti-IL6 groups compared to AA group. Additionally, daily treatment with anti-IL6 antibody and SB203580 in AA group caused significantly decrease in spinal MOR expression compared to AA control group. The results of our study can confirm that activated spinal p38 MAPK enzyme may play an important role in cellular IL-6 signaling pathways in hyperalgesia variation during different stages of AA inflammation. Also, it can be suggested that at least a part of p38 MAPK effects on hyperalgesia is mediated by spinal MOR expression variation.

Progesterone modulates the expression of spinal ephrin-B2 after peripheral nerve injury: New insights into progesterone mechanisms.

Recent studies have shown that the ephrin/Eph signaling pathway may contribute to the pathology of neuropathic pain. Drugs like progesterone may be used to counteract both thermal hyperalgesia and mechanical allodynia in different models of neuropathic pain. The present study was designed to determine progesterone's modulatory role on neuropathic pain and spinal expression of ephrin-B2 following chronic constriction nerve injury (CCI). Thirty-six adult male Wistar rats were used. The sciatic nerve was chronically constricted. Progesterone (5 mg/kg and 15 mg/kg) was administrated for 10 days (from day 1 up to day10) following sciatic constriction. Behavioral tests were performed before surgery (day 0) and on days 1, 3, 7, and 14 after CCI and before progesterone administration on the same days. Western blotting was performed on days 3, 7, and 14th post-surgery. The findings showed that after CCI, the expression of spinal cord ephrin-B2 increased significantly in parallel with mechanical allodynia and thermal hyperalgesia. Post-injury administration of progesterone (15 mg/kg but not 5) decreased mechanical allodynia, thermal hyperalgesia, and the expression of spinal ephrin-B2. It is concluded that post-injury repeated administration of progesterone could be an effective way of alleviating neuropathic pain by suppressing ephrin-B2 activation and helps to make the better design of steroid-based therapies to inhibit pain after peripheral injury.

Naloxone could limit morphine hypersensitivity: Considering the molecular mechanisms.

BACKGROUND: Naloxone has been used as an opioid antagonist to prevent multiple adverse side effects of opioid-like tolerance and hyperalgesia. This study has investigated naloxone combined with morphine to limit pain hypersensitivity. In addition, the expression of brain-derived neurotrophic factor (BDNF) and K+ Cl- cotransporter2 (KCC2) were also studied. METHODS: Forty-eight adult male Wistar rats (180-220 g) were divided into eight groups, with six rats in each group. Rats were divided into two tolerance and hyperalgesia groups; the sham group, the morphine group, the treatment group (naloxone along with morphine), and the sham group (naloxone along with saline) for eight consecutive days. Tail-flick test was performed on days 1, 5, and 8, and the plantar test on days 1 and 10. On days 8 and 10, the lumbar segments of the spinal cord were collected, and BDNF and KCC2 expression were analyzed using western blotting and immunohistochemistry, respectively. RESULTS: Results showed that tolerance and hyperalgesia developed following eight days of repeated morphine injection. BDNF expression significantly increased, but KCC2 was downregulated. Co-administration of naloxone and morphine decreased tolerance and hyperalgesia by decreasing BDNF and increasing KCC2 expression, respectively. CONCLUSION: This study suggests that BDNF and KCC2 may be candidate molecules for decreased morphine tolerance and hyperalgesia.

Pharmacological blockade of neurokinin1 receptor restricts morphine-induced tolerance and hyperalgesia in the rat.

OBJECTIVES: The most notable adverse side effects of chronic morphine administration include tolerance and hyperalgesia. This study investigated the involvement of dorsal root ganglion (DRG) protein kinase Cɛ (PKCɛ) expression during chronic morphine administration and also considered the relationship between DRG PKCɛ expression and the substance P- neurokinin1 receptor (SP- NK1R) activity. METHODS: Thirty-six animals were divided into six groups (n=6) in this study. In the morphine and sham groups, rats received 10 µg intrathecal (i.t.) morphine or saline for eight consecutive days, respectively. Behavioral tests were performed on days 1 and 8 before and after the first injections and then 48 h after the last injection (day 10). In the treatment groups, rats received NK1R antagonist (L-732,138, 25 µg) daily, either alone or 10 min before a morphine injection, Sham groups received DMSO alone or 10 min before a morphine injection. Animals were sacrificed on days 8 and 10, and DRG PKCɛ and SP expression were analyzed by western blot and immunohistochemistry techniques, respectively. RESULTS: Behavioral tests indicated that tolerance developed following eight days of chronic morphine injection. Hyperalgesia was induced 48 h after the last morphine injection. Expression of SP and PKCɛ in DRG significantly increased in rats that developed morphine tolerance on day 8 and hyperalgesia on day 10, respectively. NK1R antagonist (L-732,138) not only blocked the development of hyperalgesia and the increase of PKCɛ expression but also alleviated morphine tolerance. CONCLUSIONS: Our results provide evidence that DRG PKCɛ and SP-NK1R most likely participated in the generation of morphine tolerance and hyperalgesia. Pharmacological inhibition of SP-NK1R activity in the spinal cord suggests a role for NK1R and in restricting some side effects of chronic morphine. All experiments were performed by the National Institute of Health (NIH) Guidelines for the Care and Use of Laboratory Animals (NIH Publication No. 80-23, revised1996) and were approved by the Animal Ethics Committee of Shahid Beheshti University of Medical Sciences, Tehran, Iran (IR.SBMU.MSP.REC.1396.130).

Carbamylated erythropoietin improves recognition memory by modulating microglia in a rat model of pain.

Patients with chronic pain often complain about memory impairments. Experimental studies have shown neuroprotective effects of Carbamylated erythropoietin (Cepo-Fc) in the treatment of cognitive dysfunctions. However, little is currently known about its precise molecular mechanisms in a model of inflammatory pain. Therefore, this study aimed to investigate neuroprotective effects of Cepo-Fc against cognitive impairment induced by the inflammatory model of Complete Freund's Adjuvant (CFA). Carbamylated erythropoietin was administrated Intraperitoneally (i.p) on the day CFA injection, continued for a 21-days period. After conducting the behavioral tests (thermal hyperalgesia and novel object recognition test), western blot and ELISA were further preformed on days 0, 7, and 21. The results of this study indicate that Cepo-Fc can effectively reverse the CFA induced thermal hyperalgesia and recognition memory impairment. Additionally, Cepo-Fc noticeably decreased the hippocampal microglial expression, production of hippocampal IL-1ß, and hippocampal apoptosis and necroptosis induced by the inflammatory pain. Therefore, our findings suggest that neuroprotective effects of Cepo-Fc in the treatment of pain related recognition memory impairment may be mediated through reducing hippocampal microglial expression as well as IL-1ß production.

Analgesic effects of methanolic extracts of the leaf or root of Moringa oleifera on complete Freund's adjuvant-induced arthritis in rats.

OBJECTIVE: Moringa oleifera (family Moringaceae) has been widely used in African folk medicine, and researchers have recently revealed its anti-inflammatory effects in human. This study aimed to evaluate the analgesic properties of methanolic extracts of M. oleifera in complete Freund's adjuvant (CFA)-induced arthritis in rats. METHODS: Adult male Wistar rats, weighing 200 to 220 g, were used in this study. Adjuvant arthritis was induced on day 0 by a single subcutaneous injection of CFA. The prepared extracts from both the root and leaf (200, 300 and 400 mg/kg) of M. oleifera were administered intraperitonealy to rats in the treatment groups 0, 3 and 6 d after CFA injection and indomethacin (5 mg/kg) was used as a positive control drug. Thermal hyperalgesia and mechanical allodynia were evaluated for the analgesic effect 0, 3 and 6 d after CFA injection. Combined methanolic root and leaf extracts of M. oleifera (200 mg/kg) were also tested for the analgesic effect. RESULTS: The potency of the root or leaf extracts of M. oleifera (300 and 400 mg/kg) was similar to that of indomethacin, resulted in significant reductions in both thermal hyperalgesia and mechanical allodynia in rats with CFA-induced arthritis compared with the control group after 3 and 6 d, respectively (P<0.01 or P<0.05). Combined root and leaf extracts (200 mg/kg) of M. oleifera resulted in a significant reduction in thermal hyperalgesia compared with the control group after 3 and 6 d, respectively (P<0.01). Prophylactic injections of combined root and leaf extracts of M. oleifera (200 mg/kg) resulted in a significant reduction in thermal hyperalgesia compared with the control group, the root extracts group, and the leaf extracts group after 3 and 6 d, respectively (P<0.01). CONCLUSION: The methanolic extracts of the root or leaf of M. oleifera are effective in the reduction of pain induced by CFA in rats. A comparison of single and combination therapies of root and leaf extracts also showed a synergistic effect on pain reduction.

Evaluation of the GABAA Receptor Expression and the Effects of Muscimol on the Activity of Wide Dynamic Range Neurons Following Chronic Constriction Injury of Sciatic Nerve in Rats.

INTRODUCTION: The modality of γ-aminobutyric acid type a receptors (GABAA) controls dorsal horn neuronal excitability and inhibits sensory information. This study aimed to investigate the expression of the GABAA receptor and the effects of its agonist muscimol on Wide Dynamic Range (WDR) neuronal activity in the Chronic Constriction Injury (CCI) model of neuropathic pain. METHODS: Adult male Wistar rats weighing 200 to 250 g were used to induce CCI neuropathy. Fourteen days after surgery, muscimol (0.5, 1, and 2 mg/kg IP) was injected. Then, the behavioral tests were performed. After that, the animals were killed, and the lumbar segments of the spinal cords were collected for Western blot analysis of the GABAA receptor α1 subunit expression. The electrophysiological properties of WDR neurons were studied by single-unit recordings in separate groups 14 days after CCI. RESULTS: The outcomes indicated the development of thermal hyperalgesia and mechanical allodynia after neuropathy; nonetheless, the expression of the GABAA receptor α1 subunit did not change significantly. Moreover, the evoked responses of the WDR neurons to electrical, mechanical, and thermal stimuli increased considerably. Fourteen days after CCI, muscimol administration decreased thermal hyperalgesia, mechanical allodynia, and hyper-responsiveness of the WDR neurons in CCI rats. CONCLUSION: The modulation of the spinal GABAA receptors after nerve injury can offer further insights to design new therapeutic agents to reduce neuropathic pain symptoms.

Retraction notice to "Noopept; a nootropic dipeptide, modulates persistent inflammation by effecting spinal microglia dependent Brain Derived Neurotropic Factor (BDNF) and pro-BDNF expression throughout apoptotic process" [Heliyon (2021) e06219].

[This retracts the article DOI: 10.1016/j.heliyon.2021.e06219.].

Noopept; a nootropic dipeptide, modulates persistent inflammation by effecting spinal microglia dependent Brain Derived Neurotropic Factor (BDNF) and pro-BDNF expression throughout apoptotic process.

There are largely unknown associations between changes in pain behavior responses during persistent peripheral inflammation and spinal cell alteration such as apoptosis. Some evidence suggests that microglia and microglia related mediators play notable roles in induction and maintenance of central nervous system pathologies and inflammatory pain. By considering those relationships and microglia related nootrophic factors, such as the Brain Derived Neurotrophic Factor (BDNF) in CNS, we attempted to assess the relationship between microglia dependent BDNF and its precursor with pain behavior through spinal cell apoptosis as well as the effect of Noopept on this relationship. Persistent peripheral inflammation was induced by a single subcutaneous injection of Complete Freund's Adjuvant (CFA) on day 0. Thermal hyperalgesia, paw edema, microglial activity, microglia dependent BDNF, pro-BDNF expression, and apoptosis were assessed in different experimental groups by confirmed behavioral and molecular methods on days 0, 7, and 21 of the study. Our findings revealed hyperalgesia and spinal cell apoptosis significantly increased during the acute phase of CFA-induced inflammation but was then followed by a decrement in the chronic phase of the study. Aligned with these variations in spinal microglial activity, microglia dependent BDNF significantly increased during the acute phase of CFA-induced inflammation. Our results also indicated that daily administration of Noopept (during 21 days of the study) not only caused a significant decrease in hyperalgesia and microglia dependent BDNF expression but also changed the apoptosis process in relation to microglia activity alteration. It appears that the administration of Noopept can decrease spinal cell apoptosis and hyperalgesia during CFA-induced inflammation due to its direct effects on microglial activity and microglia dependent BDNF and pro-BDNF expression.

Achillea santolina reduces serum interleukin-6 level and hyperalgesia during complete Freund's adjuvant-induced inflammation in male Wistar rats.

OBJECTIVE: Immune system is involved in the etiology and pathophysiologic mechanisms of inflammation. Medicinal plants are an important source of substances which are claimed to induce non-specific immunomodulatory effects. In view of this and on account of the interleukin (IL)-6's role in inflammation and pain induction, this study investigated the effects of Achillea santolina extracts on inflammation which was induced by complete Freund's adjuvant (CFA) in male Wistar rats. METHODS: Both methanolic and defatted extracts prepared from aerial parts of the plant were examined. Inflammatory symptoms such as hyperalgesia and paw edema in CFA-injected rats' paw were measured by radiant heat and plethysmometer during different stages of study respectively. Serum IL-6 level was checked by rat standard enzyme-linked immunosorbent assay specific kit. RESULTS: The results indicated dose-related effects of methanolic extract on paw edema, hyperalgesia and serum IL-6 level reduction in rats. Methanolic extract of A. santolina exhibited significant antihyperalgesic and anti-inflammatory effects during pretreatment and short-term treatment at dose of 200 mg/kg and there was no significant difference between 200 and 400 mg/kg doses of this extract. Defatted extract did not show significant effect on CFA-induced inflammation during different stages of treatment (P>0.05). Short-term treatment with methanolic extract at dose of 200 mg/kg was more effective than indomethacin in edema, hyperalgesia and serum IL-6 level reduction (P<0.01, P<0.01 and P<0.05 respectively). CONCLUSION: These results suggest that methanolic extract of A. santolina possesses potent anti-inflammatory and immunomodulatory activities during pretreatment and short-term administration.

Chemical stimulation of the lateral hypothalamus induces antiallodynic and anti-thermal hyperalgesic effects in animal model of neuropathic pain: Involvement of orexin receptors in the spinal cord.

To date several circuities in supraspinal site of the central nervous system have been known to engage in pain modulation. Lateral hypothalamus (LH) is known as part of the circuit of pain modulation among supraspinal sites. Its role in several animal pain models has been well defined. In this study, we examined the role of spinal orexin receptors in antinociceptive response elicited by the LH stimulation in an animal model of neuropathic pain. Male Wistar rats were unilaterally implanted with a cannula into the LH and a catheter into the L4-L5 segments of the spinal cord followed by chronic constriction injury (CCI) surgery. Intra-LH microinjection of carbachol (500 nM; 0.5 µL) was done 5 min after intrathecal administration of the orexin receptor antagonists, SB-334867 or TCS OX2 29; control animals received DMSO. Mechanical allodynia and thermal hyperalgesia were evaluated using von Frey filaments and a thermal stimulus. The results showed that carbachol induces antiallodynic and anti-thermal hyperalgesic effects in a dose-dependent manner. The antiallodynic and anti-thermal hyperalgesic effects induced by intra-LH injection of carbachol were reversed by intrathecal administration of 10 µL-100 nM solutions of SB-334867 or TCS OX2 in neuropathic rats. However, solely intrathecal administration of both antagonists had no effect in neuropathic rats. There appears to be a neural pathway from the LH to the spinal cord, which potentially contributes to the modulation of neuropathic pain. The implications are that there may be novel therapeutic approaches for the treatment of people suffered from chronic neuropathic pain in clinic.

The Potential Role of Glycogen Synthase Kinase-3ß in Neuropathy-Induced Apoptosis in Spinal Cord.

INTRODUCTION: Glycogen Synthase Kinase-3ß (GSK-3ß) participates in several signaling pathways and plays a crucial role in neurodegenerative diseases, inflammation, and neuropathic pain. The ratio of phosphorylated GSK-3ß over total GSK-3ß (p-GSK-3ß/t-GSK-3ß) is reduced following nerve injury. Apoptosis is a hallmark of many neuronal dysfunctions in the context of neuropathic pain. Thus, this study aimed to evaluate the contribution of p-GSK-3ß/t-GSK-3ß ratio in spinal dorsal horn apoptosis following peripheral nerve injury. METHODS: In this study, adult male Wistar rats (220-250 g) underwent Spinal Nerve Ligation (SNL) surgery. Mechanical allodynia and thermal hyperalgesia were assessed before the surgery (day 0); then, every other day up to day 8. GSK-3ß selective inhibitor, AR-014418 [0.3 mg/kg, Intraperitoneal (IP)] was administrated 1 h prior to SNL on day 0, then daily up to the day 8. The GSK-3ß activity and apoptosis in the lumbar section (L4, L5, or L6) of the study rat's spinal cord were assessed by immunohistochemical and Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL) staining, respectively on day 8 post-SNL. RESULTS: Following the SNL, the mechanical allodynia and thermal hyperalgesia increased on day 2 up to day 8 post-SNL. The ratio of p-GSK-3ß/t-GSK-3ß decreased, and the number of apoptotic cells increased in the spinal dorsal horn on day 8. However, AR-A014418 administration could increase the p-GSK-3ß/t-GSK-3ß ratio and decreased apoptosis in the SNL rats. In addition, AR-A014418 decreased the mechanical allodynia from day 4 up to day 8; however, it did not affect thermal hyperalgesia. CONCLUSION: The study findings suggested that increasing the p-GSK-3ß/t-GSK-3ß ratio might be a helpful strategy for reducing the apoptotic cells and subsequent neuropathic pain during peripheral nerve injury.

Microglia dependent BDNF and proBDNF can impair spatial memory performance during persistent inflammatory pain.

Inflammatory pain is commonly associated with cognitive impairment. However, its molecular mechanisms are poorly understood. Thus, this study was conducted to investigate the molecular mechanisms of behavioral changes associated with inflammatory pain. Briefly, 36 Wistar rats were randomly divided into two main groups: CFA group treated with 100 µL of Complete Freunds' Adjuvant (CFA) and CFA + Minocycline group treated with 100 µL of CFA+40 mg/kg/day of minocycline). After that, each group was divided into three subgroups based on different time points of the study. The pain was induced using CFA and subsequent behavioral changes (i.e., hyperalgesia and learning and spatial memory) were analyzed by the Morris Water Maze (MWM) task and Radiant Heat. Then, the cellular and molecular changes were assessed using Western Blotting, Immunohistochemistry, and Terminal deoxynucleotidyl transferase dUTP Nick End Labeling (TUNEL) techniques. Results of the study indicated that CFA-induced pain impaired spatial learning and memory functions. Studying the cellular changes showed that persistent inflammatory pain increased the microglial activity in CA1 and Dentate Gyrus (DG) regions. Furthermore, an increase was observed in the percentage of TUNEL-positive cells. Also, pro-Brain-Derived Neurotrophic Factor (BDNF)/BDNF ratio, Caspase3, and Receptor-Interacting Protein kinase 3 (RIP3) levels increased in the rats' hippocampus following induction of persistent inflammatory pain. These changes were reversed following the cessation of pain as well as the injection of minocycline. Taking together, the results of the current study for the first time revealed that an increase in the microglia dependent proBDNF/BDNF ratio following persistent inflammatory pain leads to cell death of the CA1 and DG neurons that subsequently causes a cognitive deficit in the learning and spatial memory functions.

Recovery of inferior alveolar nerve by photobiomodulation therapy using two laser wavelengths: A behavioral and immunological study in rat.

Postoperative sensory disturbances of inferior alveolar nerve (IAN) are major challenges in dental procedures. We aimed to investigate the effect of photobiomodulation therapy (PBMT) with 810 nm and 980 nm diode lasers on behavioral and immunological factors in a rat IAN crush model. Seventy-two rats were randomly assigned to the four groups of 810 nm laser (crush injury+810 nm laser; 6 J/cm2, 15 sessions, every 48 h), 980 nm laser (crush injury+980 nm laser; same protocol), control (crush injury without irradiation), and sham surgery (no crush injury and no irradiation). The neurosensory response of IAN was evaluated by Von Frey behavioral test before (baseline) and post-surgery in a period of one month. Changes of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), nuclear factor-kappa B (NF-κB), TNF-α, and IL-1ß, were assessed on days 2 and 30 post injury. Data were analyzed for significant differences by repeated measures and one-way ANOVA (p < .05). One day after surgery, all rats subjected to nerve injury showed significant increase in the withdrawal threshold of von Frey test compared to the baseline (p = .02 for control and p = .03 for laser groups). The threshold gradually returned to the baseline scores in 810 nm, 980 nm, and control groups from days 11, 17, and 29, respectively. There was a significant lower withdrawal threshold in 810 nm and 980 nm laser groups compared to the control group in days 11 to 19 and 9 to 23, respectively. At both time points, the levels of NGF and BDNF were significantly higher in 810 nm laser group compared to the control group. There was a significant difference between laser and control groups regarding NF-κB expression (all p values<.001). TNF-α and IL-1ß were significantly lower in laser groups compared to the control group (all p values < .001). PBMT with 810 and 980 nm diode laser protocol used in this study, promoted the neurosensory recovery of IAN after crush injury in rats. In addition, application of 810 nm diode laser was associated with more improvement in immunological responses compared to that of 980 nm laser.

Role of spinal glial cells in excitability of wide dynamic range neurons and the development of neuropathic pain with the L5 spinal nerve transection in the rats: Behavioral and electrophysiological study.

The activation of glial cells affects the neuronal excitability in the spinal cord. Therefore, in this study, we tried to find out the modulatory role of spinal glial cells in the excitability of wide dynamic range (WDR) neurons, induction of the long-term potentiation (LTP) and development of neuropathic pain by L5 spinal nerve transection model in the rats. Forty-eight adult male Wistar rats were used to measure the paw withdrawal threshold to mechanical stimuli and also, to carry out the spinal extracellular single unit recording experiments. In these experiments, spinal nerve ligation (SNL) and a daily injection of propentofylline (1 mg/kg, ip) as a glial cell inhibitor agent, 1 h following nerve ligation during 7-day post-SNL period, were performed. Our findings showed that the mechanical allodynia, and synaptically-evoked firing were caused LTP in the Aδ-fiber, C-fiber and lesser in the Aß-fiber after high frequency stimulation. Daily injection of propentofylline considerably decreased LTP induction in the Aδ- and C-fibers (P < .001). It was concluded that glial cell activation mediates LTP induction in the spinal cord following peripheral nerve injury. It seems that pain modulatory role of glial cells is partly parallel to changes in neural excitability of the WDR neurons in the dorsal horn of spinal cord.

Thymulin treatment attenuates inflammatory pain by modulating spinal cellular and molecular signaling pathways.

Thymulin is a peptide hormone which is mainly produced by thymic epithelial cells and it has immune-modulatory and anti-inflammatory effects. In this study, we investigated the effects of different doses and various timings of thymulin intraperitoneal administration on spinal microglial activity and intracellular pathways in an inflammatory rat model of Complete Freund's adjuvant (CFA). Thymulin treatment was implemented following CFA-induced inflammation for 21 days. After conducting behavioral tests (edema and hyperalgesia), the cellular and molecular aspects were examined to detect the thymulin effect on inflammatory factors and microglial activity. We demonstrated that thymulin treatment notably reduced thermal hyperalgesia and paw edema induced by CFA. Furthermore, molecular investigations showed that thymulin reduced CFA-induced activation of microglia cells, phosphorylation of p38 MAPK and the production of spinal pro-inflammatory cytokines (TNF-α, IL-6) during the study. Our results suggest that thymulin treatment attenuates CFA-induced inflammation. This effect may be mediated by inhibition of spinal microglia and production of central inflammatory mediators which seems to be associated with the ability of thymulin to reduce p38 MAPK phosphorylation. These data provide evidence of the anti-hyperalgesic effect of thymulin on inflammatory pain and characterize some of the underlying spinal mechanisms.