Maternal sciatic nerve administered bupivacaine induces hippocampal cell apoptosis in offspring.

BACKGROUND: Bupivacaine, an amid-type local anesthetic, is widely used for clinical patients especially in pregnant women. In addition to neurotoxicity effect of bupivacaine, it can cross the placenta, accumulates in this tissue and retained in fetal tissues. Nevertheless, whether bupivacaine can cause neurotoxicity in fetus remains unclear. Hence, this study was design to investigate the effects of maternal bupivacaine use on fetus hippocampal cell apoptosis and the possible related mechanism. METHODS: On day 15 of pregnancy, sciatic nerve of pregnant wistar rat (180-200 g) were exposed by lateral incision of the right thigh and 0.2 ml of bupivacaine was injected. After their delivery, we randomly selected one male offspring of every mother. On day 30 after of their birth, the rat's hippocampi were isolated for molecular studies. Western blotting was used to examine the expression of cleaved caspase-3, caspase-8 and p-Akt in fetal hippocampus. RESULTS: Our results showed that maternal bupivacaine use caused a significant increment of cleaved caspase-3 and caspase-8 expression in fetal hippocampus compared with the sham group. In addition, maternally administered bupivacaine could significantly decrease hippocampal P.Akt/T.Akt ratio which was concurrent with an increment of cleaved caspase-3 and caspase-8 expression. CONCLUSION: Our data suggest that maternal bupivacaine use increases fetal hippocampal cell apoptosis markers such as caspase 8 and cleaved caspase 3, at least in part, via inhibiting the Akt activation.

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.

Antinociceptive and anti-inflammatory effects of the aerial parts of Artemisia dracunculus in mice.

CONTEXT: Tarragon (Artemisia dracunculus L., Asteraceae) is an ancient herb, which is widely used as a medicine, flavoring, or fragrance. OBJECTIVE: To determine the antinociceptive and anti-inflammatory effects of aerial parts of tarragon, we investigated the effects of ethanolic extract of the plant in adult male Balb/c mice. MATERIALS AND METHODS: Antinociceptive activity was determined using formalin, hot-plate, and writhing tests. The effect of the ethanolic extract on acute inflammation was evaluated by xylene-induced ear edema in mice. The ethanolic extract was administered at doses of 5, 10, 50, and 100 mg/kg, i.p. The control group received saline as vehicle of ethanolic extract. RESULTS: Our results showed that the ethanolic extract (50 and 100 mg/kg) decreased both phases of pain in the formalin test (ED50 = 109.66 and 87.13 mg/kg, respectively). In the hot-plate test, the extract (50 and 100 mg/kg) increased pain threshold during 60 min (ED50 = 81.03 mg/kg). The extract (50 and 100 mg/kg) exhibited antinociceptive activity against acetic acid-induced writhing (ED50 = 66.99 mg/kg). The extract (50 and 100 mg/kg) showed significant activity in the xylene ear edema test (ED50 = 78.20 mg/kg). Pretreatment of the animals with naloxone decreased the analgesia induced by the extract in hot-plate and formalin tests; therefore, opioid receptors may be involved, at least partly, in the analgesic effect of tarragon extract. DISCUSSION AND CONCLUSION: The results suggested that tarragon have significant analgesic and anti-inflammatory effects in mice, and, therefore, further studies are required to evaluate these effects and additional potential of the plant.

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.

Relationship between serum IL10 level and p38MAPK enzyme activity on behavioral and cellular aspects of variation of hyperalgesia during different stages of arthritis in rats.

OBJECTS AND DESIGN: Regarding to anti-inflammatory role of interleukin (IL) 10, its inhibitory effects on p38MAPK activity and, different pro and anti-inflammatory roles of activated p38MAPK in cells, this study was aimed to investigate relationship between serum IL10 level and p38MAPK enzyme activity on behavioral and cellular aspects variation of hyperalgesia during different stages of arthritis in rats. MATERIALS AND METHODS: Adjuvant arthritis (AA) was induced by a single subcutaneous injection of complete Freund's adjuvant into the rats' hind paw. Behavioral and inflammatory responses were assessed at 0, 3, 7, 14, and 21 days of study. Receptor and other protein enzyme expression variations were detected by western blotting. Anti-IL10 and p38MAPK inhibitor were administered daily during the 21 days of study. RESULT: Daily treatment with anti-IL10 antibody significantly increased paw edema and hyperalgesia in the AA group compared with the AA control group. Administration of anti-IL10 antibody caused significant increase in the ratio of phosphorylated p38 to p38MAPK enzyme level expression on 14th and 21st days of study compared with the AA control group. CONCLUSION: Our study confirmed that a part of anti- inflammatory effects of serum IL10 during AA inflammation was mediated via inhibition of p38MAPK enzyme phosphorylation. Moreover, these findings suggest that increase in the level of spinal mu opioid receptor expression during AA inflammation is not mediated via the direct effect of serum IL10 on spinal p38MAPK.

Intranasal CEPO-FC prevents attention deficits in streptozotocin-induced rat model of Alzheimer's disease: Focus on synaptic plasticity-related factors.

Alzheimer's disease remains an issue of great controversy due to its pathology. It is characterized by cognitive impairments and neuropsychiatric symptoms. The FDA approved medications for this disease, can only mitigate the symptoms. One reason for the lack of effective medications is the inaccessibility of the brain which is encompassed by the blood-brain barrier, making intranasal (IN) route of administration potentially advantageous. Male Wistar rats underwent stereotaxic surgery to induce an Alzheimer's disease model via intracerebroventricular (ICV) streptozotocin injection, and Carbamylated Erythropoietin-Fc (CEPO-FC), a derivative of Erythropoietin without its harmful characteristics, was administered intranasally for ten consecutive days. Cognition performance for memory and attention was assessed using the Novel Object Recognition Test and the Object-Based Attention Test respectively. Depression like behavior was evaluated using the Forced Swim Test. Western blotting was done on the extracted hippocampus to quantify STIM proteins. Calbindin, PSD-95, Neuroplastin, Synaptophysin and GAP-43 genes were assessed by Realtime PCR. Behavioral tests demonstrated that IN CEPO-FC could halt cognition deficits and molecular investigations showed that, STIM proteins were decreased in Alzheimer's model, and increased after IN CEPO-FC treatment. Calbindin and PSD-95 were downregulated in our disease model and upregulated when treated with IN CEPO-FC. While Neuroplastin, and GAP-43 expressions remained unchanged. This study suggests that IN CEPO-FC in low doses could be promising for improving cognition and synaptic plasticity deficits in Alzheimer's disease and since IN route of administration is a convenient way, choosing IN CEPO-FC for clinical trial might worth consideration. See also the graphical abstract(Fig. 1).

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.

Blockade of the orexin receptors in the ventral tegmental area could attenuate the stress-induced analgesia: A behavioral and molecular study.

Exposure to stressful stimuli induces various physiological and behavioral responses, affects pain perception, and alters gene expression. Stress elicits an analgesic effect in laboratory animals, termed the "stress-induced analgesia" (SIA). Orexin neuropeptides, processed from pre-pro-orexin in the hypothalamus, release during stress and are known to be antinociceptive. The current study examined the modulatory role of the ventral tegmental area (VTA) orexinergic system in the restraint SIA and extracellular signal-regulated kinase (ERK) activation in the nucleus accumbens (NAc). Adult male Wistar rats were subjected to intra-VTA injection of orexin-1 and -2 receptor antagonists (SB334867 and TCS OX2 29; 1, 3, 10, and 30 nmol/0.3 µl, respectively) five min before a 3-h period of exposure to restraint stress (RS). Western blot analysis was also used to assess the levels of ERK and phosphorylated ERK (p-ERK) in the NAc tissues. RS exposure produced an analgesic response to the thermal pain model (Tail-flick test). RS-induced antinociception was inhibited by intra-VTA administration of SB334867 and TCS OX2 29. Moreover, in the molecular study, exposure to forced swim stress (FSS) and RS significantly enhanced the p-ERK/ERK ratio. Blockade of both orexin receptors diminished the p-ERK/ERK ratio, but this decrease was significant only in the FSS group of animals that received TCS OX2 29. Collectively, the present findings suggested the functional roles of intra-VTA orexin receptors and ERK signaling in the SIA.

Dipeptide mimetic of BDNF ameliorates motor dysfunction and striatal apoptosis in 6-OHDA-induced Parkinson's rat model: Considering Akt and MAPKs signaling.

Parkinson's disease (PD) is a progressive and debilitating neurodegenerative disorder associated with motor and non-motor complaints. Dysregulation of neurotrophic factors and related signaling cascades have been reported to be common events in PD which is accompanied by dopaminergic (DA) neuron demise. However, the restoration of neurotrophic factors has several limitations. Bis-(N-monosuccinyl-L-methionyl-L-serine) heptamethylenediamide (BHME) is a dipeptide mimetic of brain-derived neurotrophic factor (BDNF) with reported anti-oxidant and neuroprotective effects in several experimental models. The current study has investigated the effect of BHME on 6-hydroxydopamine (6-OHDA)-caused motor anomalies in Wistar rats. In this regard, rats were treated daily with BHME (0.1 or 1 mg/kg) 1 h after 6-OHDA-caused damage until the twelfth day. Afterwards, motor behavior and DA neuron survival were evaluated via behavioral tests and immunohistochemistry (IHC) staining, respectively. Moreover, the activity of Akt, mitogen-activated protein kinases (MAPKs) family, and Bax/Bcl-2 ratio were evaluated by Western blotting. Our results indicated that BHME prevents motor dysfunction and DA cell death following 6-OHDA injection, and this improvement was in parallel with an enhancement in Akt activity, decrement of P38 phosphorylation, along with a reduction in Bax/Bcl-2 ratio. In conclusion, our findings indicated that BHME, as a mimetic of BDNF, can be considered for further research and is a promising therapeutic agent for PD therapy.

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.

Modulatory role of the orexin system in stress-induced analgesia: Involvement of the ventral tegmental area.

BACKGROUND: Exposure to stressful experiences is often accompanied by suppressing pain perception, referred to as stress-induced analgesia. The neuropeptides orexins are essential in regulating the mechanism that responds to stressful and painful stimuli. Meanwhile, the ventral tegmental area (VTA), as a part of descending pain inhibitory system, responds to noxious stimuli. This study aimed to investigate the role of intra-VTA administration of orexin receptor antagonists on stress-induced antinociceptive responses in the animal model of acute pain. METHOD: Ninety-three adult Wistar rats weighing 230-250 g were unilaterally implanted by a cannulae above the VTA. Animals were pretreated with different doses (1, 3, 10 and 30 nM/0.3 µl) of SB334867 as the orexin-1 receptor antagonist and TCS OX2 29 as the orexin-2 receptor antagonist into the VTA, just 5 min before 6 min exposure to forced swim stress (FSS). Nociceptive threshold was measured using the tail-flick test as a model of acute pain. RESULTS: The results showed that exposure to FSS could significantly increase analgesic responses. Moreover, intra-VTA administration of SB334768 and TCS OX2 29 blocked the antinociceptive effect of FSS in the tail-flick test. CONCLUSION: The findings suggest that OX1 and OX2 receptors in the VTA might modulate the antinociceptive behaviours induced by FSS in part. SIGNIFICANCE: Acute exposure to physical stress suppresses pain-related behaviors in the animal model of acute pain. Blockade of the OX1 and OX2 receptors in the VTA attenuates antinociceptive responses induced by FSS. The contribution of the OX2 receptors in the VTA is more predominant than OX1 receptors in stress-induced analgesia.

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.

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.