Microfluidic Synthesis of Ultrasmall Chitosan/Graphene Quantum Dots Particles for Intranasal Delivery in Alzheimer's Disease Treatment.

Nanoparticles (NPs) based therapies for Alzheimer's disease (AD) attract interest due to their ability to pass across or bypass the blood-brain barrier. Chitosan (CS) NPs or graphene quantum dots (GQDs) are promising drug carriers with excellent physicochemical and electrical properties. The current study proposes the combination of CS and GQDs in ultrasmall NP form not as drug carriers but as theranostic agents for AD. The microfluidic-based synthesis of the CS/GQD NPs with optimized characteristics makes them ideal for transcellular transfer and brain targeting after intranasal (IN) delivery. The NPs have the ability to enter the cytoplasm of C6 glioma cells in vitro and show dose and time-dependent effects on the viability of the cells. IN administration of the NPs to streptozotocin (STZ) induced AD-like models lead to a significant number of entrances of the treated rats to the target arm in the radial arm water maze (RAWM) test. It shows the positive effect of the NPs on the memory recovery of the treated rats. The NPs are detectable in the brain via in vivo bioimaging due to GQDs as diagnostic markers. The noncytotoxic NPs localize in the myelinated axons of hippocampal neurons. They do not affect the clearance of amyloid ß (Aß) plaques at intercellular space. Moreover, they showed no positive impact on the enhancement of MAP2 and NeuN expression as markers of neural regeneration. The memory improvement in treated AD rats may be due to neuroprotection via the anti-inflammation effect and regulation of the brain tissue microenvironment that needs to be studied.

Effect of multifactorial therapeutic approach on axonal regeneration and cell viability in an in-vitro model of spinal-derived neural injury.

The highly debilitated nature of spinal cord injuries (SCI) creates an inhibitory repair environment that limits the recovery rate and therefore single interventional treatment has been resulted in incomplete recovery. A multifactorial approach that combines several therapeutic approaches may address diverse aspects of SCI pathology and enhance the recovery rate over single therapy. Accordingly, in this study, we aimed to investigate the effect of combined olfactory ensheathing cells (OECs) (to transport trophic factor, mediate immunomodulation, provide a suitable environment for cell survival), G-CSF (to establish a favorable environment for cell survival) and lipopolysaccharide (LPS) (to boost the protective activity of OEC) therapy on the cell viability after a scratch injury caused by a cataract knife on cells in an in-vitro model of spinal-derived neural injury. In this study, we used mixed neuronal-glial cultures, which are widely used for an in vitro study of neuronal damage. Scratch insult was made on cells using a cataract knife. The cells were divided into 8 groups (two control groups with and without olfactory ensheathing cells (OECs) treatment, injury group, three injury groups with single therapy by using super low dose of LPS (SLD-LPS) (100 pg/ml), OEC group, and G-CSF (100 ng/ml) group, and two injury groups with combined therapy (OEC with SLD-LPS and with all three treatments)). We found a significant decrease in the survival rate of injured cells (p < 0.001) 24 h after scratching insult. Our results indicated morphological alterations in cells in the acute phase (1, 2 and 6 h) after injury, with significant increased gap size at 6 h after induction of injury. Our combined therapy, significantly prevented cell death and decreased the size of the gap over time. We found that combined therapy promoted cell survival following spinal injury by providing a neuroprotective environment for cells. Therefore, our findings provide new insight into the combined therapy, which can be considered for promising preclinical therapeutic strategy for SCI toward clinical trials.

Roles and Interaction of the MAPK Signaling Cascade in Aß25-35-Induced Neurotoxicity Using an Isolated Primary Hippocampal Cell Culture System.

Alzheimer's disease (AD) is characterized with increased formation of amyloid-ß (Aß) in the brain. Aß peptide toxicity is associated with disturbances of several intracellular signaling pathways such as mitogen activated protein kinases (MAPKs). The aim of this study was to investigate the role of MAPKs and their interactions in Aß-induced neurotoxicity using isolated hippocampal neurons from the rat. Primary hippocampal cells were cultured in neurobasal medium for 4 days. Cells were treated with Aß25-35 and/or MAPKs inhibitors for 24 h. Cell viability was determined by an MTT assay and phosphorylated levels of P38, JNK, and ERK were measured by Western blots. Aß treatment (10-40 µM) significantly decreased hippocampal cell viability in a dose-dependent manner. Inhibition of P38 and ERK did not restore cell viability, while JNK inhibition potentiated the Aß-induced neurotoxicity. Compared to the controls, Aß treatment increased levels of phosphorylated JNK, ERK, and c-Jun, while it had no effect on levels of phosphorylated P38. In addition, P38 inhibition led to decreased expression levels of phosphorylated ERK; inhibition of JNK resulted in decreased expression of c-Jun; and inhibition of ERK, decreased phosphorylated levels of JNK. These results strongly suggest that P38, ERK, and JNK are not independently involved in Aß-induced toxicity in the hippocampal cells. In AD, which is a multifactorial disease, inhibiting a single member of the MAPK signaling pathway, does not seem to be sufficient to mitigate Aß-induced toxicity and thus their interactions with each other or potentially with different signaling pathways should be taken into account.

CEPO (carbamylated erythropoietin)-Fc protects hippocampal cells in culture against beta amyloid-induced apoptosis: considering Akt/GSK-3ß and ERK signaling pathways.

The tissue-protective properties of erythropoietin (EPO) have been described in several neurodegenerative diseases models, but erythrocytosis following EPO treatment may lead to deleterious effects. Carbamylated erythropoietin, an EPO derivative lacking hematopoietic side effects, has shown protective properties in some studies. However, it is not known if CEPO protects primary hippocampal cells against Aß25-35 toxicity. The present study aimed to investigate the effect of CEPO-Fc on biochemical alterations in Akt, GSK-3ß, and ERK signaling and cell death induced by Aß25-35 in isolated hippocampal cell culture. The embryonic hippocampal cells were obtained from 18-19 day rat embryos. The cells were exposed with Aß25-35 (20 µM) in the absence or presence of CEPO-Fc (1 or 5 IU) and PI3k and ERK inhibitors. CEPO-Fc at the dose of 5 IU significantly prevented the cell loss and caspase-3 cleavage caused by Aß25-35. Additionally, CEPO-Fc noticeably reversed Aß mediated decrement of Akt and GSK-3ß phosphorylation. With exposure to LY294002, PI3 kinase inhibitor, Akt phosphorylation diminished and CEPO-Fc protective effects disappeared. Furthermore, while CEPO-Fc nullified Aß-induced increment of phospho-ERK, inhibition of ERK activity by PD98059, had no effect on Aß25-35-mediated caspase-3 cleavage and cell toxicity. These results imply that protective effects of CEPO-Fc seem to be mainly exerted through the PI3K/Akt pathway rather than ERK signaling. This study suggested that CEPO-Fc prevents Aß-induced cell toxicity as well as Akt/GSK-3ß and ERK alterations in isolated hippocampal cells. These findings might provide a new perspective on CEPO-Fc protective properties as a prospective remedial factor for neurodegenerative diseases like AD.

Correction to: CEPO (carbamylated erythropoietin)-Fc protects hippocampal cells in culture against beta amyloid-induced apoptosis: considering Akt/GSK-3ß and ERK signaling pathways.

Unfortunately, the original version of this article contained a mistake in the arrangement of representative cell images in Fig. 2. In this figure, the same representative image for Aß group was mistakenly placed for Aß + LY group. The corrected form of this figure is provided in this correction.

To be or not to be: PP2A as a dual player in CNS functions, its role in neurodegeneration, and its interaction with brain insulin signaling.

Accumulating evidence has reached the consensus that the balance of phosphorylation state of signaling molecules is a pivotal point in the regulation of cell signaling. Therefore, characterizing elements (kinases-phosphatases) in the phosphorylation balance are at great importance. However, the role of phosphatase enzymes is less investigated than kinase enzymes. PP2A is a member of serine/threonine protein phosphatase that its imbalance has been reported in neurodegenerative diseases. Therefore, we reviewed the superfamily of phosphatases and more specifically PP2A, its regulation, and physiological functions participate in CNS. Thereafter, we discussed the latest findings about PP2A dysregulation in Alzheimer and Parkinson diseases and possible interplay between this phosphatase and insulin signaling pathways. Finally, activating/inhibitory modulators for PP2A activity as well as experimental methods for PP2A study have been reviewed.

The neuroprotective effect of agmatine against amyloid ß-induced apoptosis in primary cultured hippocampal cells involving ERK, Akt/GSK-3ß, and TNF-α.

ß-Amyloid peptide (Aß), the major element of senile plaques in Alzheimer's disease (AD), has been found to accumulate in brain regions critical for memory and cognition. Deposits of Aß trigger neurotoxic events which lead to neural apoptotic death. The present study examined whether agmatine, an endogenous polyamine formed by the decarboxylation of L-arginine, possesses a neuroprotective effect against Aß-induced toxicity. Primary rat hippocampal cells extracted from the brains of 18-19-day-old embryos were exposed to 10 µM of Aß (25-35) in the absence or presence of agmatine at 150 or 250 µM. Additionally, the involvement of Akt (Protein Kinae B), GSK-3ß (glycogen synthase kinase 3-ß), ERK (Extracellular Signal-Regulated Kinase) and TNF-α (Tumor necrosis factor-α) in the agmatine protection against Aß-induced neurotoxicity was investigated. Agmatine significantly prevented the effect of Aß exposure on cell viability and caspase-3 assays. Furthermore, agmatine considerably restored Aß-induced decline of phospho-Akt and phospho-GSK and blocked Aß-induced increase of phospho-ERK and TNF-alpha. Taken together, these findings might shed light on the protective effect of agmatine as a potential therapeutic agent for AD.

Time-course study of high fat diet induced alterations in spatial memory, hippocampal JNK, P38, ERK and Akt activity.

Consumption of high fat diet (HFD) is a health concern in modern societies, which participate in wide range of diseases. One underlying mechanism in the HFD mediated pathologies is disruption of insulin signaling activity. It is believed that HFD activates several stress signaling molecules such as MAPKs signaling pathway and these molecules participate in harmful effects in different cell populations including hippocampal cells. However, the activity of MAPKs signaling molecules are time dependent, even causing some opposing effects. Given that, MAPKs activity fluctuate with time of stress, there is less cleared how different lengths of HFD consumption can affect hippocampal MAPK. To test how duration of HFD consumption affect hippocampal MAPKs and insulin signaling activity and animal's cognitive function, rats were fed with HFD for different lengths (up to 6 months) and after each point spatial memory performances of animals was tested, then the peripheral indices of insulin resistance and hippocampal MAPKs and insulin signaling activity was evaluated. Results showed that while different time courses of HFD, up to 6 months, did not bring about significant spatial memory impairment, meanwhile the peripheral insulin sensitivity as well as hippocampal insulin and MAPKs signaling showed significant fluctuations during the different time courses of high fat diet regime. These results showed that neuronal responses to HFD is not constant and differ in a time-dependent manner, it seems that in acute phase molecular responses aimed to compensate the HFD stress but in chronic states these responses failed and devastating effects of stress began.

Correction to: Agmatine Protects Against 6-OHDA-Induced Apoptosis, and ERK and Akt/GSK Disruption in SH-SY5Y Cells.

The original version of this article unfortunately contained a mistake in the unit of agmatine doses. The agmatine doses were erroneously written in nanomolar in the published article. The correct effective doses of agmatine were 150 and 250 µM.

Agmatine Protects Against 6-OHDA-Induced Apoptosis, and ERK and Akt/GSK Disruption in SH-SY5Y Cells.

6-Hydroxydopamine (6-OHDA), a metabolite of dopamine is known to induce dopaminergic cell toxicity which makes that a suitable agent inducing an experimental model of Parkinson's disease (PD). Agmatine has been shown to protect against some cellular and animal PD models. This study was aimed to assess whether agmatine prevents 6-OHDA-induced SH-SY5Y cell death and if yes, then how it affects Akt/glycogen synthesis kinase-3ß (GSK-3ß) and extracellular signal-regulated kinases (ERK) signals. The cells were treated with different drugs, and their viability was examined via MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay and morphological observation. Western blot studies were done to assess cleaved caspase-3, Akt/GSK-3ß, and ERK proteins. 6-OHDA-induced cell death and caspase-3 cleavage, while agmatine prevented those changes. 6-OHDA also decreased the amount of phosphorylated Akt (pAkt)/Akt while increased GSK-3ß activity which was prevented by agmatine. Additionally, this toxin increased pERK/ERK ratio which was averted again by agmatine. The PI3/Akt inhibitor, LY294002, impeded the changes induced by agmatine, while ERK inhibitor (PD98059) did not disturb the effects of agmatine, and by itself, it preserved the cells against 6-OHDA toxicity. This study revealed that agmatine is protective in 6-OHDA model of PD and affects Akt/GSK-3ß and ERK pathways.

Integrated sphingosine-1 phosphate signaling in the central nervous system: From physiological equilibrium to pathological damage.

Sphingosine-1 phosphate (S1P), a bioactive sphingolipid metabolite, plays an essential role in cellular homeostasis. It is well evidenced that enzymes responsible for S1P production, as well as S1P receptors are expressed in the central nervous system (CNS), implying that S1P may contribute to CNS physiology. In current review, we will present the current knowledge about developmental and neuromodulatory functions of S1P in the brain. Considering neuroprotective effects of S1P, we also review the relation between S1P and cellular autophagy, mitochondrial function, oxidative stress and apoptosis as well as molecular pathways underlying neuroprotective effects of S1P. Given these pivotal functions, in the last section, we will summarize latest findings about possible contribution of S1P dysregulation in neurological disorders like Alzheimer's disease and multiple sclerosis.

Neurosteroids; potential underpinning roles in maintaining homeostasis.

The neuroactive steroids which are synthesized in the brain and nervous system are known as "Neurosteroids". These steroids have crucial functions such as contributing to the myelination and organization of the brain connectivity. Under the stressful circumstances, the concentrations of neurosteroid products such as allopregnanolone (ALLO) and allotetrahydrodeoxycorticosterone (THDOC) alter. It has been suggested that these stress-derived neurosteroids modulate the physiological response to stress. Moreover, it has been demonstrated that the hypothalamic-pituitary-adrenal (HPA) axis mediates the physiological adaptation following stress in order to maintain homeostasis. Although several regulatory pathways have been introduced, the exact role of neurosteroids in controlling HPA axis is not clear to date. In this review, we intend to discern specific pathways associated with regulation of HPA axis in which neuroactive steroids have the main role. In this respect, we propose pathways that may be initiated after neurosteroidogenesis in different brain subregions following acute stress which are potentially capable of activating or inhibiting the HPA axis.

Glycogen synthase kinase-3 beta (GSK-3ß) signaling: Implications for Parkinson's disease.

Glycogen synthase kinase 3 (GSK-3) dysregulation plays an important role in the pathogenesis of numerous disorders, affecting the central nervous system (CNS) encompassing both neuroinflammation and neurodegenerative diseases. Several lines of evidence have illustrated a key role of the GSK-3 and its cellular and molecular signaling cascades in the control of neuroinflammation. Glycogen synthase kinase 3 beta (GSK-3ß), one of the GSK-3 isomers, plays a major role in neuronal apoptosis and its inhibition decreases expression of alpha-Synuclein (α-Synuclein), which make this kinase an attractive therapeutic target for neurodegenerative disorders. Parkinson's disease (PD) is a chronic neurodegenerative movement disorder characterized by the progressive and massive loss of dopaminergic neurons by neuronal apoptosis in the substantia nigra pars compacta and depletion of dopamine in the striatum, which lead to pathological and clinical abnormalities. Thus, understanding the role of GSK-3ß in PD will enhance our knowledge of the basic mechanisms underlying the pathogenesis of this disorder and facilitate the identification of new therapeutic avenues. In recent years, GSK-3ß has been shown to play essential roles in modulating a variety of cellular functions, which have prompted efforts to develop GSK-3ß inhibitors as therapeutics. In this review, we summarize GSK-3 signaling pathways and its association with neuroinflammation. Moreover, we highlight the interaction between GSK-3ß and several cellular processes involved in the pathogenesis of PD, including the accumulation of α-Synuclein aggregates, oxidative stress and mitochondrial dysfunction. Finally, we discuss about GSK-3ß inhibitors as a potential therapeutic strategy in PD.

Mitochondrial Transportation, Transplantation, and Subsequent Immune Response in Alzheimer's Disease: An Update.

Alzheimer's disease (AD) is a devastating neurodegenerative disease characterized by memory impairment and a progressive decline in cognitive function. Mitochondrial dysfunction has been identified as an important contributor to the development of AD, leading to oxidative stress and energy deficits within the brain. While current treatments for AD aim to alleviate symptoms, there is an urgent need to target the underlying mechanisms. The emerging field of mitotherapy, which involves the transplantation of healthy mitochondria into damaged cells, has gained substantial attention and has shown promising results. However, research in the context of AD remains limited, necessitating further investigations. In this review, we summarize the mitochondrial pathways that contribute to the progression of AD. Additionally, we discuss mitochondrial transfer among brain cells and mitotherapy, with a focus on different administration routes, various sources of mitochondria, and potential modifications to enhance transplantation efficacy. Finally, we review the limited available evidence regarding the immune system's response to mitochondrial transplantation in damaged brain regions.

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).

Nanocurcumin prevents memory impairment, hippocampal apoptosis, Akt and CaMKII-α signaling disruption in the central STZ model of Alzheimer's disease in rat.

The central route of streptozotocin (STZ) administration has been introduced as a rat model of sporadic Alzheimer's disease (AD). Curcumin was suggested to possess possible neuroprotective effects, which may be profitable in AD. However, the low bioavailability of curcumin hinders its beneficial effects in clinical studies. Earlier studies suggested that a bovine serum albumin-based nanocurcumin, produces superior neuroprotective effects compared to natural curcumin. In the present study, the protective effect of nanocurcumin in rat model of central STZ induced memory impairment was assessed. In addition, due to the importance of the hippocampus in memory, the amounts of hippocampal active caspase-3, Akt, and CaMKII-α were evaluated. Adult male Wistar rats weighing 250-300 g were used. STZ (icv) was injected during days 1 and 3 (3 mg/kg in divided), and nanocurcumin or curcumin 50 mg/kg/oral gavage was administered daily during days 4-14. Morris water maze training was performed on days 15-17, and the retention memory test was achieved on the 18th day. Following memory assessment, the rats were sacrificed and the hippocampi were used to assess caspase-3 cleavage, Akt, and CaMKII-α signaling. The findings revealed that nanocurcumin ingestion (but not natural curcumin) in the dose of 50 mg/kg was capable to prevent the impairment of water maze learning and memory induced by central STZ. Molecular assessments indicated that STZ treatment increased the caspase-3 cleavage in the hippocampus while deactivating Akt and CaMKII-α. Nanocurcumin reduced caspase-3 cleavage to a non-significant level compared to control group and restored Akt and CaMKII-α within the hippocampus while natural curcumin exerted no significant effect. These findings might suggest that nanocurcumin can restore memory deficit, hippocampal apoptosis as well as Akt and CaMKII-α signaling disruption associated with brain insulin resistance.

Sustained feeding of a diet high in fat resulted in a decline in the liver's insulin-degrading enzyme levels in association with the induction of oxidative and endoplasmic reticulum stress in adult male rats: Evaluation of 4-phenylbutyric acid.

The current study explored the impact of high fat diet (HFD) on hepatic oxidative and endoplasmic reticulum (ER) stress and its insulin degrading enzyme (IDE) content with the injection of 4-phenyl butyric acid (4-PBA) in adult male rats. Following the weaning period, male offspring were distributed among six distinct groups. The corresponding diet was used for 20 weeks, subsequently 4-PBA was administered for three consecutive days. Plasma glucose and insulin levels, HOMA-ß (homeostasis model assessment of ß-cell), hepatic ER and oxidative stress biomarkers and IDE protein content were assessed. Long-term ingestion of HFD (31 % cow butter) induced oxidative and ER stress in the liver tissue. Accordingly, a rise in the malondialdehyde (MDA) content and catalase enzyme activity and a decrease in the glutathione (GSH) content were detected within the liver of the HFD and HFD + DMSO groups. Consumption of this diet elevated the liver expression of binding immunoglobulin protein (BIP) and C/enhancer-binding protein homologous protein (CHOP) levels while reduced its IDE content. The HOMA-ß decreased significantly. The injection of the 4-PBA moderated all the induced changes. Findings from this study indicated that prolonged HFD consumption led to a reduction in plasma insulin levels, likely attributed to pancreatic ß cell malfunction, as evidenced by a decline in the HOMA-ß index. Also, the HFD appears to have triggered oxidative and ER stress in the liver, along with a decrease in its IDE content.

Kindling-induced learning deficiency and possible cellular and molecular involved mechanisms.

Hippocampus learning disturbance is a major symptom of patients with seizure, hence hippocampal dysfunction has essential role in worsening the disease. Hippocampal formation includes neurons and myelinated fibers that are necessary for acquisition and consolidation of memory, long-term potentiation and learning activity. The exact mechanism by which seizure can decrease memory and learning activity of hippocampus remains unknown. In the present study, electrical kindling-induced learning deficit in rats was evaluated by Morris water maze (MWM) test. The hippocampus was removed and changes in neurons and myelin sheaths around hippocampal fibers were investigated using histological and immunohistochemical methods. Demyelination was assessed by luxol fast blue staining, and immunohistological staining of myelin-binding protein (MBP). The TUNEL assay was used for evaluation of neuronal apoptosis and the glial fibriliary acetic protein (GFAP) was used for assessment of inflammatory reaction. The results indicated that electrical kindling of hippocampus could induce deficiency in spatial learning and memory as compared to control group. In addition, electrical kindling caused damage to the myelin sheath around hippocampal fibers and produced vast demyelination. Furthermore, an increase in the number of apoptotic cells in hippocampal slices was observed. In addition, inflammatory response was higher in kindled animals as compared to the control group. The results suggested that the decrease in learning and memory in kindled animals is likely due to demyelination and augmentation in apoptosis rate accompanied by inflammatory reaction in hippocampal neurons of kindled rats.

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.