The effects of urolithin A on poly I:C-induced microglial activation.

Neuroinflammation can be triggered by various stimuli, including viral infections. Viruses can directly invade the brain and infect neuronal cells or indirectly trigger a "cytokine storm" in the periphery that eventually leads to microglial activation in the brain. While this initial activation of microglial cells is important for viral clearance, chronic activation leads to excessive inflammation and oxidative stress, which can be neurotoxic. Remarkebly, recent studies have shown that certain viruses such as influenza A virus, coronavirus, herpes virus and Epstein-Barr virus may be involved in the development of neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, and multiple sclerosis. Therefore, it is important to find therapeutic strategies against chronic neuroinflammation triggered by viral infections. Here, we investigated the effects of urolithin A (UA) on microglial activation in vitro induced by a viral mimetic, poly I:C, in a triple co-culture system of neurons, astrocytes and microglial cells. Immunocytochemistry was used to perform a comprehensive single-cell analysis of the morphological changes of microglia as an indicator of their reactive state. Treatment with UA significantly prevented the poly I:C-induced reactive state of microglia, which was characterized by increased expression of the microglial activation markers CD68 and IBA-1. UA restored the poly I:C-induced morphology by restoring microglial ramification. In addition, UA was able to reduce the release of the pro-inflammatory mediators CCL2, TNF-α, and IL-1ß and showed a trend toward attenuation of cellular ROS production in poly I:C-treated cultures. Overall, this study suggests that UA as a component of a healthy diet may help prevent virus-induced neuroinflammation and may have therapeutic potential for future studies to prevent or treat neurodegenerative diseases by targeting the associated neuroinflammatory processes.

Deletion of p75NTR rescues the synaptic but not the inflammatory status in the brain of a mouse model for Alzheimer's disease.

Introduction: Alzheimer's disease (AD), is characterized by a gradual cognitive decline associated with the accumulation of Amyloid beta (Aß)-oligomers, progressive neuronal degeneration and chronic neuroinflammation. Among the receptors shown to bind and possibly transduce the toxic effects of Aß-oligomers is the p75 neurotrophin receptor (p75NTR). Interestingly, p75NTR mediates several crucial processes in the nervous system, including neuronal survival and apoptosis, maintenance of the neuronal architecture, and plasticity. Furthermore, p75NTR is also expressed in microglia, the resident immune cells of the brain, where it is markedly increased under pathological conditions. These observations indicate p75NTR as a potential candidate for mediating Aß-induced toxic effects at the interface between the nervous and the immune system, thereby potentially participating in the crosstalk between these two systems. Methods: Here we used APP/PS1 transgenic mice (APP/PS1tg) and compared the Aß-induced alterations in neuronal function, chronic inflammation as well as their cognitive consequences between 10 months old APP/PS1tg and APP/PS1tg x p75NTRexonIV knockout mice. Results: Electrophysiological recordings show that a loss of p75NTR rescues the impairment in long-term potentiation at the Schaffer collaterals in the hippocampus of APP/PS1tg mice. Interestingly, however loss of p75NTR does not influence the severity of neuroinflammation, microglia activation or the decline in spatial learning and memory processes observed in APP/PS1tg mice. Conclusion: Together these results indicate that while a deletion of p75NTR rescues the synaptic defect and the impairment in synaptic plasticity, it does not affect the progression of the neuroinflammation and the cognitive decline in a mouse model for AD.

Influenza vaccine is able to prevent neuroinflammation triggered by H7N7 IAV infection.

Influenza A virus (IAV) subtypes are a major cause of illness and mortality worldwide and pose a threat to human health. Although IAV infection is considered a self-limiting respiratory syndrome, an expanded spectrum of cerebral manifestations has been reported following IAV infection. Neurotropic IAVs, such as the H7N7 subtype, are capable of invading the central nervous system (CNS) and replicating in brain cells, resulting in microglia-induced neuroinflammation. Microglial cells, the brain's resident immune cells, are instrumental in the inflammatory response to viral infection. While activation of microglia is important to initially contain the virus, excessive activation of these cells leads to neuronal damage. Previous studies have shown that acute and even long-term IAV-induced neuroinflammation leads to CNS damage. Therefore, the search for possible preventive or therapeutic strategies is of great importance. In this study, we investigated the potential effect of vaccination against acute neuroinflammation induced by H7N7 infection and subsequent neuronal damage in the hippocampus, a particularly vulnerable brain region, comparing young and aged mice. Immunosenescence is one of the striking pathophysiological changes during mammalian aging that leads to "inflammaging" and critically limits the protection by vaccines in the elderly. The results suggest that formalin-inactivated H7N7 vaccine has a preventive effect against the inflammatory responses in the periphery and also in the CNS after H7N7 infection. Cytokine and chemokine levels, increased microglial density, and cell volume after H7N7 infection were all attenuated by vaccination. Further structural analysis of microglial cells also revealed a change in branching complexity after H7N7 infection, most likely reflecting the neuroprotective effect of the vaccination. In addition, synapse loss was prevented in vaccinated mice. Remarkably, engulfment of post-synaptic compartments by microglia can be proposed as the underlying mechanism for spine loss triggered by H7N7 infection, which was partially modulated by vaccination. Although young mice showed better protection against neuroinflammation and the resulting deleterious neuronal effects upon vaccination, a beneficial role of the vaccine was also observed in the brains of older mice. Therefore, vaccination can be proposed as an important strategy to prevent neurological sequelae of H7N7 infection.

How viral infections cause neuronal dysfunction: a focus on the role of microglia and astrocytes.

In recent decades, a number of infectious viruses have emerged from wildlife or reemerged that pose a serious threat to global health and economies worldwide. Although many of these viruses have a specific target tissue, neurotropic viruses have evolved mechanisms to exploit weaknesses in immune defenses that eventually allow them to reach and infect cells of the central nervous system (CNS). Once in the CNS, these viruses can cause severe neuronal damage, sometimes with long-lasting, life-threatening consequences. Remarkably, the ability to enter the CNS and cause neuronal infection does not appear to determine whether a viral strain causes neurological complications. The cellular mechanisms underlying the neurological consequences of viral infection are not fully understood, but they involve neuroimmune interactions that have so far focused mainly on microglia. As the major immune cells in the brain, reactive microglia play a central role in neuroinflammation by responding directly or indirectly to viruses. Chronic reactivity of microglia leads to functions that are distinct from their beneficial roles under physiological conditions and may result in neuronal damage that contributes to the pathogenesis of various neurological diseases. However, there is increasing evidence that reactive astrocytes also play an important role in the response to viruses. In this review article, we summarize the recent contributions of microglia and astrocytes to the neurological impairments caused by viral infections. By expanding knowledge in this area, therapeutic approaches targeting immunological pathways may reduce the incidence of neurological and neurodegenerative disorders and increase the therapeutic window for neural protection.

Repeated performance of spatial memory tasks ameliorates cognitive decline in APP/PS1 mice.

BACKGROUND: Alzheimer's disease (AD) is a burden on the public health system because it is a neurodegenerative disease that is incurable and for which there is no successful treatment. AD patients suffer from symptoms for many years, with progressive loss of cognitive and functional abilities. In addition to the features of AD, described as amyloid plaques and neurofibrillary tangles, neuroinflammatory processes, genetic factors, and lifestyle also play important roles. Increasing evidence for lifestyle factors includes possible changes due to smoking, social engagement, and physical activity. METHODS: Morris water maze behavioral tasks were performed to analyze the formation of spatial memory. APPswe/PS1dE9 mice with a remarkable increase in amyloid-ß production associated with certain behavioral abnormalities comparable to AD symptoms and age-matched wild-type littermates were trained several times at 3, 6, 9, and 12 months of age and compared with untrained groups at 9 and 12 months of age. Performance during the acquisition phase, in the reference memory test, and in searching strategies were analyzed. RESULTS: 9- and 12-month-old APP/PS1 mice showed cognitive impairment, especially in the reference memory test and searching strategies. This cognitive deterioration was reversed in 9- and 12-month-old APP/PS1 mice that had been previously trained several times. Even in the reversal test, in which memory formation must be adapted to the new platform position, several trained APP/PS1 mice performed better. CONCLUSION: Repeated spatial memory training in the water maze showed positive effects on memory formation in APP/PS1 mice. Interestingly, the cohort that had been previously trained several times was able to use increased hippocampus-dependent strategies, similar to the WT mice. This may suggest that cognitively demanding and physically active tasks can improve cognitive function.

The role of α-tubulin tyrosination in controlling the structure and function of hippocampal neurons.

Microtubules (MTs) are central components of the neuronal cytoskeleton and play a critical role in CNS integrity, function, and plasticity. Neuronal MTs are diverse due to extensive post-translational modifications (PTMs), particularly detyrosination/tyrosination, in which the C-terminal tyrosine of α-tubulin is cyclically removed by a carboxypeptidase and reattached by a tubulin-tyrosine ligase (TTL). The detyrosination/tyrosination cycle of MTs has been shown to be an important regulator of MT dynamics in neurons. TTL-null mice exhibit impaired neuronal organization and die immediately after birth, indicating TTL function is vital to the CNS. However, the detailed cellular role of TTL during development and in the adult brain remains elusive. Here, we demonstrate that conditional deletion of TTL in the neocortex and hippocampus during network development results in a pathophysiological phenotype defined by incomplete development of the corpus callosum and anterior commissures due to axonal growth arrest. TTL loss was also associated with a deficit in spatial learning, impaired synaptic plasticity, and reduced number of spines in hippocampal neurons, suggesting that TTL also plays a critical role in hippocampal network development. TTL deletion after postnatal development, specifically in the hippocampus and in cultured hippocampal neurons, led to a loss of spines and impaired spine structural plasticity. This indicates a novel and important function of TTL for synaptic plasticity in the adult brain. In conclusion, this study reveals the importance of α-tubulin tyrosination, which defines the dynamics of MTs, in controlling proper network formation and suggests TTL-mediated tyrosination as a new key determinant of synaptic plasticity in the adult brain.

IL-37 expression reduces acute and chronic neuroinflammation and rescues cognitive impairment in an Alzheimer's disease mouse model.

The anti-inflammatory cytokine interleukin-37 (IL-37) belongs to the IL-1 family but is not expressed in mice. We used a human IL-37 (hIL-37tg) expressing mouse, which has been subjected to various models of local and systemic inflammation as well as immunological challenges. Previous studies reveal an immunomodulatory role of IL-37, which can be characterized as an important suppressor of innate immunity. Here, we examined the functions of IL-37 in the central nervous system and explored the effects of IL-37 on neuronal architecture and function, microglial phenotype, cytokine production and behavior after inflammatory challenge by intraperitoneal LPS-injection. In wild-type mice, decreased spine density, activated microglial phenotype and impaired long-term potentiation (LTP) were observed after LPS injection, whereas hIL-37tg mice showed no impairment. In addition, we crossed the hIL-37tg mouse with an animal model of Alzheimer's disease (APP/PS1) to investigate the anti-inflammatory properties of IL-37 under chronic neuroinflammatory conditions. Our results show that expression of IL-37 is able to limit inflammation in the brain after acute inflammatory events and prevent loss of cognitive abilities in a mouse model of AD.

Itaconate controls its own synthesis via feedback-inhibition of reverse TCA cycle activity at IDH2.

Macrophages undergo extensive metabolic reprogramming during classical pro-inflammatory polarization (M1-like). The accumulation of itaconate has been recognized as both a consequence and mediator of the inflammatory response. In this study we first examined the specific functions of itaconate inside fractionated mitochondria. We show that M1 macrophages produce itaconate de novo via aconitase decarboxylase 1 (ACOD1) inside mitochondria. The carbon for this reaction is not only supplied by oxidative TCA cycling, but also through the reductive carboxylation of α-ketoglutarate by isocitrate dehydrogenase (IDH). While macrophages are capable of sustaining a certain degree of itaconate production during hypoxia by augmenting the activity of IDH-dependent reductive carboxylation, we demonstrate that sufficient itaconate synthesis requires a balance of reductive and oxidative TCA cycle metabolism in mouse macrophages. In comparison, human macrophages increase itaconate accumulation under hypoxic conditions by augmenting reductive carboxylation activity. We further demonstrated that itaconate attenuates reductive carboxylation at IDH2, restricting its own production and the accumulation of the immunomodulatory metabolites citrate and 2-hydroxyglutarate. In line with this, reductive carboxylation is enhanced in ACOD1-depleted macrophages. Mechanistically, the inhibition of IDH2 by itaconate is linked to the alteration of the mitochondrial NADP+/NADPH ratio and competitive succinate dehydrogenase inhibition. Taken together, our findings extend the current model of TCA cycle reprogramming during pro-inflammatory macrophage activation and identified novel regulatory properties of itaconate.

SiMeEx, a simplified method for metabolite extraction of adherent mammalian cells.

A reliable method for metabolite extraction is central to mass spectrometry-based metabolomics. However, existing methods are lengthy, mostly due to the step of scraping cells from cell culture vessels, which restricts metabolomics in broader application such as lower cell numbers and high-throughput studies. Here, we present a simplified metabolite extraction (SiMeEx) method, to efficiently and quickly extract metabolites from adherent mammalian cells. Our method excludes the cell scraping step and therefore allows for a more efficient extraction of polar metabolites in less than 30 min per 12-well plate. We demonstrate that SiMeEx achieves the same metabolite recovery as using a standard method containing a scraping step, in various immortalized and primary cells. Omitting cell scraping does not compromise the performance of non-targeted and targeted GC-MS analysis, but enables metabolome analysis of cell culture on smaller well sizes down to 96-well plates. Therefore, SiMeEx demonstrates advantages not only on time and resources, but also on the applicability in high-throughput studies.

Long-Term Consequence of Non-neurotropic H3N2 Influenza A Virus Infection for the Progression of Alzheimer's Disease Symptoms.

Influenza viruses until today are a leading cause of worldwide severe pandemics and represent a major threat to human and animal health. Although the primary target of influenza viruses is the lung, infection may manifest with acute and even chronic neurological complications (e.g., status epilepticus, encephalopathies, and encephalitis) potentially increasing the long-term risk for neurodegenerative diseases. We previously described that a peripheral influenza A virus (IAV) infection caused by non-neurotropic H3N2 (maHK68) variant leads to long-term neuroinflammation and synapse loss together with impaired memory formation in young adult mice. Processes of neuroinflammation have been associated with neurodegenerative diseases such as Alzheimer's disease (AD) and prolonged or excessive innate immune responses are considered a risk factor for AD. Here, the role of purely peripheral IAV infection for the development and progression of AD in a transgenic mouse model (APP/PS1) was investigated. At 2 months of age, mice were infected with H3N2 IAV and the detailed analysis of microglia morphology revealed neuroinflammation in the hippocampus already of 6 months old non-infected APP/PS1 mice together with impaired spatial learning, however, microglia activation, amyloid-ß plaques load and cognitive impairments were even more pronounced in APP/PS1 mice upon H3N2 infection. Moreover, CA1 hippocampal dendritic spine density was reduced even at 120 dpi compared to wild-type and also to non-infected APP/PS1 mice, whereas neuronal cells number was not altered. These findings demonstrate that non-neurotropic H3N2 IAV infection as a peripheral immune stimulation may exacerbate AD symptoms possibly by triggering microglial hyperactivation.

The NLRP3 inflammasome inhibitor OLT1177 rescues cognitive impairment in a mouse model of Alzheimer's disease.

Numerous studies demonstrate that neuroinflammation is a key player in the progression of Alzheimer's disease (AD). Interleukin (IL)-1ß is a main inducer of inflammation and therefore a prime target for therapeutic options. The inactive IL-1ß precursor requires processing by the the nucleotide-binding oligomerization domain-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome into a mature and active form. Studies have shown that IL-1ß is up-regulated in brains of patients with AD, and that genetic inactivation of the NLRP3 inflammasome improves behavioral tests and synaptic plasticity phenotypes in a murine model of the disease. In the present study, we analyzed the effect of pharmacological inhibition of the NLRP3 inflammasome using dapansutrile (OLT1177), an oral NLRP3-specific inhibitor that is safe in humans. Six-month-old WT and APP/PS1 mice were fed with standard mouse chow or OLT1177-enriched chow for 3 mo. The Morris water maze test revealed an impaired learning and memory ability of 9-mo-old APP/PS1 mice (P = 0.001), which was completely rescued by OLT1177 fed to mice (P = 0.008 to untreated APP/PS1). Furthermore, our findings revealed that 3 mo of OLT1177 diet can rescue synaptic plasticity in this mouse model of AD (P = 0.007 to untreated APP/PS1). In addition, microglia were less activated (P = 0.07) and the number of plaques was reduced in the cortex (P = 0.03) following NLRP3 inhibition with OLT1177 administration. We also observed an OLT1177 dose-dependent normalization of plasma metabolic markers of AD to those of WT mice. This study suggests the therapeutic potential of treating neuroinflammation with an oral inhibitor of the NLRP3 inflammasome.

Langat virus infection affects hippocampal neuron morphology and function in mice without disease signs.

BACKGROUND: Tick-borne encephalitis virus (TBEV) is an important human pathogen that can cause the serious illness tick-borne encephalitis (TBE). Patients with clinical symptoms can suffer from severe meningoencephalitis with sequelae that include cognitive disorders and paralysis. While less than 30% of patients with clinical symptoms develop meningoencephalitis, the number of seropositive individuals in some regions indicates a much higher prevalence of TBEV infections, either with no or subclinical symptoms. The functional relevance of these subclinical TBEV infections and their influence on brain functions, such as learning and memory, has not been investigated so far. METHODS: To compare the effect of low and high viral replication in the brain, wildtype and Irf-7-/- mice were infected with Langat virus (LGTV), which belongs to the TBEV-serogroup. The viral burden was analyzed in the olfactory bulb and the hippocampus. Open field, elevated plus maze, and Morris water maze experiments were performed to determine the impact on anxiety-like behavior, learning, and memory formation. Spine density of hippocampal neurons and activation of microglia and astrocytes were analyzed. RESULTS: In contrast to susceptible Irf-7-/- mice, wildtype mice showed no disease signs upon LGTV infection. Detection of viral RNA in the olfactory bulb revealed CNS infections in wildtype and Irf-7-/- mice. Very low levels of viral replication were detectable in the hippocampus of wildtype mice. Although wildtype mice develop no disease signs, they showed reduced anxiety-like behavior and impaired memory formation, whereas Irf-7-/- mice were not affected. This impairment was associated with a significant decrease in spine density of neurons in the hippocampal CA1 region of wildtype mice. Microglia activation and astrogliosis were detected in the hippocampus. CONCLUSION: In this study, we demonstrate that subclinical infections by viruses from the TBEV-serogroup affected anxiety-like behavior. Virus replication in the olfactory bulb induced far-reaching effects on hippocampal neuron morphology and impaired hippocampus-dependent learning and memory formation.

Type I Interferon Receptor Signaling in Astrocytes Regulates Hippocampal Synaptic Plasticity and Cognitive Function of the Healthy CNS.

Type I interferon receptor (IFNAR) signaling is a hallmark of viral control and host protection. Here, we show that, in the hippocampus of healthy IFNAR-deficient mice, synapse number and synaptic plasticity, as well as spatial learning, are impaired. This is also the case for IFN-ß-deficient animals. Moreover, antibody-mediated IFNAR blocking acutely interferes with neuronal plasticity, whereas a low-dose application of IFN-ß has a positive effect on dendritic spine structure. Interfering with IFNAR signaling in different cell types shows a role for cognitive function and synaptic plasticity specifically mediated by astrocytes. Intriguingly, levels of the astrocytic glutamate-aspartate transporter (GLAST) are reduced significantly upon IFN-ß treatment and increase following inhibition of IFNAR signaling. These results indicate that, besides the prominent role for host defense, IFNAR is important for synaptic plasticity as well as cognitive function. Astrocytes are at the center stage of this so-far-unknown signaling cascade.

Long-Term Neuroinflammation Induced by Influenza A Virus Infection and the Impact on Hippocampal Neuron Morphology and Function.

Acute influenza infection has been reported to be associated with neurological symptoms. However, the long-term consequences of an infection with neurotropic and non-neurotropic influenza A virus (IAV) variants for the CNS remain elusive. We can show that spine loss in the hippocampus after infection with neurotropic H7N7 (rSC35M) and non-neurotropic H3N2 (maHK68) in female C57BL/6 mice persists well beyond the acute phase of the disease. Although spine number was significantly reduced at 30 d postinfection (dpi) with H7N7 or H3N2, full recovery could only be observed much later at 120 dpi. Infection with H1N1 virus, which was shown previously to affect spine number and hippocampus-dependent learning acutely, had no significant long-term effects. Spine loss was associated with an increase in the number of activated microglia, reduced long-term potentiation in the hippocampus, and impairment in spatial memory formation, indicating that IAV-associated inflammation induced functional and structural alterations in hippocampal networks. Transcriptome analyses revealed regulation of many inflammatory and neuron- and glia-specific genes in H3N2- and H7N7-infected mice at day 18 and in H7N7-infected mice at day 30 pi that related to the structural and functional alterations. Our data provide evidence that neuroinflammation induced by neurotropic H7N7 and infection of the lung with a non-neurotropic H3N2 IAV result in long-term impairments in the CNS. IAV infection in humans may therefore not only lead to short-term responses in infected organs, but may also trigger neuroinflammation and associated chronic alterations in the CNS.SIGNIFICANCE STATEMENT In the acute phase of influenza infection, neuroinflammation can lead to alterations in hippocampal neuronal morphology and cognitive deficits. The results of this study now also provide evidence that neuroinflammation induced by influenza A virus (IAV) infection can induce longer-lasting, virus-specific alterations in neuronal connectivity that are still detectable 1 month after infection and are associated with impairments in spatial memory formation. IAV infection in humans may therefore not only lead to short-term responses in infected organs, but may also trigger neuroinflammation and associated chronic alterations in the CNS.

Single Dose Corticosteroid Therapy After Surgical Repair of Fallot's Tetralogy; A Randomized Controlled Clinical Trial.

BACKGROUND: Inflammatory reaction can produce several complications after cardiac surgery. Many attempts have been made to reduce these complications; perioperative corticosteroid therapy is one of the simplest methods. OBJECTIVES: We conducted a randomized study to evaluate the efficacy of single dose methylprednisolone, prescribed after surgery, for reducing the complications. Repair of Tetralogy of Fallot was chosen as a homogenous large group for the study. PATIENTS AND METHODS: One hundred children who underwent total repair of Tetralogy of Fallot were enrolled in this study. After the surgery, all patients were transferred to pediatric ICU and were randomized (in a double-blind fashion) in 2 groups (A and B); a single dose of methylprednisolone (30 mg/kg of body weight) was injected to participants of group "A" just at the time of ICU entrance. Group "B" received no drug. Then, clinical outcomes and laboratory data were compared between the two groups. RESULTS: The only significant differences were lower incidence of bacteremia and higher incidence of hyperglycemia in the group who were used methylprednisolone. CONCLUSIONS: Using a single postsurgical dose of methylprednisolone does not significantly alter the clinical outcome after repairing Tetralogy of Fallot.

Effect of obesity on mortality and morbidity after coronary artery bypass grafting surgery in Iranian patients.

BACKGROUND: Recent years have witnessed the emergence of obesity as a major public health concern. The drastic rise in obesity and its concomitant co-morbidities is a reflection of the recent changes in dietary habits in Iran and many other developing countries. A recent large population study in Tehran reported that 58% and 75% of middle-aged Iranian men and women, respectively, were either overweight or obese. OBJECTIVES: Considering the impact of obesity on mortality and morbidity after coronary artery bypass graft surgery (CABG), we sought to investigate the association between central obesity and the body mass index (BMI) and the post-CABG mortality and morbidity in Iranian patients. PATIENTS AND METHODS: This prospective study was on 235 adult patients scheduled for isolated CABG in a university hospital. The patients were divided in two groups according to BMI ≥ 30 (obese; n = 60) and BMI < 30 (non-obese; n = 175). In-hospital and late (after 3 months) morbidity and mortality rates were compared between obese and non-obese patients. RESULTS: A total of 235 patients (135 women) with a mean age of 59 ± 9.2 years (range = 29 to 79 years), mean BMI of 27.3 ± 4.2 (range = 17 to 40), and mean waist circumference of 101.2 ± 14.7 cm (range = 55 to 145 cm) were included. By the third postoperative month, wound infection had significantly increased in patients with BMI ≥ 30 (P = 0.022). In-hospital and late morbidity and mortality rates were comparable between the two groups (P > 0.05). CONCLUSIONS: In our patients obesity was a risk factor for wound infection but not atelectasis or the need for intra-aortic balloon pump or re-exploration. Obesity was not associated with increased in-hospital or 3 months mortality rates after CABG.

Role of C-fibers in pain and morphine induced analgesia/hyperalgesia in rats.

BACKGROUND: Usual dosage of morphine (10 mg/kg) induces analgesia and ultra-low dose (ULD) of morphine (1 µg/kg); hyperalgesia, and C-fibers are also bearing µ-opioid receptors; here the importance of C-fibers on pain and morphine induced analgesia/hyperalgesia is questioned and investigated using pain evaluation methods and infant capsaicin treating for C-fibers lesioning. METHODS: Wistar male rats (200-250 grams) were assigned to three categories i.e. control, sham (receiving neonatal capsaicin vehicle) and c-lesion (receiving neonatal capsaicin), each one with three groups (n=7). They were injected intraperitoneally with single dosage of saline, 10 mg/kg or 1 µg/kg morphine, respectively. Thermal pain threshold was evaluated using the tail flick test before and 30 minutes after the injections. Chemical pain was assessed using the formalin test (FT) 30 minutes after the administrations. RESULTS: Results indicated that thermal (P < 0.001) and chemical pains in both neurogenic and inflammatory phases of FT (P < 0.05) were reduced in C-lesion animals. In the C-normal and C-lesion animals, 10 mg/kg morphine exerted analgesia both in thermal (P < 0.001) and two phases of FT (P < 0.01), but it was more potent in C-lesion animals (P < 0.05). Although ULD of morphine in C-normal animals produced hyperalgesic effect in thermal and chemical pains (P < 0.001), in C-lesion animals, it produced analgesia (P < 0.05) at the neurogenic phase of FT. CONCLUSION: Results can raise the C-fibers involvement for a significant portion of nociceptive transmission, because C-lesioning potentiated morphine induced analgesia and eliminated ULD of morphine induced hyperalgesia. Therefore C and Aδ fibers can be involved in morphine analgesia; while, just C-fibers are possibly responsible for only presynaptically hyperalgesic/excitatory action of ULD in morphine.

The effects of trypsin on rat brain astrocyte activation.

BACKGROUND: Astrocytes are cells within the central nervous system which are activated in a wide spectrum of infections, and autoimmune and neurodegenerative diseases. In pathologic states, they produce inflammatory cytokines, chemokines, and nitric oxide (NO), and sometimes they induce apoptosis. Their protease-activated receptors (PARs) can be activated by proteases, e.g. thrombin and trypsin, which are important in brain inflammation. The current study aimed to investigate the effects of different concentrations of trypsin (1 to 100U/ml) on cultured astrocytes. METHODS: In the present study, two-day rat infants' brains were isolated and homogenized after meninges removal, then cultivated in DMEM + 10% FBS medium. 10 days later, astrocytes were harvested and recultivated for more purification (up to 95%), using Immunocytochemistry method, in order to be employed for tests. They were affected by different concentrations of trypsin (1, 5, 10, 15, 20, 40, 60, 80, and 100 U/ml). To reveal the inflammation progress, NO concentrations (the Griess test) were assessed after 24 and 48 hours. RESULTS: The results showed that trypsin concentration up to 20 U/ml caused a significant increase in NO, in a dose-dependent manner, on cultured astrocytes (P < 0.001). Trypsin 20 U/ml increased NO production fivefold the control group (P < 0.001). At higher concentrations than 20 U/ml, NO production diminished (P < 0.001). At 100 U/ml, NO production was less than the control group (P < 0.001). CONCLUSION: Inflammatory effects of trypsin 5-20 U/ml are probably due to the stimulation of astrocytes' PAR-2 receptors and the increasing of the activation of NF-κB, PKC, MAPKs. Stimulation of astrocytes' PAR-2 receptors causes an increase in iNOS activation which in turn leads to NO production. However, higher trypsin concentration possibly made astrocyte apoptosis; therefore, NO production diminished. These assumptions need to be further investigated.