Corticosteroid Treatment During Sepsis Alters Hippocampal Function in Male and Female Survivors.

Background: Millions of sepsis survivors annually face neuropsychiatric sequelae of their illness. Corticosteroids are frequently administered for sepsis, and their use improves neuropsychiatric outcomes, but the mechanisms are unknown. In light of prior work that has shown persistent inflammation in sepsis survivors, we hypothesized that short-term corticosteroid treatment during illness would reverse the long-term impact of sepsis on inflammatory gene expression in the hippocampus and rescue associated changes to affective behaviors. Methods: Male and female mice underwent cecal ligation and puncture or a sham surgery to induce acute infection and were treated for 5 days with corticosterone or vehicle. Starting 2 weeks after the surgery, we performed functional phenotyping in the survivor mice followed by hippocampal RNA sequencing to identify underlying mechanisms. Results: Long-term cecal ligation and puncture survivors exhibited anxiety-like behavior, increased central hypothalamic-pituitary-adrenal axis activity, and persistent systemic and neuroinflammation. Corticosterone treatment during illness did not reverse anxiety-like behavior or inflammation in survivors. Instead, corticosterone treatment impaired object memory and increased active coping behavior in females. History of corticosterone treatment influenced the expression of >10% of detectable transcripts in the dorsal and ventral hippocampus, including a coordinated downregulation of activity-dependent genes. Conclusions: Corticosterone treatment during sepsis impaired memory formation in survivors and caused a lasting decrease in hippocampal neural activity, which could underlie its effect on memory. Future studies should focus on how this lasting effect of corticosteroid treatment on hippocampal activity and memory translates into improved neuropsychiatric outcomes in human sepsis survivors.

Forebrain Glucocorticoid Receptor Overexpression Alters Behavioral Encoding of Hippocampal CA1 Pyramidal Cells in Mice.

Glucocorticoid signaling influences hippocampal-dependent behavior and vulnerability to stress-related neuropsychiatric disorders. In mice, lifelong overexpression of glucocorticoid receptor (GR) in forebrain excitatory neurons altered exploratory behavior, cognition, and dorsal hippocampal gene expression in adulthood, but whether GR overexpression alters the information encoded by hippocampal neurons is not known. We performed in vivo microendoscopic calcium imaging of 1359 dorsal CA1 pyramidal cells in freely behaving male and female wild-type (WT) and GR-overexpressing (GRov) mice during exploration of a novel open field, where most CA1 neurons are expected to respond to center location and mobility. Most neurons showed sensitivity to center location and/or mobility based on single-neuron calcium amplitude and event rate, but these sensitivity patterns differed between genotypes. GRov neurons were more likely than WT neurons to display center sensitivity and less likely to display mobility sensitivity. More than one-third of these responsive GRov neurons were sensitive only to center location and not mobility, while uniquely center-sensitive neurons were rare in WT. Most center-sensitive neurons exhibited anticipatory activity, suggesting they could drive behavior. We conclude that, compared with wild-type, dorsal CA1 pyramidal cells in GRov mice preferentially respond to center location rather than mobility in a novel open field. Such changes in the information encoded by individual hippocampal neurons in an aversive environment could underlie changes in stress vulnerability because of genetic or epigenetic variations in glucocorticoid receptor signaling.

Effect of dural inflammatory soup application on activation and sensitization markers in the caudal trigeminal nucleus of the rat and the modulatory effects of sumatriptan and kynurenic acid.

BACKGROUND: The topical inflammatory soup can model the inflammation of the dura mater causing hypersensitivity and activation of the trigeminal system, a phenomenon present in migraineurs. Calcitonin gene-related peptide, transient receptor potential vanilloid-1 receptor, and neuronal nitric oxide synthase are important in the sensitization process there. 5-HT1B/1D receptor agonists, triptans are used as a treatment of migraine. Kynurenic acid an NMDA antagonist can act on structures involved in trigeminal activation. AIM: We investigated the effect of inflammatory soup induced dural inflammation on the calcitonin gene-related peptide, transient receptor potential vanilloid-1 receptor, and neuronal nitric oxide synthase levels in the caudal trigeminal nucleus. We also tested whether pretreatment with a well-known antimigraine drug, such as sumatriptan and kynurenic acid, a compound with a different mechanism of action, can affect these changes and if their modulatory effects are comparable. MATERIAL AND METHODS: After subcutaneous sumatriptan or intraperitoneal kynurenic acid the dura mater of adult male Sprague-Dawley rats (n = 72) was treated with inflammatory soup or its vehicle (synthetic interstitial fluid). Two and a half or four hours later perfusion was performed and the caudal trigeminal nucleus was removed for immunohistochemistry. RESULTS AND CONCLUSION: Inflammatory soup increased calcitonin gene-related peptide, transient receptor potential vanilloid-1 receptor, and neuronal nitric oxide synthase in the caudal trigeminal nucleus compared to placebo, which was attenuated by sumatriptan and kynurenic acid. This suggests the involvement of 5-HT1B/1D and NMDA receptors in neurogenic inflammation development of the dura and thus in migraine attacks.

Corrigendum: The Effect of Systemic Nitroglycerin Administration on the Kynurenine Pathway in the Rat.

[This corrects the article DOI: 10.3389/fneur.2017.00278.].

Trigeminal activation patterns evoked by chemical stimulation of the dura mater in rats.

BACKGROUND: Although migraine is one of the most common primary headaches, its therapy is still limited in many cases. The use of animal models is crucial in the development of novel therapeutic strategies, but unfortunately, none of them show all aspects of the disease, therefore, there is a constant need for further improvement in this field. The application of inflammatory agents on the dura mater is a widely accepted method to mimic neurogenic inflammation in rodents, which plays a key role in the pathomechanism of migraine. Complete Freund's Adjuvant (CFA), and a mixture of inflammatory mediators, called inflammatory soup (IS) are often used for this purpose. METHODS: To examine the activation pattern that is caused by chemical stimulation of dura mater, we applied CFA or IS over the right parietal lobe. After 2 h and 4 h (CFA groups), or 2.5 h and 4 h (IS groups), animals were perfused, and c-Fos immunoreactive cells were counted in the caudal trigeminal nucleus. To explore every pitfall, we examined whether our surgical procedure (anesthetic drug, stereotaxic apparatus, local lidocaine) can alter the results under the same experimental settings. c-Fos labeled cells were counted in the second-order neuron area based on the somatotopic organization of the trigeminal nerve branches. RESULTS: We could not find any difference between the CFA and physiological saline group neither 2 h, nor 4 h after dural stimulation. IS caused significant difference after both time points between IS treated and control group, and between treated (right) and control (left) side. Stereotaxic frame usage had a substantial effect on the obtained results. CONCLUSIONS: Counting c-Fos immunoreactive cells based on somatotopic organization of the trigeminal nerve helped to examine the effect of chemical stimulation of dura in a more specific way. As a result, the use of IS over the parietal lobe caused activation in the area of the ophthalmic nerve. To see this effect, the use of lidocaine anesthesia is indispensable. In conclusion, application of IS on the dura mater induces short-term, more robust c-Fos activation than CFA, therefore it might offer a better approach to model acute migraine headache in rodents.

Sepsis survivor mice exhibit a behavioral endocrine syndrome with ventral hippocampal dysfunction.

Severe acute stressors are known to trigger mood disorders in humans. Sepsis represents one such stressor, and survivors often suffer long term from psychiatric morbidity. We hypothesized that sepsis leads to lasting changes in neural circuits involved in stress integration, altering affective behavior and the stress response. To investigate this hypothesis, sepsis was induced in male C57Bl/6 mice using cecal ligation and puncture (CLP), and control mice underwent sham surgery. Mice recovered from acute illness within 2 weeks, after which they exhibited increased avoidance behavior and behavioral despair compared with sham, with behavioral changes observed more than 5 weeks after recovery. Sepsis survivors also showed evidence of enhanced hypothalamic-pituitary-adrenal (HPA) axis activity, with increased corticosterone after a novel stressor and increased adrenal weight. In the brain, sepsis survivor mice showed decreased stress-induced cfos mRNA and increased glucocorticoid receptor immunoreactivity specifically in the ventral hippocampus, a brain region known to coordinate emotional behavior and HPA axis activity. We conclude that murine sepsis survivors exhibit a behavioral neuroendocrine syndrome of negative affective behavior and HPA axis hyperactivity, which could be explained by ventral hippocampal dysfunction. These findings could contribute to our understanding of the human post-intensive care syndrome.

Persistent Neuroinflammation and Brain-Specific Immune Priming in a Novel Survival Model of Murine Pneumosepsis.

Pneumonia is the leading cause of sepsis and septic shock. Patients who survive pneumonia are vulnerable to long-term complications including increased risk of neurocognitive dysfunction. This study investigated the immune response and long-term complications of a non-surgical mouse model of Klebsiella pneumoniae pneumosepsis with antibiotic treatment. Pneumosepsis resulted in acutely enhanced expression of inflammatory cytokines, chemokines, and damage-associated molecular patterns in the brain and spleen. Despite resolution of infection, murine pneumosepsis survivors demonstrated a deficit in exploratory locomotor behavior at 2 weeks. This was associated with brain-specific persistent inflammatory gene expression and infiltrating myeloid cells in the brain. The brain inflammatory response was also primed in response to secondary challenge with lipopolysaccharide. The findings of this study demonstrate behavioral and inflammatory outcomes that parallel observations in other models of sepsis, but that have not previously been described in antibiotic-treated pneumonia models, highlighting a common pathway to the development of chronic brain dysfunction in sepsis survival.

Chronic 17ß-estradiol pretreatment has pronociceptive effect on behavioral and morphological changes induced by orofacial formalin in ovariectomized rats.

BACKGROUND: The prevalence of craniofacial pain disorders show sexual dimorphism with generally more common appearance in women suggesting the influence of estradiol, but the exact cause remains unknown. The common point in the pathogenesis of these disorders is the activation of trigeminal system. One of the animal experimental models of trigeminal activation is the orofacial formalin test, in which we investigated the effect of chronic 17ß-estradiol pretreatment on the trigeminal pain-related behavior and activation of trigeminal second-order neurons at the level of spinal trigeminal nucleus pars caudalis (TNC). METHODS: Female Sprague Dawley rats were ovariectomized and silicone capsules were implanted subcutaneously containing cholesterol in the OVX group and 17ß-estradiol and cholesterol in 1:1 ratio in the OVX+E2 group. We determined 17ß-estradiol levels in serum after the implantation of capsules. Three weeks after operation, 50 µL of physiological saline or 1.5% of formalin solution was injected subcutaneously into the right whisker pad of rats. The time spent on rubbing directed to the injected area and c-Fos immunoreactivity in TNC was measured as the formalin-induced pain-related behavior, and as the marker of pain-related neuronal activation, respectively. RESULTS: The chronic 17ß-estradiol pretreatment mimics the plasma levels of estrogen occurring in the proestrus phase and significantly increased the formalin-induced pain-related behavior and neuronal activation in TNC. CONCLUSION: Our results demonstrate that the chronic 17ß-estradiol treatment has strong pronociceptive effect on orofacial formalin-induced inflammatory pain suggesting modulatory action of estradiol on head pain through estrogen receptors, which are present in the trigeminal system.

S100A8/A9 Drives Neuroinflammatory Priming and Protects against Anxiety-like Behavior after Sepsis.

Sepsis commonly results in acute and chronic brain dysfunction, which dramatically increases the morbidity associated with this common disease. Chronic brain dysfunction in animal models of sepsis survival is linked to persistent neuroinflammation and expression of multiple cytokines. However, we have found previously that microglia predominantly upregulate the damage associated molecule S100A8/A9 after sepsis. In this article, we show that S100A8/A9 is increased in the brains of patients who died of sepsis and that S100A8 is expressed in astrocytes and myeloid cells. Using a mouse model of sepsis survival, we show that S100A8/A9 is persistently expressed in the brain after sepsis. S100A9 expression is necessary for recruitment of neutrophils to the brain and for priming production of reactive oxygen species and TNF-α secretion in microglia and macrophages. However, despite improving these indices of chronic inflammation, S100A9 deficiency results in worsened anxiety-like behavior 2 wk after sepsis. Taken together, these results indicate that S100A8/A9 contributes to several facets of neuroinflammation in sepsis survivor mice, including granulocyte recruitment and priming of microglial-reactive oxygen species and cytokine production, and that these processes may be protective against anxiety behavior in sepsis survivors.

Nitroglycerin increases serotonin transporter expression in rat spinal cord but anandamide modulated this effect.

Migraine is one of the most prevalent neurological diseases, which affects 16% of the total population. The exact pathomechanism of this disorder is still not well understood, but it seems that serotonin and its transporter have a crucial role in the pathogenesis. One of the animal models of migraine is the systemic administration of nitroglycerin (NTG), a nitric oxide (NO) donor. NO can initiate a central sensitization process in the trigeminal system, which is also present in migraineurs. Recent studies showed that the endocannabinoid system has a modulatory role on the trigeminal activation and sensitization. Our aim was to investigate the effect of an endogenous cannabinoid, anandamide (AEA) on the NTG-induced changes on serotonin transporter (5-HTT) expression in the upper cervical spinal cord (C1-C2) of the rat, where most of the trigeminal nociceptive afferents convey. The animals were divided into four groups. Rats in the first group, called placebo, received only the vehicle solution as treatment. In the second group, they were treated with an intraperitoneal (i.p.) injection of NTG (10mg/kg). Rats in the third and fourth groups received i.p. AEA (2×5mg/kg) half hour before and one hour after the placebo or NTG treatment. Four hours after placebo/NTG injection, the animals were perfused and the cervical spinal cords were removed for immunohistochemistry and Western blotting. Our results show that both NTG and AEA alone are able to increase 5-HTT expression in the C1-C2 segments. Combination of NTG and AEA has an opposing effect on this marker, nullifying the effects of non-combined administration, probably by negative feedback mechanisms.

The Effect of Systemic Nitroglycerin Administration on the Kynurenine Pathway in the Rat.

The primary headache disorders include migraine, which is one of the most frequent neurological disorders, which influences more than 14% of the whole population. Despite the research efforts, its exact pathomechanism is not fully revealed, but evidence points to the role of glutamate and its receptors. Kynurenic acid is an endogenous glutamate receptor antagonist produced by the kynurenine pathway (KP). Tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO) convert l-tryptophan to N-formyl-l-kynurenine, to be further transformed to l-kynurenine. Kynurenine aminotransferase-II (KAT-II), l-kynurenine hydrolase (KYNU), and l-kynurenine 3-monooxygenase (KMO) are key enzymes in the later steps of the KP. Nitroglycerin (NTG) administration serves as both human and animal model of migraine, causing the activation and sensitization in the trigeminal system. A previous study demonstrated a reduction of KAT-II expression following NTG administration in animals. The goal of current tests was to identify the potential modulatory effect of NTG on other metabolizing enzymes of the KP in the caudal trigeminal nucleus (TNC) of rats. Four hours following the intraperitoneal injection of NTG (10 mg/kg), the rats were perfused transcardially and the TNC was extracted for Western blotting. Western blot studies revealed that the expression of TDO2, IDO1, KYNU, and KMO decreased in the TNC. The results demonstrated that NTG is able to downregulate the KP, with a potential influence on the glutamatergic system as well, contributing to the development of trigeminal activation and sensitization in animals.

A comparative assessment of two kynurenic acid analogs in the formalin model of trigeminal activation: a behavioral, immunohistochemical and pharmacokinetic study.

Kynurenic acid (KYNA) has well-established protective properties against glutamatergic neurotransmission, which plays an essential role in the activation and sensitization process during some primary headache disorders. The goal of this study was to compare the effects of two KYNA analogs, N-(2-N,N-dimethylaminoethyl)-4-oxo-1H-quinoline-2-carboxamide hydrochloride (KA-1) and N-(2-N-pyrrolidinylethyl)-4-oxo-1H-quinoline-2-carboxamide hydrochloride (KA-2), in the orofacial formalin test of trigeminal pain. Following pretreatment with KA-1 or KA-2, rats were injected with subcutaneous formalin solution in the right whisker pad. Thereafter, the rubbing activity and c-Fos immunoreactivity changes in the spinal trigeminal nucleus pars caudalis (TNC) were investigated. To obtain pharmacokinetic data, KA-1, KA-2 and KYNA concentrations were measured following KA-1 or KA-2 injection. Behavioral tests demonstrated that KA-2 induced larger amelioration of formalin-evoked alterations as compared with KA-1 and the assessment of c-Fos immunoreactivity in the TNC yielded similar results. Although KA-1 treatment resulted in approximately four times larger area under the curve values in the serum relative to KA-2, the latter resulted in a higher KYNA elevation than in the case of KA-1. With regard to TNC, the concentration of KA-1 was under the limit of detection, while that of KA-2 was quite small and there was no major difference in the approximately tenfold KYNA elevations. These findings indicate that the differences between the beneficial effects of KA-1 and KA-2 may be explained by the markedly higher peripheral KYNA levels following KA-2 pretreatment. Targeting the peripheral component of trigeminal pain processing would provide an option for drug design which might prove beneficial in headache conditions.

The modulatory effect of anandamide on nitroglycerin-induced sensitization in the trigeminal system of the rat.

BACKGROUND: One of the human and animal models of migraine is the systemic administration of the nitric oxide donor (NO) nitroglycerin (NTG). NO can provoke migraine-like attacks in migraineurs and initiates a self-amplifying process in the trigeminal system, probably leading to central sensitization. Recent studies suggest that the endocannabinoid system is involved in nociceptive signal processing and cannabinoid receptor (CB) agonists are able to attenuate nociception in animal models of pain. AIM: The purpose of the present study was to investigate the modulatory effects of a CB agonist anandamide (AEA) on the NTG-induced expression of transient receptor potential vanilloid type 1 (TRPV1), neuronal nitric oxide synthase (nNOS), nuclear factor kappa B (NF-κB), cyclooxygenase-2 (COX-2) and kynurenine aminotransferase-II (KAT-II) in the upper cervical spinal cord (C1-C2) of the rat, where most of the trigeminal nociceptive afferents convey. METHODS: A half hour before and one hour after NTG (10 mg/kg) or placebo injection, adult male Sprague-Dawley rats (n = 44) were treated with AEA (2 × 5 mg/kg). Four hours after placebo/NTG injection, the animals were perfused and the cervical spinal cords were removed for immunohistochemistry and Western blotting. RESULTS AND CONCLUSION: Our results show that NTG is able to increase TRPV1, nNOS, NF-κB and COX-2 and decrease KAT-II expression in the C1-C2 segments. On the other hand, we have found that AEA modulates the NTG-induced changes, thus it influences the activation and central sensitization process in the trigeminal system, probably via CBs.