Regulation of Inflammasomes by Application of Omega-3 Polyunsaturated Fatty Acids in a Spinal Cord Injury Model.

Omega-3 polyunsaturated fatty acids (PUFA n3) ameliorate inflammation in different diseases and potentially improve neurological function after neuronal injury. Following spinal cord injury (SCI), inflammatory events result in caspase-1 mediated activation of interleukin-1 beta (IL-1b) and 18. We aim to evaluate the neuroprotective potency of PUFA n3 in suppressing the formation and activation of inflammasomes following SCI. Male Wistar rats were divided into four groups: control, SCI, SCI+PUFA n3, and SCI+Lipofundin MCT (medium-chain triglyceride; vehicle). PUFA n3 or vehicle was intravenously administered immediately after SCI and every 24 h for the next three days. We analyzed the expression of NLRP3, NLRP1, ASC, caspase-1, IL-1b, and 18 in the spinal cord. The distribution of microglia, oligodendrocytes, and astrocytes was assessed by immunohistochemistry analysis. Behavioral testing showed significantly improved locomotor recovery in PUFA n3-treated animals and the SCI-induced upregulation of inflammasome components was reduced. Histopathological evaluation confirmed the suppression of microgliosis, increased numbers of oligodendrocytes, and the prevention of demyelination by PUFA n3. Our data support the neuroprotective role of PUFA n3 by targeting the NLRP3 inflammasome. These findings provide evidence that PUFA n3 has therapeutic effects which potentially attenuate neuronal damage in SCI and possibly also in other neuronal injuries.

Administration of 17ß-Estradiol Improves Motoneuron Survival and Down-regulates Inflammasome Activation in Male SOD1(G93A) ALS Mice.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease manifested by the progressive loss of upper and lower motoneurons. The pathomechanism of ALS is complex and not yet fully understood. Neuroinflammation is believed to significantly contribute to disease progression. Inflammasome activation was recently shown in the spinal cord of human sporadic ALS patients and in the SOD1(G93A) mouse model for ALS. In the present study, we investigated the neuroprotective and anti-inflammatory effects of 17ß-estradiol (E2) treatment in pre-symptomatic and symptomatic male SOD1(G93A) mice. Symptomatic mice with E2 substitution exhibited improved motor performance correlating with an increased survival of motoneurons in the lumbar spinal cord. Expression of NLRP3 inflammasome proteins and levels of activated caspase 1 and mature interleukin 1 beta were significantly reduced in SOD1(G93A) mice supplemented with E2.

Thalamus Degeneration and Inflammation in Two Distinct Multiple Sclerosis Animal Models.

There is a broad consensus that multiple sclerosis (MS) represents more than an inflammatory disease: it harbors several characteristic aspects of a classical neurodegenerative disorder, i.e., damage to axons, synapses, and nerve cell bodies. While several accepted paraclinical methods exist to monitor the inflammatory-driven aspects of the disease, techniques to monitor progression of early and late neurodegeneration are still in their infancy and have not been convincingly validated. It was speculated that the thalamus with its multiple reciprocal connections is sensitive to inflammatory processes occurring in different brain regions, thus acting as a "barometer" for diffuse brain parenchymal damage in MS. To what extent the thalamus is affected in commonly applied MS animal models is, however, not known. In this article we describe direct and indirect damage to the thalamus in two distinct MS animal models. In the cuprizone model, we observed primary oligodendrocyte stress which is followed by demyelination, microglia/astrocyte activation, and acute axonal damage. These degenerative cuprizone-induced lesions were found to be more severe in the lateral compared to the medial part of the thalamus. In MOG35-55-induced EAE, in contrast, most parts of the forebrain, including the thalamus were not directly involved in the autoimmune attack. However, important thalamic afferent fiber tracts, such as the spinothalamic tract were inflamed and demyelinated on the spinal cord level. Quantitative immunohistochemistry revealed that this spinal cord inflammatory-demyelination is associated with neuronal loss within the target region of the spinothalamic tract, namely the sensory ventral posterolateral nucleus of the thalamus. This study highlights the possibility of trans-neuronal degeneration as one mechanism of secondary neuronal damage in MS. Further studies are now warranted to investigate involved cell types and cellular mechanisms.

Neurodegeneration Triggers Peripheral Immune Cell Recruitment into the Forebrain.

Brain-intrinsic degenerative cascades have been proposed to be an initial factor driving lesion formation in multiple sclerosis (MS). Here, we identify neurodegeneration as a potent trigger for peripheral immune cell recruitment into the mouse forebrain. Female C57BL/6 mice were fed cuprizone for 3 weeks, followed by a period of 2 weeks on normal chow to induce the formation of lesion foci in the forebrain. Subsequent immunization with myelin oligodendrocyte glycoprotein 35-55 peptide, which induces myelin autoreactive T cells in the periphery, resulted in massive immune cell recruitment into the affected forebrain. Additional adoptive transfer experiments together with flow cytometry analysis underline the importance of brain-derived signals for immune cell recruitment. This study clearly illustrates the significance of brain-intrinsic degenerative cascades for immune cell recruitment and MS lesion formation. Additional studies have to address the signaling cascades and mechanistic processes that form the top-down communication between the affected brain area, neurovascular unit, and peripheral immune cells. SIGNIFICANCE STATEMENT: We identify neurodegeneration as a potent trigger for peripheral immune cell recruitment into the forebrain. Thus, immune cell recruitment might be a second step during the formation of new inflammatory lesions in multiple sclerosis. A better understanding of factors regulating neurodegeneration-induced immune cell recruitment will pave the way for the development of novel therapeutic treatment strategies.

Comparative analysis of gonadal steroid-mediated neuroprotection after transient focal ischemia in rats: route of application and substrate composition.

Progesterone (P) and 17ß-estradiol (E2) mitigate neuronal damage after experimentally induced traumatic brain injury (TBI) and ischemic stroke. Fish oil components such as omega-3 polyunsaturated fatty acids (PUFA n3) also provide neuroprotection in these traumatic models. Steroids and PUFA n3 dampen neuroinflammatory processes and regulate glial function in the affected brain areas. Using a transient focal ischemic rat model, we demonstrate that the co-application of PUFA n3 and P/E2 and the choice of the application route have a clear impact on the prevention of ischemia-induced infarct volume and behavioral recovery. A combinatory PUFA n3 plus P/E2 emulsion intravenously administered was most effective in reducing the infarct size and in restoring behavioral reconstitution compared to other oil emulsions and subcutaneous depot medication. These data encourage to refining clinical treatment protocols for TBI and stroke with gonadal steroids and to establishing combinatory drugs of steroids and fish oil-enriched emulsions thereby creating a win-win situation with two effective components.

Thalamus pathology in multiple sclerosis: from biology to clinical application.

There is a broad consensus that MS represents more than an inflammatory disease: it harbors several characteristic aspects of a classical neurodegenerative disorder, i.e. damage to axons, synapses and nerve cell bodies. While the clinician is equipped with appropriate tools to dampen peripheral cell recruitment and, thus, is able to prevent immune-cell driven relapses, effective therapeutic options to prevent the simultaneously progressing neurodegeneration are still missing. Furthermore, while several sophisticated paraclinical methods exist to monitor the inflammatory-driven aspects of the disease, techniques to monitor progression of early neurodegeneration are still in their infancy and have not been convincingly validated. In this review article, we aim to elaborate why the thalamus with its multiple reciprocal connections is sensitive to pathological processes occurring in different brain regions, thus acting as a "barometer" for diffuse brain parenchymal damage in MS. The thalamus might be, thus, an ideal region of interest to test the effectiveness of new neuroprotective MS drugs. Especially, we will address underlying pathological mechanisms operant during thalamus degeneration in MS, such as trans-neuronal or Wallerian degeneration. Furthermore, we aim at giving an overview about different paraclinical methods used to estimate the extent of thalamic pathology in MS patients, and we discuss their limitations. Finally, thalamus involvement in different MS animal models will be described, and their relevance for the design of preclinical trials elaborated.

Omega-3 polyunsaturated fatty acids ameliorate neuroinflammation and mitigate ischemic stroke damage through interactions with astrocytes and microglia.

Omega-3 polyunsaturated fatty acids (PUFA n3) provide neuroprotection due to their anti-inflammatory and anti-apoptotic properties as well as their regulatory function on growth factors and neuronal plasticity. These qualities enable PUFA n3 to ameliorate stroke outcome and limit neuronal damage. Young adult male rats received transient middle cerebral artery occlusion (tMCAO). PUFA n3 were intravenously administered into the jugular vein immediately after stroke and 12h later. We analyzed stroke volume and behavioral performance as well as the regulation of functionally-relevant genes in the penumbra. The extent of ischemic damage was reduced and behavioral performance improved subject to applied PUFA n3. Expression of Tau and growth-associated protein-43 genes were likewise restored. Ischemia-induced increase of cytokine mRNA levels was abated by PUFA n3. Using an in vitro approach, we demonstrate that cultured astroglial and microglia directly respond to PUFA n3 administration by preventing ischemia-induced increase of cyclooxygenase 2, hypoxia-inducible factor 1alpha, inducible nitric oxide synthase, and interleukin 1beta. Cultured cortical neurons also appeared as direct targets, since PUFA n3 shifted the Bcl-2-like protein 4 (Bax)/B-cell lymphoma 2 (Bcl 2) ratio towards an anti-apoptotic constellation. Thus, PUFA n3 reveal a high neuroprotective and anti-inflammatory potential in an acute ischemic stroke model by targeting astroglial and microglial function as well as improving neuronal survival strategies. Our findings signify the potential clinical feasibility of PUFA n3 therapeutic treatment in stroke and other acute neurological diseases.

Pharmacological and histological effects of Centaurea bruguierana ssp. belangerana on indomethacin-induced peptic ulcer in rats.

The species Centaurea bruguierana (DC.) Hand.-Mazz. ssp. belangerana (DC.) Bornm. (CBB) (Asteraceae), known as "Baad-Avard" in Borazjan, Bushehr Province, southern Iran, is used in folk medicine as a hypoglycemic herb in diabetes and as a remedy for peptic ulcer disorders. Total 80% EtOH extract and petroleum ether, CHCl(3), EtOAc, n-BuOH, and remaining fractions obtained by solvent-solvent fractionation of dried aerial flowering parts of the plant were investigated for anti-ulcer activity against indomethacin-induced ulcerogenesis in rats. Anti-ulcer activity was evaluated by measuring the ulcer index (UI) and ulcer inhibition. The UI was significantly reduced in all treated animals. A dramatic decrease in the UI was observed following the administration of total extract (100 mg/kg, p < 0.001) and CHCl(3) fraction (42 mg/kg, ***p < 0.001) in comparison with the control group. The percentage ulcer inhibition with total extract at a dose of 100 mg/kg (97.66%) and CHCl(3) fraction at a dose of 42 mg/kg (96.96%) was found to be higher (p < 0.001) than the reference group (cimetidine 100 mg/kg) (87.08%). The pharmacological and histological results of the present study proved that the aerial flowering parts of CBB possess preventive activity against peptic ulcer, supporting the traditional assertion in southern Iranian folk medicine.

Sedative effects of Iranian Artemisia annua in mice: possible benzodiazepine receptors involvement.

CONTEXT: Artemisia annua L. (Asteraceae), commonly known as sweet wormwood or Qinghao, is an annual herb/shrub native of Asia. The plant grows broadly in Caspian Sea shores in North of Iran. In China, the aerial parts of this plant are source of artemisinin, which is an antimalarial compound. OBJECTIVE: This study aimed to establish the scientific basis of reported ethnomedicinal use of A. annua as sedative agent. MATERIAL AND METHODS: The plants were gathered from Gilan Province in Iran. Plant aerial parts were extracted with methanol and concentrated in vacuum. Methanol extract was partitioned into chloroform, petroleum ether, and ethyl acetate. Each fraction was administered intraperitoneally (i.p.) in male mice with different concentrations (50, 100, and 200 mg/kg), and for evaluation of sedative activity, immobility time was determined. In effort to clarify the mechanism of action, flumazenil (3 mg/kg, i.p.) as a benzodiazepine (BZD) receptor antagonist was injected 15 min before chloroform fraction (200 mg/kg, i.p.). RESULTS: Compared with control group (saline-treated mice), the chloroform fraction significantly increased immobility time in a dose-dependent manner. Flumazenil decreased immobility time induced by chloroform fraction significantly. DISCUSSION AND CONCLUSION: The results of the present study suggest that A. annua growing in Iran has sedative effects, which are probably mediated via BZD receptors pathways.

Evaluation of systemic administration of Boswellia papyrifera extracts on spatial memory retention in male rats.

Time-dependent effects of ethanolic extract of Boswellia papyrifera, administered systemically, on spatial memory retention in the Morris water maze were investigated in male rats. A total extract of Boswellia papyrifera (300 mg/kg) was administered every eight hours to three groups of rats by gavage for 1, 2 and 4 weeks. In a separate set of experiments, three doses of a fraction of the extract, called the boswellic acids (100, 200 and 300 mg/kg) were administered by gavage to three groups of rats three times a day for 2 weeks. Following these applications, animals were trained for 4 days. Behavioral testing for evaluation of spatial memory retention was performed 48 h after completion of training. Boswellia papyrifera extracts and boswellic acids caused a significant reduction in escape latency and distance traveled but had no influence on swimming speed. These findings provide evidence that Boswellia papyrifera extracts affect spatial memory retention irrespective of the treatment period. In addition our data show that systemic administration of the boswellic acids fraction enhanced spatial memory retention in a dose-dependent manner. These improving effects may be due to some extent to the interactions of these products with inflammatory mediators, neurotransmitter signaling cascades or protein kinase pathways in the brain.

Estrogen regulates tyrosine hydroxylase expression in the neonate mouse midbrain.

Estrogen plays an important role during differentiation of midbrain dopaminergic neurons. This is indicated by the presence of estrogen receptors and the transient expression of the estrogen-forming enzyme aromatase within the dopaminergic cell groups. We have previously shown that estrogen regulates the plasticity of dopamine cells through the stimulation of neurite growth/arborization. In this study, we have analyzed the capability of estrogen to influence the activity of developing mouse dopamine neurons. The expression of tyrosine hydroxylase (TH) was assessed by competitive RT-PCR and Western blotting. The developmental expression of TH in the ventral midbrain was studied from embryonic day 15 until postnatal day 15 and revealed highest TH levels early postnatally. This profile coincides with the transient aromatase expression in this brain area. Using cultured midbrain cells, we found that estrogen increased TH mRNA/protein levels. The application of the estrogen receptor antagonist ICI 182,780 resulted in a complete inhibition of estrogen effects. To verify these data in vivo, fetuses were exposed in utero from E15 until birth to the aromatase inhibitor CGS 16949A or to CGS supplemented with estrogen. CGS caused a robust reduction in TH mRNA/protein levels in the midbrain, which could be restored by estrogen substitution. Taken together, our data strongly suggest that estrogen controls dopamine synthesis in the developing nigrostriatal dopaminergic system and support the concept that estrogen is implicated in the regulation of ontogenetic steps but also in the function of midbrain dopamine neurons.

Estradiol stimulates GDNF expression in developing hypothalamic neurons.

Estrogens stimulate the differentiation of neurons and neural networks in the CNS. The concordance of the cellular responses of estrogens and growth factors suggests that both factors may interact on the cellular level to ensure their developmental role. We have put forward this hypothesis and analyzed the effect of estrogens on the expression of glial cell line-derived neutrotrophic factor (GDNF) in developing hypothalamic cells. Using Western blotting and competitive RTPCR, we have demonstrated that 17beta-estradiol (E2) increases the expression of GDNF in hypothalamic cell cultures. E2-induced GDNF expression was seen in neurons but not astrocytes. GDNF induction by E2 appeared to be transmitted through nonclassical estrogen action, since the application of the nuclear estrogen receptor antagonists ICI 182, 780 did not abolish this effect. Only inhibitors of intracellular Ca(2+) and cAMP/protein kinase A signaling were effective in preventing E2 effects. We conclude that E2 is capable of influencing GDNF expression in the developing hypothalamus. Thus, it is conceivable that developmental E2 effects in the hypothalamus are partially mediated through the regulation of other important developmental signals such as growth factors.