Acupuncture reduces peripheral and brainstem cytokines in rats subjected to lipopolysaccharide-induced inflammation.

BACKGROUND: Lipopolysaccharide (LPS) endotoxins are activators of innate immunity inducing infection and inflammatory responses. Anti-inflammatory drugs can have undesirable side effects. Acupuncture may be an alternative for the treatment of inflammatory processes. OBJECTIVE: We investigated the potential anti-inflammatory effect of manual acupuncture (MA) at SP6 upon LPS-induced peritonitis in rats. METHODS: Peritonitis was induced in rats with an intraperitoneal injection of LPS (0.002, 0.02, 0.2 or 2 µg/kg) in four experimental groups (n = 6 each). A fifth group was injected with sterile saline solution (saline group, n = 6). Four hours after the procedure, peritoneal fluid was collected to determine total cell counts for inflammatory cells, differential leukocyte counts and peritoneal capillary permeability. The LPS dose of 0.02 µg/kg was used in the subsequent experiments as it most successfully induced peritoneal inflammation. Subsequently, five experimental groups (n = 12 rats each) were used: (1) saline, (2) control (untreated LPS group), (3) indomethacin (LPS group treated with indomethacin), (4) NA (LPS group treated with MA at a location not corresponding to any traditional acupuncture point), and (5) SP6 (LPS group treated with verum MA at SP6). Ten minutes after MA or 30 min after indomethacin treatment, the rats received an intraperitoneal injection of LPS. After 4 h, total leukocyte and differential cell counts, myeloperoxidase (MPO) activity, vascular permeability and cytokine levels were evaluated in the peritoneal fluid. Cytokine levels were additionally evaluated in the brainstem. RESULTS: SP6 MA and indomethacin treatments reduced inflammatory cell infiltration, vascular permeability and MPO activity in the LPS-exposed rats. Pre-treatment with indomethacin and SP6 MA decreased tumor necrosis factor (TNF)-α levels and preserved interleukin (IL)-10 in the peritoneal fluid. Indomethacin also reduced IL-6 in the peritoneal fluid. In the brainstem, indomethacin reduced IL-1ß, IL-6, TNFα and IL-10, whereas SP6 MA reduced only TNFα and IL-6 levels. CONCLUSIONS: This study clearly demonstrates the anti-inflammatory effect of acupuncture, which we believe may involve the activation of anti-inflammatory neural reflexes in the regulation of peritonitis.

Scolopendra subspinipes mutilans attenuates neuroinflammation in symptomatic hSOD1(G93A) mice.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a progressive, adult-onset neurodegenerative disorder characterized by selective motor neuron death in the spinal cord, brainstem, and motor cortex. Neuroinflammation is one of several pathological causes of degenerating motor neurons and is induced by activated microglial cells and astrocytes in ALS.Scolopendra subspinipes mutilans (SSM) is utilized in traditional Chinese and Korean medicine for the treatment of a variety of diseases, such as cancer, apoplexy, and epilepsy. However, the mechanisms underlying the effects of SSM are currently unclear, even though SSM increases immune and antibiotic activity. METHODS: To determine the effects of SSM on symptomatic hSOD1G93A transgenic mice, SSM (2.5 µâ„“/g) was injected bilaterally at the Zusanli (ST36) acupoint three times per week for two weeks. The effects of SSM treatment on anti-neuroinflammation in the brainstem and spinal cord of hSOD1G93A mice were assessed via Nissl and Fluoro-Jade B (FJB) staining, and immunohistochemistry using Iba-1, CD14, HO1, and NQO1 proteins was evaluated by Western blotting. RESULTS: In this study, we investigated whether SSM affects neuroinflammation in the spinal cord of symptomatic hSOD1G93A transgenic mice. We found that SSM treatment attenuated the loss of motor neurons and reduced the activation of microglial cells and astrocytes. Furthermore, we demonstrated that SSM administration in this animal model of ALS suppressed oxidative stress in the brainstem and spinal cord by 1.6- and 1.8-fold, respectively. CONCLUSIONS: Our findings suggest that SSM, which has previously been used in complementary and alternative medicine (CAM), might also be considered as an anti-neuroinflammatory therapy for neurodegenerative diseases.

Double step paraneoplastic brainstem encephalitis: a clinicopathological study.

A case of brainstem encephalitis in a man positive for both anti-Hu and anti-Ri antibodies is reported. This case had an unusual double step evolution and progressive involvement of different CNS subdivisions at MRI. Brainstem encephalitis developed abruptly, mimicking a posterior vascular deficit with vertigo and dizziness. These symptoms transiently remitted completely after a few days to relapse acutely 1 month later with sudden loss of consciousness, followed by confusion, disorientation, dysarthria, dysphagia and reduced thermic sensation on the right side. Within another few days, the patient developed acute respiratory failure and died some weeks later. MRI was negative at the beginning but later showed a progressive ascending involvement of the brainstem and thalamus. At autopsy, this picture corresponded to lymphocytic infiltration, preferentially B cells into the perivascular spaces and T cells in the brainstem parenchyma, confirming that T cells could be the effector of cytotoxicity, probably in the presence of cooperation with B cells that were well represented in this setting.

Uncoupling of interleukin-6 from its signalling pathway by dietary n-3-polyunsaturated fatty acid deprivation alters sickness behaviour in mice.

Sickness behaviour is an adaptive behavioural response to the activation of the innate immune system. It is mediated by brain cytokine production and action, especially interleukin-6 (IL-6). Polyunsaturated fatty acids (PUFA) are essential fatty acids that are highly incorporated in brain cell membranes and display immunomodulating properties. We hypothesized that a decrease in n-3 (also known as omega3) PUFA brain level by dietary means impacts on lipopolysaccharide (LPS)-induced IL-6 production and sickness behaviour. Our results show that mice exposed throughout life to a diet containing n-3 PUFA (n-3/n-6 diet) display a decrease in social interaction that does not occur in mice submitted to a diet devoid of n-3 PUFA (n-6 diet). LPS induced high IL-6 plasma levels as well as expression of IL-6 mRNA in the hippocampus and cFos mRNA in the brainstem of mice fed either diet, indicating intact immune-to-brain communication. However, STAT3 and STAT1 activation, a hallmark of the IL-6 signalling pathway, was lower in the hippocampus of LPS-treated n-6 mice than n-3/n-6 mice. In addition, LPS did not reduce social interaction in IL-6-knockout (IL-6-KO) mice and failed to induce STAT3 activation in the brain of IL-6-KO mice. Altogether, these findings point to alteration in brain STAT3 as a key mechanism for the lack of effect of LPS on social interaction in mice fed with the n-6 PUFA diet. The relative deficiency of Western diets in n-3 PUFA could impact on behavioural aspects of the host response to infection.

Brainstem and cervical spinal cord Fos immunoreactivity evoked by nerve growth factor injection into neck muscles in mice.

Although myofascial tenderness is thought to play a key role in the pathophysiology of tension-type headache, very few studies have addressed neck muscle nociception. The neuronal activation pattern following local nerve growth factor (NGF) administration into semispinal neck muscles in anaesthetized mice was investigated using Fos protein immunohistochemistry. In order to differentiate between the effects of NGF administration on c-fos expression and the effects of surgical preparation, needle insertion and intramuscular injection, the experiments were conducted in three groups. In the sham group (n=7) cannula needles were only inserted without any injection. In the saline (n=7) and NGF groups (n=7) 0.9% physiological saline solution or 0.8 microm NGF solution were injected in both muscles, respectively. In comparison with sham and saline conditions, NGF administration induced significantly stronger Fos immunoreactivity in the mesencephalic periaqueductal grey (PAG), the medullary lateral reticular nucleus (LRN), and superficial layers I and II of cervical spinal dorsal horns C1, C2 and C3. This activation pattern corresponds very well to central nervous system processing of deep noxious input. A knowledge of the central anatomical representation of neck muscle pain is an essential prerequisite for the investigation of neck muscle nociception in order to develop a future model of tension-type headache.

Distribution of beacon immunoreactivity in the rat brain.

Beacon is a novel peptide isolated from the hypothalamus of Israeli sand rat. In the present study, we determined the distribution of beacon in the rat brain using immunohistochemical approach with a polyclonal antiserum directed against the synthetic C-terminal peptide fragment (47-73). The hypothalamus represented the major site of beacon-immunoreactive (IR) cell bodies that were concentrated in the paraventricular nucleus (PVN) and the supraoptic nucleus (SON). Additional immunostained cells were found in the septum, bed nucleus of the stria terminalis, subfornical organ and subcommissural organ. Beacon-IR fibers were seen with high density in the internal layer of the median eminence and low to moderate density in the external layer. Significant beacon-IR fibers were also seen in the nucleus of the solitary tract and lateral reticular formation. The beacon neurons found in the PVN were further characterized by double label immunohistochemistry. Several beacon-IR neurons that resided in the medial PVN were shown to coexpress corticotrophin-releasing hormone (CRH) and most labeled beacon fibers in the external layer of median eminence coexist with CRH. The topographical distribution of beacon-IR in the brain suggests multiple biological activities for beacon in addition to its proposed roles in modulating feeding behaviors and pituitary hormone release.

Changes in expressions of proinflammatory cytokines IL-1beta, TNF-alpha and IL-6 in the brain of senescence accelerated mouse (SAM) P8.

The senescence-accelerated mouse (SAM) is known to be a murine model for accelerated aging. The SAMP8 strain shows age-related deterioration of learning and memory at an earlier age than control mice (SAMR1). In the present study, we investigated the changes in expressions of interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) in the brain of SAMP8. In the hippocampus of 10 months old SAMP8, the expression of IL-1 mRNA was significantly elevated in comparison with that of SAMR1. In both strains of SAMs, increases in IL-1beta protein in the brain were observed at 10 months of age compared with 2 and 5 months. The only differences found between the strain in protein levels were at 10 months and were elevations in IL-1beta in the hippocampus and hypothalamus, and in TNF-alpha and IL-6 in the cerebral cortex and the hippocampus in SAMP8 as compared with SAMR1. However, lipopolysaccharide-induced increases in the expression of these cytokines in brain did not differ between SAMP8 and SAMR1. Increases in expression of proinflammatory cytokines in the brain may be involved in the age-related neural dysfunction and/or learning deficiency in SAMP8.

Sound stimulation induces Fos-related antigens in cells with common morphological properties throughout the auditory brainstem.

Cells within the auditory brainstem of cat that respond to sound by producing the transcription factor Fos or related proteins were identified by immunostaining with antisera against Fos and Zif/268. Within the cochlear nucleus, all antisera showed similar staining patterns, however, in the superior olive and inferior colliculus, staining patterns differed between antisera. Immunostained cells were characterized by their size, location, by the presence of perisomatic terminals that immunostained for glutamate decarboxylase or synaptophysin, or by electron microscopy. Most cell classes were not immunopositive. In the ventral cochlear nucleus, roughly 99% of Fos-positive cells had few perisomatic terminals. Within the superior olivary complex (SOC), the majority of immunopositive cells had few perisomatic terminals. Lateral olivocochlear cells were identified as Fos positive by their size, location, lack of perisomatic terminals, and positive costaining for acetylcholinesterase as evidenced by a novel reaction product. This report appears to be the first demonstration of these cells responding to sound stimulation. Within the inferior colliculus, bands of positive cells produced by tonal stimulation extended from the central nucleus throughout the dorsal cortex and the posterior pericentral region, a finding unexpected on the basis of previous electrophysiological recordings and anatomical studies of ascending inputs to the colliculus. Approximately 35% of Fos-positive cells in the inferior colliculus had plentiful perisomatic terminals. Results demonstrate a high degree of specificity of auditory cell types that respond to sound by producing Fos-like proteins and show that previously intractable physiological questions can be addressed by assaying for sound-induced production of these antigens.

The developmental regulation of the L2/HNK-1 and L3 carbohydrate epitopes in mouse brain. Evidence for separate control of lipid- and protein-bound epitopes.

The carbohydrate epitopes L2/HNK-1 and L3 have previously been identified on various neural cell adhesion molecules and have been suggested to play a role in the mediation of cell-cell adhesion. In this study, the developmental expression of the two epitopes in soluble, membrane-bound and chloroform/methanol-extracted fractions of the constituent mouse brain regions was examined by enzyme-linked immunosorbent assay (ELISA). The protein-bound epitopes were shown to be uniformly developmentally regulated, with levels peaking at postnatal day 20 (P20). The epitopes in a crude chloroform/methanol fraction, however, demonstrated a different pattern, with L2 peaking earlier at postnatal day zero (P0). These results suggest a possible interaction between the control of the two pools of the epitope.

Analysis of Ia induction on Lewis rat astrocytes in vitro by virus particles and bacterial adjuvants.

Viral particles of a neurotropic murine hepatitis virus (JHM) and various substances known to have immunoregulatory effects, including bacterial lipopolysaccharide (LPS) and synthetic adjuvant peptide (muramyl dipeptide) (AP), were tested for their ability to induce Ia antigen expression on Lewis rat astrocytes in vitro. JHM virus, LPS and AP are all capable of inducing Ia molecules on astrocytes, however, in a pattern and kinetics distinct from recombinant rat gamma interferon (gamma-IFN). Whereas gamma-IFN induced Ia expression on astrocytes and all macrophages after 48 h treatment, JHM virus, LPS and AP required 4-7 days for maximal induction of Ia on astrocytes, but had little to no effect on the macrophage population. This indicates that astrocytes are uniquely reactive to components derived from infectious agents and that these components are immunoregulatory with respect to Ia expression on astrocytes. We have also attempted to determine possible mechanisms by which these agents induce astrocyte Ia and show that phorbol myristate acetate and Ca2+ ionophore A23187 have similar effects. These findings suggest that infectious agents may directly stimulate antigen presenting functions of astrocytes in the brain through gamma-IFN-independent mechanisms.

A monoclonal antibody to limbic system neurons.

A monoclonal antibody produced against hippocampal cell membranes labeled the surface of neurons in the rat limbic system. With a few exceptions, all nonlimbic components were unstained. This specific distribution of immunopositive neurons provides strong evidence of molecular specificity among functionally related neurons in the mammalian brain and supports the concept of a limbic system.

The immune response evokes changes in brain noradrenergic neurons.

A decreased noradrenaline turnover in the hypothalami of rats was observed at the peak of the immune response to sheep red blood cells. The decrease in noradrenergic neuronal activity was mimicked by injection of soluble r mediators released by immunological cells activated in vitro. Noradrenaline also tended to decrease in the brainstem but not in the residual brain. It is suggested that products released from activated immunological cells during the immune response may induce the previously described autonomic and endocrine mechanisms that contribute to immunoregulation.