A. Yu. Kozlov

Cytokine Levels in Rat Blood and Brain Structures after Administration of Lipopolysaccharide

We compared cytokine profi le of rat serum and brain structures after immune status modulation by LPS (30 μg/kg intraperitoneally). The content of infl ammatory (IL-1α, IL-1β, IL-2, IL-6, IFN-γ, and TNF-α) and anti-infl ammatory (IL-4 and IL-10) cytokines in biological samples of animals was measured on days 1 and 7 after antigenic stimulation. LPS administration reduced the levels of both infl ammatory and anti-infl ammatory cytokines in the peripheral blood of the rats, especially on the 1st day. LPS administration was also accompanied by specifi c changes in cytokine content in the dorsal hippocampus and anterior cingulate cortex. Antigenic stimulation increased the level of anti-infl ammatory cytokines IL-4 and IL-10 in the examined brain tissues, the changes were most pronounced on day 1 after LPS injection. No signifi cant changes in the levels of proinfl ammatory cytokines in the brain tissue of animals were found at the above terms after LPS injection. Thus, peripheral LPS administration to rats shifts the balance between the infl ammatory and anti-infl ammatory cytokines in the CNS structures towards the latter.

Dynamics of changes in nociceptive reactions of rats after peripheral administration of lipopolysaccharide.

The type of changes in nociceptive reactions of rats was studied at various time intervals after intraperitoneal injection of LPS in a dose of 30 μg/kg. The perceptual component of nociception in animals was evaluated from the tail-flick latency in response to light-heat stimulation. The emotional component of nociception in rats was determined from the vocalization threshold during electrocutaneous stimulation of the tail. The tail-flick latency of animals under conditions of light-heat stimulation was shown to decrease 1 day after peripheral administration of LPS, which illustrates an increase in the perceptual component of nociception at the relatively early stage of antigenic stimulation. A significant decrease in the tail-flick latency and vocalization threshold in rats in response to nociceptive stimulation was observed on day 7 after LPS injection. These changes demonstrate an increase in the perceptual and emotional components of nociception at the late period after antigen exposure. Our results indicate that antigenic stimulation due to peripheral administration of LPS is followed by the increase in nociceptive sensitivity of rats. LPS-induced variations in the emotional and perceptual components of nociception in animals suggest the existence of specific mechanisms for systemic regulation of pain. They probably depend on the period of study and type of immune reactions during antigenic stimulation.

Nociceptive Thresholds of the Response to Lipopolysaccharide Injection into the Limbic Structures of the Brain in Rats

Microinjections of LPS into the limbic structures of rat brain (dorsal hippocampus and caudal compartment of the cingulate fascicle) caused opposite effects on the nociceptive thresholds: injection into the dorsal hippocampus enhanced perception and reduced the emotional affective perception of pain, while injection into the cingulate fascicle reduced the perceptual and enhanced the emotional components of the nociceptive reaction. These results indicated specific involvement of these limbic structures in nociception modulation during induction of the immune response in CNS.

The Effect of the Tumor Necrosis Factor DNA on the Immune Response in DNA Immunization against the Herpes Simplex Virus

A study was made of the adjuvant effect of the mouse tumor necrosis factor α (mTNFα) on DNA immunization against the herpes simplex virus type 1 (HSV1). The HSV1 gD gene (pDNAgD) served as an immunogen; mTNFα or its gene cloned in an eukaryotic expression vector (pDNAmTNF) were used to modulate the immune response. Double immunization with pDNAgD led to a sixfold increase in the in vitro T-cell response, a high (1:2000) titer of anti-HSV1 antibodies (including virus-neutralizing antibodies), an increase in IgG2a/IgG1 (suggesting a shift of the immune response to the Th1 type), and no change in CD4/CD8 T-cell ratio. A single injection of mTNFα along with inactivated HSV1 allowed a twice higher antibody titer and a fourfold higher T-cell response as compared with immunization with HSV1 alone. Double immunization with both pDNAgD and pDNAmTNF increased the titer of anti-HSV1 antibodies and the T-cell response by factors of 8 and 1.5, respectively, as compared with immunization with pDNAgD alone. However, the protective effect was significantly lower with the two plasmids than with pDNAgD (73 vs. 100%). Thus, DNA immunization with pDNAgD induced both B- and T-cell responses and completely protected mice from a lethal doze of HSV1. The adjuvant properties of mTNFα and pDNAmTNF need further investigation.

Comparison of the Adjuvant Activity for the Glucosaminyl-Muramyl Dipeptide and the Granulocyte-Macrophage Colony-Stimulating Factor Gene in Gene Immunization against the Herpes Simplex Virus

The adjuvant activity in DNA immunization against herpes simplex virus type 1 (HSV1) was studied for the granulocyte-macrophage colony-stimulating factor (GM-CSF) synthesized from an eukaryotic expression plasmid (pDNAGM-CSF) and for the synthetic glucosaminyl-muramyl dipeptide (GMDP). A plasmid containing the HSV gD gene (pDNAgD) was used as an immunogen. GMDP and pDNAGM-CSF each enhanced the T-cell immune response to DNA immunization. The protective effect of DNA immunization increased from 63 to 100% when the two plasmids were injected simultaneously and to 96% when pDNAgD was injected one day after injecting GMDP. The results showed that DNA vaccines combined with genetic or peptide adjuvants are promising for DNA immunization against HSV.

Nociceptive Reactions during Stimulation of Immunity in Rats with Different Individual Sensitivities to Stress

Different nociceptive reactions and individual resistance were studied in rats in the initial state and in conditions of stimulation of immune processes by various methods. The aims were to study the effect of the preparation Imunofan, an immune stimulator, on various behavioral manifestations of pain reactions in animals with different individual resistances to stress and to compare the results with similar data in a natural model of immune activation. Detection of the central, immune-dependent component of the regulation of nociceptive reactions was addressed using intracerebroventricular administration of the study compound. Studies were performed on 43 male Wistar rats. Individual resistance to stress was assessed by recording the free behavior of the animals in an open field test. The following nociceptive reactions were assessed: 1) the tail withdrawal reaction using the standard tail flick method; and 2) running, twitching, jumping, and vocalization reactions to electrical stimulation of the limbs. These studies demonstrated that i.m. administration of Imunofan (10 μl of 0.005% solution) suppressed the active behavior of the animals in the open field and produced hyperalgesia, with decreases in the thresholds of nociceptive reactions. Hyperalgesia in stress-sensitive rats were significantly greater than in resistant animals. Similar results were obtained in conditions of natural activation of immunity produced by the operative intervention needed for injection of the agent into the cerebral ventricle. Intracerebroventricular administration of Imunofan was accompanied by more marked and complex changes in pain sensitivity.

Role of caudomedial portion in left and right cingulum bundle in perceptual and emotional components of nociception in rats

The effects of electrocoagulation of caudomedial portion of the left and right cingulum bundles on tail-flick latency and vocalization threshold were examined on rats. We revealed a tendency to a decrease in the tail-flick latency and more pronounced elevation of the vocalization threshold after destruction of the right cingulum bundle. These findings indicate functional asymmetry of the cingulum bundle in the realization of the emotional nociceptive reaction.

Comparative study of macrophage response in mice after DNA immunization and infection with herpes simplex virus type 1.

Functional activity of macrophages and intensity of T cell immune response in mice were studied after intravaginal and intraperitoneal infection with herpes simplex virus type 1 and DNA vaccination in combination with adjuvant treatment (recombinant granulocytemacrophage colony-stimulating factor and glucosaminylmuramyl dipeptide). DNA vaccination induced a virus-specific T cell immune response with no macrophagic inflammatory reaction. Infection with herpes simplex virus type 1 was accompanied by sustained inflammation, but not by the T cell immune response.

Nociceptive Thresholds in Rats in Response to Lipopolysaccharide Injection into the Specific Nuclei of the Thalamus of the Brain

Microinjections of LPS into the specific nuclei of rat thalamus (ventrobasal thalamic nuclei VPL and VPM) slightly increased perceptual component and significantly decreased emotional component of systemic nociceptive response.

Nociceptive Sensitivity and Lymphocytic Index of Peripheral Blood in Rats with Different Behavioral Activity in Model of Infl ammatory Pain Provoked by Injection of Freund’s Complete Adjuvant and Bovine Serum Albumin

Nociceptive thresholds decreased in rats at the early stage of infl ammatory reaction induced by subcutaneous injection of BSA and complete Freund’s adjuvant. At the later stage of this reaction, there was a trend of restoring nociceptive parameters in behaviorally passive rats in contrast to active animals, which demonstrated further decrease in the nociceptive thresholds. During the late infl ammatory period, the lymphocytic index (by Shaganin) changed unidirectionally in the rats with different behavioral parameters. Probably, the changes in nociceptive thresholds were not triggered by the shift in lymphocyte/segmented neutrophil ratio, but resulted from production of yet not established biologically active agents with proalgesic and analgesic nature.