Plasma concentrations of arginine and related amino acids following traumatic brain injury: Proline as a promising biomarker of brain damage severity.

The aim of this study was to find a plasma biomarker, in relation with nitric oxide (NO), as a sign of brain damage severity following traumatic brain injury (TBI). We first investigated the post-traumatic evolution of the plasma concentrations of NO via the level of NO end-products metabolites (nitrite plus nitrate, NO(x)), that of l-arginine (Arg) and amino acids involved in its metabolism as well as the time course of neurological score in a rat model of lateral fluid percussion brain injury. First, the level of NO(x) was increased in plasma at 24 and 48 h post-TBI with a marked increase at 72 h. In contrast, this elevation was neither accompanied by a modification of plasma concentrations of Arg, nor of amino acids involved in NO and Arg metabolism, l-ornithine (Orn), l-glutamate (Glu), and l-glutamine (Gln). Second, TBI induced a fall of plasma l-proline (Pro) concentrations. The time course of post-TBI neurological deficit showed also a decrease of neurological score at 24, 48, and 72 h. Furthermore, there is a weak negative correlation between the neurological score and the plasma level of NO(x) (r=-0.305; P<0.05), while a marked positive correlation has been found between the neurological score and the plasma level of Pro (r=0.563; P<0.001). In conclusion, the plasma concentrations of Pro could be a promising marker of post-traumatic neurological deficit.

Selective inhibition of inducible nitric oxide synthase reduces neurological deficit but not cerebral edema following traumatic brain injury.

The role of inducible nitric oxide synthase (iNOS) in cerebral edema and neurological deficit following traumatic brain injury (TBI) is not yet clear-cut. Therefore, the aim of this study was to investigate the effect of three different iNOS inhibitors on cerebral edema and functional outcome after TBI. First, the time courses of blood--brain barrier (BBB) breakdown, cerebral edema, and neurological deficit were studied in a rat model of fluid percussion-induced TBI. The permeability of BBB to Evans blue was increased from 1 h to 24 h after TBI. Consistently, a significant increase in brain water content (BWC) was observed at 6 and 24 h post-TBI. A deficit in sensorimotor neurological functions was also observed from 6 h to 7 days with a maximum 24 h after TBI. Second, a single dose of aminoguanidine (AG; 100 mg/kg, i.p.), L-N-iminoethyl-lysine (L-NIL; 20 mg/kg, i.p.), or N-[3-(aminomethyl)benzyl]acetamide (1400W; 20 mg/kg, s.c.) was administered at 6 h post-TBI. Treatment with AG reduced by 71% the increase in BWC evaluated at 24 h, while L-NIL and 1400W had no effect. In contrast, the three iNOS inhibitors reduced the neurological deficit from 30% to 40%. Third, 1400W (20 mg/kg, s.c.) was administered at 5 min, 8 and 16 h post-TBI. Although this treatment paradigm had no effect on cerebral edema evaluated at 24 h, it significantly reduced the neurological deficit and iNOS activity. In conclusion, iNOS contributes to post-TBI neurological deficit but not to cerebral edema. The beneficial effect of iNOS inhibitors is not due to their anti-edematous effect, and the reduction of cerebral edema by AG is unlikely related to iNOS inhibition. The 6 h therapeutic window of iNOS inhibitors could allow their use in the treatment of functional deficit at the acute phase of TBI.

1400W, a potent selective inducible NOS inhibitor, improves histopathological outcome following traumatic brain injury in rats.

There are conflicting data regarding the role of nitric oxide (NO) produced by inducible NO synthase (iNOS) in the pathophysiology of traumatic brain injury (TBI). In this report, we evaluated the effect of a potent selective (iNOS) inhibitor, 1400W, on histopathological outcome following TBI in a rat model of lateral fluid percussion brain injury. First, to design an appropriate treatment protocol, the parallel time courses of iNOS and neuronal NOS (nNOS) gene expression, protein synthesis, and activity were investigated. Early induction of iNOS gene was observed in the cortex of injured rats, from 6 to 72 h with a peak at 24 h. Similarly, iNOS protein was detected from 24 to 72 h and de novo synthesized iNOS was functionally active, as measured by Ca2+-independent NOS activity. The kinetic studies of nNOS showed discrepancies, since nNOS gene expression and protein synthesis were constant in the cortex of injured rats from 24 to 72 h, while Ca2+-dependent constitutive NOS activity was markedly decreased at 24 h, persisting up to 72 h. Second, treatment with 1400W, started as a bolus of 20 mg kg-1 (s.c.) at 18 h post-TBI, followed by s.c.-infusion at a rate of 2.2 mg kg-1 h-1 between 18 and 72 h, reduced by 64% the brain lesion volume at 72 h. However, the same treatment paradigm initiated 24 h post-TBI did not have any effect. In conclusion, administration of a selective iNOS inhibitor, 1400W, even delayed by 18 h improves histopathological outcome supporting a detrimental role for iNOS induction after TBI.

Exclusion of angiotensin I-converting enzyme as a candidate gene involved in exudative inflammatory resistance in F344/N rats.

BACKGROUND: Inbred LEW/N and F344/N rats respectively, are susceptible and relatively resistant to a broad range of inflammatory/autoimmune diseases. We recently identified a quantitative trait locus (QTL) on chromosome 10 that protects the F344/N rat from carrageenan-induced exudation in a dominant fashion. Angiotensin I-converting enzyme (ACE) is one of the candidate genes located in this QTL region that plays an important role in inflammation. MATERIALS AND METHODS: RNA was extracted from both LEW/N and F344/N rat strains and used to produce full length cDNA by reverse transcription polymerase chain reaction (RT-PCR). Both strands of the PCR products were entirely sequenced to determine nucleotide differences between strains. ACE activity was measured using the synthetic substrate 3H-hippuryl-glycylglycine. ACE protein levels were determined by Western blot using a specific ACE antibody. ACE kinetic and inhibition studies were performed using specific substrates (Hip-His-Leu and Acetyl-Seryl-Aspartyl-Acetyl-Lysyl-Proline) and inhibitors (lisinopril, captopril and quinaprilat) for each C- and N-terminal active site. Finally, the dose-effects of lisinopril treatment on carrageenen-induced exudate volume and ACE activity was studied. RESULTS: In this study, we report for the first time a missense mutation in the coding region of ACE cDNA at 5' 1021 from C to T, resulting in a Leu-341 to Phe substitution, close to the N-domain active site in the F344/N rats. Full characterization of soluble and tissue ACE in both LEW/N and F344/N rat strains showed that soluble ACE levels in serum and exudate were 1.5 fold higher in the F344/N rats than those in LEW/N rats. In addition, the soluble ACE level was inversely correlated with the exudate volume. However, the specific ACE activity and its catalytic properties were identical in both strains. Furthermore, the chronic inhibition of serum and exudate ACE levels by lisinopril treatment did not affect the exudate volume in F344/N rats, indicating that several factors besides ACE were involved in the control of carrageenan-induced exudation. CONCLUSIONS: This report describes a complete molecular, biochemical, enzymatic and pharmacologic study of a missense mutation in the ACE cDNA in F344/N rats, that taken together, excludes ACE as a candidate gene involved with resistance to carrageenan-induced exudation in F344/N rats.

Neuroendocrine and other factors in the regulation of inflammation. Animal models.

A variety of animal models have been used to study the role of neuroendocrine responses in various aspects of autoimmune/inflammatory disease. Complex models of autoimmune disease, such as inflammatory arthritis in rats and thyroiditis in chickens, indicate a role for blunted HPA axis and dysregulated sympathoneuronal responses in susceptibility to autoimmune disease. A variety of approaches including pharmacological, surgical (ablation, transplantation), genetic linkage and segregation studies have been used to identify factors contributing to the phenotypes of susceptibility or resistance to inflammatory/autoimmune disease. Innate inflammation, or the earliest nonspecific form of the inflammatory response, which is characterized by fluid exudation and migration of immune cells to inflammatory sites, is a subtrait of these forms of inflammatory disease. Genetic linkage and segregation studies in inflammatory susceptible and resistant rat strains indicate that this subtrait is multigenic and polygenic; that is, that multiple loci on multiple chromosomes, each with a weak effect, control this trait, and that there is a large environmental component to the variability of this trait. Such information derived from animal studies can be used to target candidate genes for further study and to inform the design of human studies.

Increased sensitivity of prediabetic nonobese diabetic mouse to the behavioral effects of IL-1.

The nonobese diabetic (NOD) mouse is a model of spontaneous insulin-dependent diabetes mellitus (IDDM) or type I diabetes. In humans, and in animal models of IDDM, the progression of the disease is modulated by various environmental factors, particularly infectious agents. Interleukin-1 (IL-1) plays a pivotal role in the development of IDDM, and modulation of its synthesis may be a mechanism by which environmental modulation of disease progression occurs. Since various alterations at the level of the gene, number, and sensitivity of IL-1 receptors have been described in different animal models of autoimmune disease, we investigated, in the prediabetic NOD mouse, the presence of IL-1 receptors and their functional behavioral characteristics. Here we present evidence that prediabetic NOD mice exhibit a normal distribution and density of functional brain IL-1 receptors, but are more sensitive to the behavioral effects of IL-1 than the control ICR strain.

Animal models of neuroimmune interactions in inflammatory diseases.

Animal models have been used successfully to study various aspects of neural-immune interactions. Although different approaches carry certain advantages and disadvantages, current high sensitivity screening and manipulation methods coupled with molecular and genetic approaches can be successfully used to tease out the neural pathways that regulate inflammatory disease and the effects of immune molecules, such as interleukins, on neuronal function and pathology. Newer methodologies that measure gene expression of thousands of genes will in the future add to the ability to evaluate complex systems interactions in whole animal models. This review addresses the advantages and disadvantages of some of these approaches in the context of application to neural-immune interactions.

Interleukin-1 receptor defect in autoimmune NZB mouse brain.

Interleukin-1 receptors (IL-1R type I and II) have been characterized in murine nervous structures (hippocampus and frontal cortex), in vascular structures (vessels, choroid plexus), and in the anterior pituitary. Because interleukin-1 (IL-1), injected or induced in the brain, is a powerful regulator of the stress axis and immune functions, it was of interest to investigate IL-1Rs and IL-1 in autoimmune mice. In control mice, bacterial lipopolysaccharide (LPS), administered i.p. or i.c.v., induces a sharp decrease in available brain IL-1 receptors, in spite of a moderate increase in mRNAs for both receptor types. This is concomitant with an increase in IL-1 alpha, beta, and ra mRNA. Ligand production clearly overcomes receptor turnover. In autoimmune mice (NZB and NZB/NZW F1), a strong defect in IL-1R (type I) is demonstrated in the dentate gyrus. This tissue-specific defect cannot be explained by increased occupancy by endogeneous ligands as for LPS-treated mice. The transmission of the defect is Mendelian and suggests the involvement of a single gene. However patterns of IL-1R mRNAs (evaluated by RT-PCR) are similar in NZB and in controls, suggesting a translational or post-translational abnormality. The contribution of this genetic disorder in the development of autoimmunity remains to be clarified. Because the brain IL-1 system sends inhibitory signals towards immune functions, this lack of functional IL-1 binding sites might participate in the disregulations observed in NZB autoimmune mice.

Interleukin-1 receptor deficiency in the hippocampal formation of (NZB x NZW)F2 mice: genetic and molecular studies relating to autoimmunity.

Interleukin-1 receptor (IL-1R) deficiency has been previously described in the dentate gyrus of autoimmune NZB and (NZB x NZW) F1 (or BWF1) mice. In this study, the genetic and molecular characterization of this defect were investigated in BWF2 mice in relation to anti-DNA antibody production and microsatellite D1Nds4 (near the IL1r1 gene) polymorphism. IL-1R density was quantified in the brain, spleen and pancreas, using in vitro quantitative autoradiography with recombinant human [125I]-IL-1alpha as the ligand. This study of the dentate gyrus of F2 mice revealed three phenotypes: NZW-like, NZB-like and F1-like, which occurred in a ratio of 1:1:2, with IL-1R densities of 100%, 17% and 59%, respectively as compared to control NZW mice (100%). In contrast, IL-1R densities observed in the choroid plexus and peripheral organs were similar. Moreover a high production of IgG2a anti-DNA antibodies was observed in F2 mice, as in their parents, particularly those with the NZB-like phenotype. Microsatellite mapping of D1Nds4 revealed polymorphism in both parents and BWF2 mice in relation to the level of IL-1R density in the dentate gyrus. In spite of the acute defect in IL-1 binding in the dentate gyrus of NZB mice, molecular analysis of IL-1R mRNA (type I, II and accessory protein) showed similar amounts of mRNA, measured following RT-PCR amplification, in the hippocampal formation of both NZB and control C3H/He mice. In conclusion, the transmission of the IL-1R defect in the dentate gyrus of NZB mice is monofactorial and the defect appears to be at the post-transcriptional level of IL-1R synthesis. The lack of IL-1R in the dentate gyrus seems to correlate with some autoimmune characteristics. Correlation of D1Nds4 polymorphism with the level of IL-1R density suggests that it could be a predisposing gene to disease or a marker for other closely linked predisposing genes.

Disturbed immunoendocrine communication via the hypothalamo-pituitary-adrenal axis in murine lupus.

Immune reactions and mitogen stimulation of mammals and chickens lead to an increase of glucocorticoid (GC) plasma levels concomitant with the immune response. Interleukin (IL) 1, one of the most important glucocorticoid increasing factors produced by cells of the immune system, acts via the hypothalamo-pituitary-adrenal (HPA) axis. This pattern of immunoendocrine feedback communication is altered in autoimmune disease (AID) and represents a possible site of action for GC therapy. In the present study we investigated the role and possible underlying mechanisms of a disturbed immunoendocrine communication via the HPA axis in murine lupus. We analyzed the response to recombinant human (rhu) IL-1alpha in AID-prone mice [NZB, NZW, (NZB/NZW)F1, MRL/MP-lpr] in comparison to nonautoimmune, normal control mice (Swiss, C3H/HeJ, MRL/MP-+/+) at different levels of the HPA axis. To this end, we quantified the plasma levels of ACTH, corticosterone, and corticosterone-binding globulin (CBG) and determined various pathology parameters for autoimmunity. AID-prone mice produced nearly the same levels of plasma corticosterone after injection of rhu IL-1alpha as normal mice, but had baseline corticosterone levels consistently higher, thus resulting in significantly lower corticosterone increasing ratios. ACTH levels increased after rhu IL-1alpha injection, but there was no clearcut difference in the increasing ratios of AID-prone and normal strains. CBG levels showed no difference. As expected, there was a correlation of pathology parameters for autoimmunity and the altered immunomodulatory response to rhu IL-1alpha per group. On an individual basis, there was no such correlation. In conclusion, our results confirm the existence of a disturbed immunoendocrine communication in AID-prone mice. This disturbance clearly differs from individual to individual and also among different types of AID.

Radioimagers as an alternative to film autoradiography for in situ quantitative analysis of 125I-ligand receptor binding and pharmacological studies.

Three radioimagers, the mu-imager, the beta-imager and the phosphorimager, were tested as alternatives to quantitative autoradiography on film, for receptor imaging and pharmacological in situ quantitative analysis. Two iodinated ligands 125I-interleukin-1 alpha and 125I-gonadotropin releasing hormone agonist were used for receptor characterization in mouse brain and pituitary sections. Due to the high number of the agonist receptors in rat pituitary gland, this tissue was used to compare measurements obtained from digital autoradiograms with classical gamma detector determination. This permits the evaluation of radioimager efficiency and absolute quantification. Radioimagers represent an improvement in terms of time of image acquisition. All the radioimagers are more sensitive than film for the detection of low levels of radioactivity. The spatial resolution provided by the mu-imager compares favourably with that obtained on film autoradiograms while digital autoradiograms from the phosphorimager and beta-imager did not show precise definition under our experimental conditions. Superimposition of histological structures from the stained sections with radiolabelled areas in the autoradiograms remains, at this time, the unique advantage of film. In conclusion, radioimagers represent an alternative to autoradiography on film or emulsion for in situ quantitative studies on tissue sections. They combine precise imaging for in situ binding studies with easy and direct access to counts in cpm. The improvement in radioimaging technology has, therefore, brought in situ analysis of iodinated ligand binding to the level of accuracy that is obtained with classical detectors of radioactivity.

Interleukin-1 receptor accessory protein transcripts in the brain and spleen: kinetics after peripheral administration of bacterial lipopolysaccharide in mice.

Receptors for IL-1, type I (IL-1R1) and type II (IL-1R2), have been characterized by pharmacological and molecular techniques in the mouse brain. High densities are mainly found in the cortex, dentate gyrus and choroid plexus. It was therefore of interest to investigate the expression of mRNA IL-1 receptor accessory protein (IL-1R AcP), which is a part of the IL-1 receptor complex and has been shown to interact specifically with IL-1R1. IL-1R AcP transcripts were detected under basal conditions following RT-PCR amplification in the mouse brain, as well as in the pituitary, spleen, adrenal and liver. IL-1R AcP transcripts were found in higher amounts than IL-1R1 transcripts in all tissues except the spleen, where their expression was minor. Following bacterial lipopolysaccharide (LPS) stimulation (3-48 h), IL-1R AcP transcripts were not changed in the brain, while IL-1R1 transcripts were increased for 3-6 h. In the spleen, a slight increase in IL-1R AcP and IL-1R1 was observed during the first hours following LPS stimulation. In conclusion, IL-1R AcP mRNA is expressed in the brain and in other tissues where IL-1R1 transcripts are found. However, the regulation of its expression is distinct from IL-1R1. The high level of expression and the lack of regulation of IL-1R AcP transcripts in the brain under inflammatory conditions suggest that the protein might be constitutively expressed in excess.

Interleukin-1 effect on glycemia in the non-obese diabetic mouse at the pre-diabetic stage.

Cytokines, particularly interleukin 1 (IL-1) and tumor necrosis factor, are known to induce hypoglycemia in normal rodents or different experimental models of type II diabetes. We investigated, at the pre-diabetic stage, the effect of short-term administration of murine recombinant interleukin-1 alpha (mrIL-1 alpha) on the levels of glucose, insulin and corticosterone in the non-obese diabetic (NOD) mouse, a spontaneous model of type I diabetes. Two-month-old, pre-diabetic NOD mice of both sexes were insensitive to mrIL-1 alpha (12.5 and 50 micrograms/kg) 2 h after administration, the time at which the maximal decrease (around 50%) was observed in the C57BL/6 mouse strain. Kinetic studies however showed that mrIL-1 alpha lowered glycemia in both sexes of NOD mice, but the effect was limited and delayed. In the NOD and C57BL/6 strains, mrIL-1 alpha had no influence on insulin levels in females, but significantly increased them in males (P < 0.0001). Castration of NOD males abrogated the stimulatory effect of mrIL-1 alpha on insulin secretion. Corticosterone secretion was stimulated by mrIL-1 alpha in both sexes of NOD and C57BL/6 mice, and this effect was faster and greater in NOD females than in C57BL/6 females. The incomplete hypoglycemic response to mrIL-1 alpha in females may be attributed to the anti-insulin effect of glucocorticoids, an effect which can be demonstrated when mrIL-1 alpha is administered to adrenalectomized animals or when mrIL-1 alpha is administered together with the glucocorticoid antagonist RU38486. In NOD males, in contrast, glucocorticoids did not play a major role in the limited hypoglycemic response to mrIL-1 alpha, since RU38486 and adrenalectomy were not able to unmask a hypoglycemic effect. Moreover, NOD mice of both sexes were less sensitive than C57BL/6 mice to the hypoglycemic effect of insulin (2.5 U/kg), which suggests some degree of insulin-resistance in NOD mice. With regard to the effect of IL-1 on NOD mouse glycemia, therefore, these results suggest that glucocorticoids and/or androgens, according to the animal's sex, may induce a state of insulin-resistance.

Localization and characterization of interleukin-1 receptors in the islets of Langerhans from control and nonobese diabetic mice.

Numerous in vivo and in vitro studies have shown the effects of interleukin-1 (IL-1) on insulin and glucagon secretion. To understand the mechanism of these effects, we performed localization and characterization of IL-1 receptors (IL-1R) in pancreas using a quantitative autoradiography method and recombinant human (rh) [125I]IL-1 alpha as a ligand. Frozen sections of pancreas were studied in control (C3H/He) and nonobese diabetic (NOD) mice (a model of autoimmune type I diabetes). Compared to splenic IL-1R, a very high density of specific IL-1R (> 4-fold that in spleen) was found on the islets of Langerhans in both strains. In C3H/He mice, competition experiments demonstrated the presence of one high affinity binding site (Ki = 3.4 and 3.2 x 10(-10) M; binding capacity, 137 and 122 fmol/mg protein for rhIL-1 alpha and rhIL-1 beta, respectively), comparable to type I IL-1R described on T-lymphocytes. In prediabetic NOD mice, these IL-1R were expressed with the same density, affinity, and specificity as in the control strain. Before the onset of diabetes, the expression of IL-1R protein on the islet cells appears to be entirely normal. In contrast, in diabetic NOD mice, IL-1R are sharply decreased, correlating with the intensity of islet destruction. In conclusion, the localization and high density of IL-1R on the mouse islets of Langerhans complement previous studies showing the presence of messenger RNA for type I IL-1R on the islets of Langerhans. These results support a direct physiological effect of IL-1 on pancreatic hormones, such as insulin and glucagon, and a potential role of IL-1R in the pathogenesis of type I diabetes.