Glial cell activity is maintained during prolonged inflammatory challenge in rats

We evaluated the expression of glial fibrillary acidic protein (GFAP), glutamine synthetase (GS), ionized calcium binding adaptor protein-1 (Iba-1), and ferritin in rats after single or repeated lipopolysaccharide (LPS) treatment, which is known to induce endotoxin tolerance and glial activation. Male Wistar rats (200-250 g) received ip injections of LPS (100 µg/kg) or saline for 6 days: 6 saline (N = 5), 5 saline + 1 LPS (N = 6) and 6 LPS (N = 6). After the sixth injection, the rats were perfused and the brains were collected for immunohistochemistry. After a single LPS dose, the number of GFAP-positive cells increased in the hypothalamic arcuate nucleus (ARC; 1 LPS: 35.6 ± 1.4 vs control: 23.1 ± 2.5) and hippocampus (1 LPS: 165.0 ± 3.0 vs control: 137.5 ± 2.5), and interestingly, 6 LPS injections further increased GFAP expression in these regions (ARC = 52.5 ± 4.3; hippocampus = 182.2 ± 4.1). We found a higher GS expression only in the hippocampus of the 6 LPS injections group (56.6 ± 0.8 vs 46.7 ± 1.9). Ferritin-positive cells increased similarly in the hippocampus of rats treated with a single (49.2 ± 1.7 vs 28.1 ± 1.9) or repeated (47.6 ± 1.1 vs 28.1 ± 1.9) LPS dose. Single LPS enhanced Iba-1 in the paraventricular nucleus (PVN: 92.8 ± 4.1 vs 65.2 ± 2.2) and hippocampus (99.4 ± 4.4 vs 73.8 ± 2.1), but had no effect in the retrochiasmatic nucleus (RCA) and ARC. Interestingly, 6 LPS increased the Iba-1 expression in these hypothalamic and hippocampal regions (RCA: 57.8 ± 4.6 vs 36.6 ± 2.2; ARC: 62.4 ± 6.0 vs 37.0 ± 2.2; PVN: 100.7 ± 4.4 vs 65.2 ± 2.2; hippocampus: 123.0 ± 3.8 vs 73.8 ± 2.1). The results suggest that repeated LPS treatment stimulates the expression of glial activation markers, protecting neuronal activity during prolonged inflammatory challenges.

Glial cell activity is maintained during prolonged inflammatory challenge in rats.

We evaluated the expression of glial fibrillary acidic protein (GFAP), glutamine synthetase (GS), ionized calcium binding adaptor protein-1 (Iba-1), and ferritin in rats after single or repeated lipopolysaccharide (LPS) treatment, which is known to induce endotoxin tolerance and glial activation. Male Wistar rats (200-250 g) received ip injections of LPS (100 µg/kg) or saline for 6 days: 6 saline (N = 5), 5 saline + 1 LPS (N = 6) and 6 LPS (N = 6). After the sixth injection, the rats were perfused and the brains were collected for immunohistochemistry. After a single LPS dose, the number of GFAP-positive cells increased in the hypothalamic arcuate nucleus (ARC; 1 LPS: 35.6 ± 1.4 vs control: 23.1 ± 2.5) and hippocampus (1 LPS: 165.0 ± 3.0 vs control: 137.5 ± 2.5), and interestingly, 6 LPS injections further increased GFAP expression in these regions (ARC = 52.5 ± 4.3; hippocampus = 182.2 ± 4.1). We found a higher GS expression only in the hippocampus of the 6 LPS injections group (56.6 ± 0.8 vs 46.7 ± 1.9). Ferritin-positive cells increased similarly in the hippocampus of rats treated with a single (49.2 ± 1.7 vs 28.1 ± 1.9) or repeated (47.6 ± 1.1 vs 28.1 ± 1.9) LPS dose. Single LPS enhanced Iba-1 in the paraventricular nucleus (PVN: 92.8 ± 4.1 vs 65.2 ± 2.2) and hippocampus (99.4 ± 4.4 vs 73.8 ± 2.1), but had no effect in the retrochiasmatic nucleus (RCA) and ARC. Interestingly, 6 LPS increased the Iba-1 expression in these hypothalamic and hippocampal regions (RCA: 57.8 ± 4.6 vs 36.6 ± 2.2; ARC: 62.4 ± 6.0 vs 37.0 ± 2.2; PVN: 100.7 ± 4.4 vs 65.2 ± 2.2; hippocampus: 123.0 ± 3.8 vs 73.8 ± 2.1). The results suggest that repeated LPS treatment stimulates the expression of glial activation markers, protecting neuronal activity during prolonged inflammatory challenges.

MHC class I upregulation is not sufficient to rescue neonatal alpha motoneurons after peripheral axotomy.

Associated with neuronal death, profound synaptic changes occur in the spinal cord during the apoptotic process triggered after axotomy in neonatal rats. With respect to this, the major histocompatibility complex of class I (MHC class I) has recently emerged as a new mechanism related to synaptic stripping and plasticity. The present study investigated the impact of upregulating MHC class I expression by treatment with beta interferon (beta INF) on motoneuron survival, synaptic plasticity and astrogliosis after neonatal sciatic nerve injury. P2 rats were subjected to unilateral axotomy followed by three days of beta INF treatment. The results were analyzed by counting Nissl stained motoneurons, immunohistochemistry (anti-synaptophysin, MHC class I, GFAP and Iba-1) and transmission electron microscopy. INF treatment induced an increased expression of MHC class I, which resulted in a stronger synaptic elimination process in the spinal cord, as seen by the synaptophysin labeling. GFAP and Iba-1 upregulation were not significantly altered by the INF treatment, displaying the same degree of enhanced reactivity as compared to the placebo group. The ultrastructural analysis showed that, apart from the overall reduction of inputs in the neuropil, no statistical differences were present when comparing the INF and placebo treated animals. Also, neuronal survival was not altered by cytokine administration. The present results provide evidence that MHC class I upregulation after neonatal injury does not change the fate of lesioned motoneurons. In this way, the lack of neurotrophic support may cause broader synaptic loss, which superposes the more subtle effects of the upregulation of MHC class I.

Mechanisms involved in the acidosis enhancement of the isoproterenol-induced phosphorylation of phospholamban in the intact heart.

Previous experiments have shown that acidosis enhances isoproterenol-induced phospholamban (PHL) phosphorylation (Mundiña-Weilenmann, C., Vittone, L., Cingolani, H. E., Orchard, C. H. (1996) Am. J. Physiol. 270, C107-C114). In the present experiments, performed in isolated Langendorff perfused rat hearts, phosphorylation site-specific antibodies to PHL combined with the quantitative measurement of 32P incorporation into PHL were used as experimental tools to gain further insight into the mechanism involved in this effect. At all isoproterenol concentrations tested (3-300 nM), phosphorylation of Thr17 of PHL was significantly higher at pHo 6.80 than at pHo 7.40, without significant changes in Ser16 phosphorylation. This increase in Thr17 phosphorylation was associated with an enhancement of the isoproterenol-induced relaxant effect. In the absence of isoproterenol, the increase in [Ca]o at pHo 6.80 (but not at pHo 7.40) evoked an increase in PHL phosphorylation that was exclusively due to an increase in Thr17 phosphorylation and that was also associated with a significant relaxant effect. This effect and the phosphorylation of Thr17 evoked by acidosis were both offset by the Ca2+/calmodulin-dependent protein kinase II inhibitor KN-62. In the presence of isoproterenol, either the increase in [Ca]o or the addition of a 1 microM concentration of the phosphatase inhibitor okadaic acid was able to mimic the increase in isoproterenol-induced Thr17 phosphorylation produced by acidosis. In contrast, these two interventions have opposite effects on phosphorylation of Ser16. Whereas the increase in [Ca]o significantly decreased phosphorylation of Ser16, the addition of okadaic acid significantly increased the phosphorylation of this residue. The results are consistent with the hypothesis that the increase in phospholamban phosphorylation produced by acidosis in the presence of isoproterenol is the consequence of two different mechanisms triggered by acidosis: an increase in [Ca2+]i and an inhibition of phosphatases.