Macrophages can be both beneficial and detrimental after CNS injury. We previously showed rapid accumulation of macrophages in injured immature brain acutely after ischemia-reperfusion. To determine whether these macrophages are microglia or invading monocytes, we subjected post-natal day 7 (P7) rats to transient 3 h middle cerebral artery (MCA) occlusion and used flow cytometry at 24 and 48 h postreperfusion to distinguish invading monocytes (CD45high/ CD11b+) from microglia (CD45low/medium/CD11b+). Inflammatory cytokines and chemokines were determined in plasma, injured and contralateral tissue 1-24 h post-reperfusion using ELISA-based cytokine multiplex assays. At 24 h, the number of CD45+/CD11b+ cells increased 3-fold in injured compared to uninjured brain tissue and CD45 expression shifted from low to medium with less than 10% of the population expressing CD45high. MCA occlusion induced rapid and transient asynchronous increases in the pro-inflammatory cytokine IL-b and chemokines cytokine-induced neutrophil chemoattractant protein 1 (CINC-1) and monocyte-chemoattractant protein 1 (MCP-1), first in systemic circulation and then in injured brain. Double immunofluorescence with celltype specific markers showed that multiple cell types in the injured brain produce MCP-1. Our findings show that despite profound increases in MCP-1 in injured regions, monocyte infiltration is low and the majority of macrophages in acutely injured regions are microglia. Keywords: cytokines, flow cytometry, inflammation, macrophage, microglia, neonatal stroke. Inflammation is a significant contributing factor to neurodegenerative diseases. Depending on injury setting, the relative involvement of systemic and local inflammation, and communication between the two compartments can vary (Carson and Sutcliffe 1999;Baggiolini 2001;Perry 2004). It is well known that stroke in adult triggers a robust inflammatory reaction, largely involving an influx of peripheral leukocytes into the brain parenchyma (for reviews, see Feuerstein et al. 1997;Dirnagl et al. 1999;Han and Yenari 2003) and disruption of the blood-brain barrier (BBB) (Gidday et al. 2005). In the adult, neutrophils are typically the first leukocyte type to infiltrate (Barone et al. 1991;Garcia et al. 1994;Matsuo et al. 1995) and are followed by macrophages and lymphocytes (Barone et al. 1991;Garcia et al. 1994). The damaging role of infiltrating neutrophils in ischemia was shown by studies in which neutrophil depletion (Garcia et al. 1994) or administration of anti-adhesion molecules (Kishimoto and Rothlein 1994;Fassbender et al. 1999) reversed the reduced local tissue perfusion, BBB disruption, and the release of free radicals, proteinases, and other cytotoxins seen after stroke. The key role of macrophages in cerebral ischemic injury has also been firmly established (Feuerstein et al. 1997;Dirnagl et al. 1999;Han and Yenari 2003). Macrophage populations in the injured brain, however, are diverse (Carson et al. 1998;Dalmau et al. 2003) Abbreviations used: BBB, blood-bra...

The incidence of neonatal stroke is high and currently there are no strategies to protect the neonatal brain from stroke or reduce the sequelae. Agents capable of modifying inflammatory processes hold promise. We set out to determine whether delayed administration of one such agent, minocycline, protects the immature brain in a model of transient middle cerebral artery (MCA) occlusion in 7-day-old rat pups. Injury volume in minocycline (45 mg/kg/dose, beginning at 2 h after MCA occlusion) and vehicle-treated pups was determined 24 h and 7 days after onset of reperfusion. Accumulation of activated microglia/macrophages, phosphorylation of mitogen-activated protein kinase (MAPK) p38 in the brain, and concentrations of inflammatory mediators in plasma and brain were determined at 24 h. Minocycline significantly reduced the volume of injury at 24 h but not 7 days after transient MCA occlusion. The beneficial effect of minocycline acutely after reperfusion was not associated with changed ED1 phenotype, nor was the pattern of MAPK p38 phosphorylation altered. Minocycline reduced accumulation of IL-1beta and CINC-1 in the systemic circulation but failed to affect the increased levels of IL-1beta, IL-18, MCP-1 or CINC-1 in the injured brain tissue. Therefore, minocycline provides early but transient protection, which is largely independent of microglial activation or activation of the MAPK p38 pathway.

The objective of this study was to establish the developmental pattern and tissue specificity of porcine myostatin expression and to evaluate expression in skeletal muscle during circumstances in which muscle growth was altered. Northern blot analysis revealed two transcripts (1.5 and 0.8 kb). Myostatin mRNA was detected in whole fetuses at 21 and 35 days and was markedly increased ( P < 0.05) by 49 days. At birth, mRNA abundance in longissimus muscle had declined significantly ( P < 0.05) from that at day 105 of gestation and continued to decrease ( P < 0.05) to its lowest level 2 wk postnatally (4 kg body wt). Myostatin expression was higher ( P < 0.05) at 55, 107, and 162 kg body wt than at 4 kg body wt. Postnatally, myostatin mRNA was detected in skeletal muscle and mammary gland. Expression at birth was 65% higher ( P < 0.04) in longissimus muscle of low-birth-weight piglets (0.57 ± 0.052 kg body wt) vs. normal (1.37 ± 0.077 kg body wt) littermates, irrespective of gender. However, suppression of longissimus muscle growth by food deprivation (3 days) did not alter ( P > 0.15) myostatin expression in either 4- or 7-wk-old piglets. Additionally, myostatin mRNA abundance was not changed by porcine growth hormone administration in growing animals. These data indicate that myostatin expression in skeletal muscle peaks prenatally and that greater expression is associated with low birth weight. Expression in mammary gland indicates a possible role for myostatin in mammary gland development and/or lactation.

Leptin, the product of the ob gene, is secreted from white adipocytes and regulates food intake and whole-body energy metabolism. In rodents and humans, leptin gene expression is under complex endocrine and metabolic control, and is strongly influenced by energy balance. Growth hormone (GH) has myriad effects on adipose tissue metabolism. The primary aim of this study was to determine the ability of GH to regulate leptin mRNA expression in bovine adipose tissue in vitro and in vivo. Incubation of subcutaneous adipose tissue explants for 24 h with GH alone had no effect on bovine leptin gene expression, whereas high concentrations of insulin or dexamethasone (DEX) potently stimulated bovine leptin mRNA abundance. GH, in combination with high concentrations of insulin, DEX, or both, attenuated the ability of insulin or DEX to stimulate leptin expression in vitro.These data indicate that GH can indirectly regulate leptin expression in vitro by altering the adipose tissue response to insulin or DEX. We extended these studies to examine the ability of GH to regulate leptin expression in vivo, using young castrate male cattle treated with no hormone (control) or GH (200 µg/kg body weight per day) for 3 days. GH increased plasma GH and insulin concentrations, but not those of cortisol or non-esterified fatty acid (NEFA) concentrations. GH treatment increased adipose tissue leptin and IGF-1 mRNA concentrations (n=9, P>0·001). In addition, leptin abundance was highly correlated with adipose tissue IGF-1 mRNA in GH-treated animals (P>0·001). The timing of GHinduced changes in leptin gene expression preceded measurable GH effects on adiposity.

We have evaluated the effect of feeding ractopamine (Rac), a phenethanolamine lean enhancer being developed for commercial use in finishing pigs, on beta-adrenoceptor (beta-AR) number and ligand-receptor binding affinity in adipose and muscle tissues. Pigs weighing 78 +/- 1 kg were fed Rac (20 mg/kg of diet) for 0 (control), 1, 8, or 24 d before being killed at 105 +/- 1 kg BW. beta-adrenoceptor density (per milligram of protein) was decreased by Rac up to approximately 50% in both the middle and the outer layers of subcutaneous (SQ) adipose tissue. Orthogonal contrasts indicated significant (P < or = .05) linear effects of Rac in middle and outer SQ adipose tissue, and also a significant (P < or = .05) quadratic effect of Rac in the middle layer. Ractopamine did not affect the maximal binding (Bmax) of longissimus muscle. The relative affinity with which the beta-AR population of the tissues examined bound the radioligand ([3H]dihydroalprenolol) was not influenced by Rac. Likewise, feeding Rac had no effect on the affinity of the beta-AR for Rac. The data indicate that a Rac-induced reduction in the Bmax of adipose tissue may account for the diminished in vitro lipolytic potency of exogenous Rac after prolonged periods of Rac feeding, and that Rac-induced desensitization differs between adipose and skeletal muscle tissues.