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.

The product of the leptin (i.e., obese) gene may be an important regulator of energy metabolism, adiposity, and reproduction, and is perhaps linked to meat quality determinants such as marbling. Molecular probes were developed using polymerase chain reaction (PCR) technology to evaluate leptin expression in adipose depots and to evaluate the tissue-dependent nature of expression reported in other species. A 438 bp fragment representing the coding region of the bovine leptin gene excluding the N-terminal secretory signal was amplified, cloned into a plasmid vector (pASK75), and expressed in E. coli. Sequence analysis of the cDNA and the corresponding polypeptide indicate that, overall, both share approximately 87% homology with the mouse and human leptin genes and polypeptides. Amino terminal sequencing (30 amino acid residues) of the recombinant bovine leptin (rBL) protein revealed 100% homology with mouse and human leptin. The bovine leptin gene is expressed as a 3,090 nt mRNA which is detected in adipose tissue, but is not found in brain (despite the appreciable fat content and lipid metabolism) or other tissues. Leptin gene expression in several adipose depots (subcutaneous, renal, and omental) was similar (P = .73) in finished cattle.

The product of the obese gene, leptin, may be an important regulator of adiposity via its regulation of feed intake and energy metabolism. Probes were developed using the polymerase chain reaction to analyze gene expression and determine the structure of the porcine ob gene. Porcine ob was expressed in adipose tissue as a 3,100 bp mRNA. Finished pigs (136 kg) had higher (P<.01) levels of ob mRNA (per unit of 6-actin mRNA) in subcutaneous adipose tissue than did growing pigs (60 kg). Obese gene expression was not detected in tissues other than adipose depots. A genomic DNA fragment containing the ob gene was isolated from a cosmid DNA library. Sequence analysis indicates that the ob gene has three exons. A short untranslated sequence was identified as exon 1 and the amino acid coding sequence was located in the second and third exons. The gene structure, intron/exon boundaries, and the amino acid sequence was highly conserved in mammalian species. The porcine leptin amino acid sequence was 95%, 92% and 89% similar to cattle, human and mouse leptin sequences, respectively.

The relationship between obese gene expression and energy intake was determined in pigs of various body weights. With ad libitum consumption, expression increased (P < 0.001) with body weight from 55 to 163 kg. Obese mRNA relative abundance was correlated with fat mass (r = 0.74, P < 0.0001) and percentage of fat (r = 0.72, P < 0. 0001). Obese expression was also evaluated at 159 kg (initial weight) and ad libitum, maintenance or 23% of maintenance intake for 28 d. Obese mRNA was independent of treatment (P > 0.78) despite considerable weight differences. Obese mRNA abundance was then compared at 136 kg (initial weight) and ad libitum or maintenance intake for 3 or 28 d. Abundance was not influenced by either duration of treatment or intake, despite a small increase (P < 0.01) in serum nonesterified fatty acids (NEFA) and a reduction (P < 0.02) in insulin attributable to maintenance intake. Finally, mRNA abundance was determined at 60 and 136 kg and conditions of food deprivation or ad libitum intake for 3 d. Food deprivation reduced (P < 0.01) serum insulin and increased (4- to 5-fold) NEFA concentrations. Obese mRNA abundance was greater (P < 0.01) in the heavier pigs and was reduced (P < 0.01) by food deprivation. We conclude that obese mRNA abundance in pigs correlates with fat mass and percentage of body fat under conditions of ad libitum intake. Furthermore, obese mRNA abundance is reduced by food deprivation, whereas lesser degrees of intake restriction do not change obese mRNA abundance, even when accompanied by appreciable weight loss.