SLPI expression was increased 3

SLPI expression was increased 3.9-fold in the microarray, making it probably one of the most highly upregulated genes in the analysis. Chen et al., 2000; GrandPr et al., 2000; Prinjha et al., 2000; Wang et al., 2002). One effective strategy for countering these effects has been to manipulate gene manifestation within neurons and therefore confer resistance to myelin-associated inhibitors. The prototypical example of this is the conditioning lesion effect, in which transection of the sciatic nerve 7 d before a dorsal column lesion significantly enhances regeneration of dorsal root ganglion (DRG) central processes (Neumann and Woolf, 1999). Subsequent studies have established that elevation of intracellular cyclic AMP (cAMP) levels and CREB-mediated transcription are required for the conditioning lesion effect (Neumann et al., 2002; Qiu et al., 2002; Gao et al., 2004). To identify genes that are transcribed in response to elevation of cAMP, we performed a microarray analysis, which revealed significantly increased manifestation of secretory leukocyte protease inhibitor (SLPI). SLPI is an 11.7 kDa serine protease inhibitor belonging to the family of whey acidic protein motif-containing proteins (Thompson and Ohlsson, 1986; Eisenberg et al., 1990). It is commonly found in the secretions lining the surfaces of the oral mucosa, bronchial epithelium, and urogenital tract (Thompson and Ohlsson, 1986; Fritz, 1988; Sallenave et al., 1994). Little is known about the function of SLPI in the nervous system; however, two studies possess found that SLPI manifestation is definitely improved after cerebral ischemia. SLPI was strongly induced in neurons, astrocytes, and microglia after middle cerebral artery occlusion (MCAO) in the rat (Wang et al., 2003), and related raises in SLPI levels were reported in the sera of human being stroke individuals (I?zecka and Stelmasiak, 2002). More importantly, adenoviral manifestation of SLPI in the cerebral cortex before MCAO significantly reduced infarct size, which suggests that SLPI may be neuroprotective (Wang et al., 2003). This hypothesis is definitely supported by a recent study by Ghasemlou et al. (2010), which reported that treatment with SLPI prospects to improved locomotor Pax6 recovery, decreased lesion volume, and reduced myelin loss 1 week after spinal cord contusion. Here we describe a new part for SLPI in axonal regeneration. We statement that administration of exogenous SLPI overcomes MAG inhibition for a number of neuronal populations and that this effect can be clogged by overexpression of Smad2. Materials and Methods All animal methods were authorized by the Institutional Animal Care and Use Committee of Hunter College, City University or college of New York, and the Protocol Management and Review Committee of the University or college of Manitoba. The experiments were performed in accordance with all institutional and national regulations. Neuronal preparations. For cortical or hippocampal neurons, cortices and hippocampi were dissected from postnatal day time 1 (P1) LongCEvans rat pups of both sexes and incubated twice with 0.5 mg/ml papain in plain Neurobasal-A media (Invitrogen). Cell suspensions were layered on an Optiprep denseness gradient (Sigma) and centrifuged at 2000 for 15 min. The purified neurons were then collected and counted. For cerebellar granule neurons (CGNs), cerebellar cortex was isolated from P5CP6 rats of both sexes and treated with papain and soybean trypsin inhibitor as explained above. After tritutation, cells were diluted in Sato’s press and counted. For DRG neurons, DRGs were isolated from P5CP6 rats of both sexes and treated with Trolox 0.015% collagenase in Neurobasal-A media for 45 min at 37C. This was followed by a second incubation in collagenase for 30 min at 37C, with the help of 0.1% trypsin and 50 g/ml DNase I. Trypsin was inactivated with DMEM comprising 10% dialyzed fetal bovine serum, and the ganglia were triturated in Sato’s press. Microarray analysis and quantitative real-time PCR. For the RNA preparations, P21CP23 LongCEvans rats of both sexes were anesthetized with isoflurane, and the right sciatic nerve was transected in the midpoint of the thigh. Animals were killed 18 h later on, and the lesioned and unlesioned L4 and L5 DRGs were collected and snap freezing. P5 DRG neurons were also prepared as explained and incubated for 18 h at 37C in the presence or absence of 1.5 mm dibutyryl cAMP (dbcAMP). In both cases, the cells were homogenized in TRIzol (Invitrogen), and RNA was purified using the RNeasy RNA isolation kit (Qiagen). Microarray hybridization and quantitative real-time PCR were then performed as explained previously (Cao et al., 2006). The full results of the microarray can be viewed on the following website: http://genome.rutgers.edu/slpi/. Endpoint PCR. Neonatal rat CGNs, DRGs, and cortical.Recently, we reported that upregulation of arginase I-mediated polyamine synthesis contributes to the conditioning lesion effect and that polyamines can promote axonal regeneration (Deng et al., 2009). et al., 1994; Trolox Mukhopadhyay et al., 1994; Chen et al., 2000; GrandPr et al., 2000; Prinjha et al., 2000; Wang et al., 2002). One effective strategy for countering these effects has been to manipulate gene expression within neurons and thereby confer resistance to myelin-associated inhibitors. The prototypical example of this is the conditioning lesion effect, in which transection of the sciatic nerve 7 d before a dorsal column lesion significantly enhances regeneration of dorsal root ganglion (DRG) central processes (Neumann and Woolf, 1999). Subsequent studies have established that elevation of intracellular cyclic AMP (cAMP) levels and CREB-mediated transcription are required for the conditioning lesion effect (Neumann et al., 2002; Qiu et al., 2002; Gao et al., 2004). To identify genes that are transcribed in response to elevation of cAMP, we performed a microarray analysis, which revealed significantly increased expression of secretory leukocyte protease inhibitor (SLPI). SLPI is an 11.7 kDa serine protease inhibitor belonging to the family of whey acidic protein motif-containing proteins (Thompson and Ohlsson, 1986; Eisenberg et al., 1990). It is commonly found in the secretions lining the surfaces of the oral mucosa, bronchial epithelium, and urogenital tract (Thompson and Ohlsson, 1986; Fritz, 1988; Sallenave et al., 1994). Little is known about the function of SLPI in the nervous system; however, two studies have found that SLPI expression is usually increased after cerebral ischemia. SLPI was strongly induced in neurons, astrocytes, and microglia after middle cerebral artery occlusion (MCAO) in the rat (Wang et al., 2003), and comparable increases in SLPI levels were reported in the sera of human stroke patients (I?zecka and Stelmasiak, 2002). More importantly, adenoviral expression of SLPI in the cerebral cortex before MCAO significantly reduced infarct size, which suggests that SLPI may be neuroprotective (Wang et al., 2003). This hypothesis is usually supported by a recent study by Ghasemlou et al. (2010), which reported that treatment with SLPI prospects to improved locomotor recovery, decreased lesion volume, and reduced myelin loss 1 week after spinal cord contusion. Here we describe a new role for SLPI in axonal regeneration. We statement that administration of exogenous SLPI overcomes MAG inhibition for several neuronal populations and that this effect can be blocked by overexpression of Smad2. Materials and Methods All animal procedures were approved by the Institutional Animal Care and Use Committee of Hunter College, City University or college of New York, and the Protocol Management and Review Committee of the University or college of Manitoba. The experiments were performed in accordance with all institutional and national regulations. Neuronal preparations. For cortical or hippocampal neurons, cortices and hippocampi were dissected from postnatal day 1 (P1) LongCEvans rat pups of both sexes and incubated twice with 0.5 mg/ml papain in plain Neurobasal-A media (Invitrogen). Cell suspensions were layered on an Optiprep density gradient (Sigma) and centrifuged at 2000 for 15 min. The purified neurons were then collected and counted. For cerebellar granule neurons (CGNs), cerebellar cortex was isolated from P5CP6 rats of both sexes and treated with papain and soybean trypsin Trolox inhibitor as explained above. After tritutation, cells were diluted in Sato’s media and counted. For DRG neurons, DRGs were isolated from P5CP6 rats of both sexes and treated with 0.015% collagenase in Neurobasal-A media for 45 min at 37C. This was followed by a second incubation in collagenase for 30 min at 37C, with the addition of 0.1% trypsin and 50 g/ml DNase I. Trypsin was inactivated with DMEM made up of 10% dialyzed fetal bovine serum, and the ganglia were triturated in Sato’s media. Microarray analysis and quantitative real-time PCR. For the RNA preparations, P21CP23 LongCEvans rats of both sexes were anesthetized with isoflurane,.