Gel matrix vehicles for growth factor application in nerve gap injuries repaired with tubes: a comparison of biomatrix, collagen, and methylcellulose.

The repair of nerve gap injuries with tubular nerve guides has been used extensively as an in vivo test model in identifying substances which may enhance nerve regeneration. The model has also been used clinical nerve repair. The objective of this study was to compare three different gel matrix-forming materials as potential vehicles for growth factors in this system. The vehicles included a laminin containing extracellular matrix preparation (Biomatrix), collagen, and a 2% methylcellulose gel. The growth factor test substance consisted of a combination of platelet-derived growth factor BB (PDGF-BB) and insulin-like growth factor I (IGF-I). An 8-mm gap in rat sciatic nerve was repaired with a silicone tube containing each of the vehicles alone or with a combination of each vehicle plus PDGF-BB and IGF-I. At 4 weeks after injury, the application of the growth factor combination significantly stimulated axonal regeneration when applied in methylcellulose or collagen, but not in Biomatrix. A similar trend was present between the vehicle control groups. By 8 weeks after injury, nerves repaired with methylcellulose as a vehicle had significantly greater conduction velocity than either collagen or Biomatrix. It was concluded that a 2% methylcellulose gel was the best of the three matrices tested, both in its effects on nerve regeneration and flexibility of formulation.

Evidence for hypoxia-induced, programmed cell death of cultured neurons.

Apoptosis, a form of cell death ("programmed" cell death) in which the nucleus and cytoplasm shrink and often fragment, serves to eliminate excessive or unwanted cells during remodeling of embryonic tissues, during organ involution, and in tumor regression. In acute pathological states, such as ischemia, the cells tend to swell and lyse--a process called necrosis. We hypothesize that the delayed neural death clinically associated with hypoxia may, in part, represent apoptosis. A tissue culture model of 24 hours of hypoxia was employed using sympathetic neurons. Pretreatment with an endonuclease inhibitor (aurintricarboxylic acid) decreased cell death by 53%, depolarizing conditions (55 mM potassium chloride) decreased cell death by 33%, and an RNA synthesis inhibitor (actinomycin D) by 26% (all have been shown to prevent apoptosis). Pretreatment with antisense c-myc had no effect. Fluorescent staining with propidium iodide (a DNA marker) demonstrated chromatin condensation and agarose gel electrophoresis demonstrated a DNA "ladder." These data suggest that apoptosis may play a role in hypoxic cell death and that in this paradigm, expression of c-myc is unnecessary. This would suggest a new approach to our understanding of hypoxia and open new strategies to lessen neuronal damage secondary to this process.

Cytokine-induced programmed death of cultured sympathetic neurons.

Programmed cell death (PCD) of sympathetic neurons is inhibited by nerve growth factor. However, factors that induce PCD of these cells are unknown. Leukemia inhibitory factor (LIF) and ciliary neurotrophic factor, neuropoietic cytokines known to regulate sympathetic neuron gene expression, were examined for effects on survival of cultured sympathetic neurons. Treatment with LIF or ciliary neurotrophic factor caused neuronal death in a dose-dependent fashion. Inhibition of RNA or protein synthesis, or treatment with potassium, all of which prevent PCD after nerve growth factor deprivation, prevented LIF-induced death. The morphologic and ultrastructural characteristics of the neuronal death induced by LIF and by nerve growth factor deprivation were similar. Furthermore, LIF treatment resulted in DNA fragmentation with a characteristic "ladder" on Southern blot analysis. These observations suggest that neuron numbers may be regulated by factors which initiate PCD, as well as by factors which prevent it.

Heterogeneity in gap junction expression in astrocytes cultured from different brain regions.

Heterogeneity among astrocytes suggests that their role in the central nervous system is more complex than is commonly recognized. This paper describes just such a functional difference, comparing gap junctions in astrocytes derived from two brain regions. Astrocytes, both in situ and in culture, employ gap junctions as a means of intercellular communication. Recent evidence utilizing cultured rat cortical and striatal astrocytes has shown that these channels consist of subunits of connexin 43, the same protein as that composing cardiac gap junctions. Here we report that astrocytes cultured from neonatal rat hypothalamus contain a greater number of functional channels than astrocytes from the striatum, a difference reflected in both connexin 43 protein and mRNA. Specifically, in hypothalamic astrocytes the level of connexin 43 protein was approximately four times that found in comparable cultures from the striatum, as determined by immunoblotting. Complementary results from immunocytochemical experiments using an antibody specific for connexin 43 reveal significantly greater fluorescence in astrocytes cultured from the hypothalamus as compared to those from the striatum. Northern blot analysis showed that connexin 43 mRNA levels were also approximately 4-fold greater in the hypothalamic cultures, consistent with the difference seen by immunoblotting. Finally, dye coupling studies using confluent cultures consistently showed that within 1 min Lucifer Yellow injected into striatal astrocytes spread to immediately surrounding cells while in hypothalamic astrocytes dye often spread to apparent third or fourth order neighbors within the same time period. Thus, the higher level of connexin 43 expression seen in hypothalamic astrocytes results in cells with greater numbers of functional channels.(ABSTRACT TRUNCATED AT 250 WORDS)

Cultured astrocytes release proenkephalin.

Astrocytes as well as neurons express the mRNA encoding the opioid peptide precursor, proenkephalin. In neurons proenkephalin is cleaved intracellularly to yield smaller, bioactive peptides such as Met-enkephalin and Leu-enkephalin. By contrast, utilizing a combination of radioimmunoassay and chromatographic analysis, we report here that astrocytes cultured from neonatal rat brain contain primarily unprocessed proenkephalin and only small amounts of Met-enkephalin. Further, similar experiments with and without the inclusion of several peptidase inhibitors indicate that cultured astrocytes release proenkephalin itself into the medium where it may be subsequently cleaved to smaller peptide products. The release of intact proenkephalin by astrocytes suggests that the glial propeptide subserves a different function than neuronal proenkephalin and that opioid peptides may play novel roles in the central nervous system.

Region-specific regulation of preproenkephalin mRNA in cultured astrocytes.

Regulation of preproenkephalin (PPE) mRNA was examined in astrocytes cultured from several regions of the neonatal rat brain. Astrocytes from these regions expressed differing levels of PPE mRNA, with higher levels in astrocytes from the hypothalamus followed by frontal cortex and striatum. Further, PPE mRNA was regulated differently in hypothalamic than in striatal glia. Treatment of striatal astrocytes with the beta-adrenergic agonist, isoproterenol, or with agents which directly increased intracellular cAMP (forskolin or 8-bromo-cAMP) elevated levels of PPE mRNA. By contrast, none of these treatments altered levels of PPE mRNA in hypothalamic astrocytes despite increasing cAMP levels 60-fold. These observations indicate that there is striking regional heterogeneity in the expression and regulation of PPE mRNA by astrocytes, suggesting that proenkephalin or its derived peptides help to mediate region-specific brain functions.

The complete nucleotide sequence and structure of the gene encoding bovine phenylethanolamine N-methyltransferase.

A cDNA clone for bovine adrenal phenylethanolamine N-methyltransferase (PNMT) was used to screen a Charon 28 genomic library. One phage was identified, designated lambda P1, which included the entire PNMT gene. Construction of a restriction map, with subsequent Southern blot analysis, allowed the identification of exon-containing fragments. Dideoxy sequence analysis of these fragments, and several more further upstream, indicates that the bovine PNMT gene is 1,594 base pairs in length, consisting of three exons and two introns. The transcription initiation site was identified by two independent methods and is located approximately 12 base pairs upstream from the ATG translation start site. The 3' untranslated region is 88 base pairs in length and contains the expected polyadenylation signal (AATAAA). A putative promoter sequence (TATA box) is located about 25 base pairs upstream from the transcription initiation site. Computer comparison of the nucleotide sequence data with the consensus sequences of known regulatory elements revealed potential binding sites for glucocorticoid receptors and the Sp1 regulatory protein in the 5' flanking region of the gene. Additionally, comparison of the sequence of the exons of the PNMT gene with cDNA sequences for other enzymes involved in biogenic amine synthesis revealed no significant homology, indicating that PNMT is not a member of a multigene family of catecholamine biosynthetic enzymes.

Purification and partial amino acid sequence of bovine adrenal phenylethanolamine N-methyltransferase: a comparison of nucleic acid and protein sequence data.

Recently, we have reported the isolation and characterization of a putative genomic DNA clone encoding bovine adrenal phenylethanolamine N-methyltransferase (PNMT) (Batter et al., 1988). However, the lack of primary amino-acid sequence data for this enzyme precluded a definitive proof of the authenticity of this clone. To establish identity, the amino acid sequence of several peptides generated by chemical and enzymatic hydrolysis of purified PNMT was compared to that predicted from the nucleotide sequence of the exons of the putative PNMT gene. Bovine adrenomedullary PNMT was purified by ammonium sulfate precipitation, gel filtration, and ion exchange chromatography. Treatment with 70% formic acid cleaved the protein at a single Asp-Pro bond near the N-terminus. The purified protein was also cleaved at a single methionine residue near the C-terminus by treatment with cyanogen bromide. N-terminal amino acid sequence analysis identified 8 and 10 amino acid residues, respectively, following each of the scissile peptide bonds. Four tryptic peptides, generated by complete enzymatic digestion, were isolated by reverse-phase HPLC and subjected to sequence analysis. Combined, the amino acid sequences of these six peptides represent 20% of the PNMT protein. These amino acid sequences matched exactly the sequences predicted from the exons of the putative PNMT genomic clone.

Sulfanilic acid: behavioral change related to azo food dyes in developing rats.

The effects of sulfanilic acid, a major azo food dye metabolite, were studied in normal developing rat pups and pups treated with 6-hydroxydopamine (60HDA). Chronic daily intraperitoneal injection of sulfanilic acid during the first postnatal month elicited hyperactivity and impaired shock escape performance in vehicle pups. No differences were noted in 60HDA treated rat pups receiving sulfanilic acid. These findings, which are similar to the results of our study of chronic administration of a food dye mix, suggest that sulfanilic acid may be one of the causative agents in food dye-induced behavioral changes in developing rats. While our work suggests a significant effect of azo food dyes on the developing rat central nervous system, species differences in parameters such as absorption, metabolism, and blood-brain barrier properties do not permit any extrapolation of these observations to proposed effects in children.

Effects of bromocriptine in developing rat pups after 6-hydroxydopamine.

The effects of low (0.5 mg/kg) and high (2.0 mg/kg) doses of bromocriptine (BCR) on activity and escape performance were examined during the first month of postnatal life in normal developing rat pups and littermates treated at 5 days of age with a combination of desmethylimipramine and 6-hydroxydopamine (6-OHDA). Such a procedure resulted in significant reductions in brain dopamine to concentrations 10-20% of vehicle controls while norepinephrine was unaffected. BCR increased general motor activity in vehicle pups at 13 and 19 days but had little effect on more mature animals. Pups who had not received BCR exhibited a decline in activity over the hour long observation period (habituation of activity) but this decline was abolished by both low (0.5 mg/kg) and high (2.0 mg/kg) doses of the agent. Stereotyped activity, particularly at 19 days was increased by BCR in 6-OHDA pups but not in vehicle animals, an effect suggesting denervation supersensitivity. Head dips in a hole box at 30 days of age were not influenced by BCR in vehicle pups but significantly reduced by BCR in 6-OHDA pups, suggesting that BCR might be acting to stimulate inhibitory dopaminergic mechanisms. Escape learning in a T-maze at 20 days and shuttle box at 28 days was disrupted by high doses of BCR in vehicle pups and both doses of BCR in 6-OHDA animals. The similarity with the behaviors observed in the clinical syndrome of attention deficit disorder with hyperactivity prompted a number of investigative groups including our own to suggest that the 6-OHDA model might serve as a useful and convenient paradigm to evaluate pharmacological agents that offer potential in the treatment of this most common disorder. From this perspective we would predict that BCR would have little clinical utility since it both failed to attenuate 6-OHDA induced hyperactivity and tended to disrupt performance in an avoidance learning task.

Determination of indoles and catechols in rat brain and pineal using liquid chromatography with fluorometric and amperometric detection.

Tryptophan, serotonin, 5-hydroxyindoleacetic acid, and homovanillic acid were determined in rat brain by the direct injection of a centrifuged tissue homogenate into a liquid chromatographic-fluorometric/amperometric system. The above indoles, along with melatonin, were also determined in single rat pineal glands. The utility of the system in determining several additional catechols and idoles in brain was examined.