Optimal neural differentiation and extension of hybrid neuroblastoma cells (NDC) for nerve-target evaluations using a multifactorial approach.

In vitro models of tissues, such as the cornea, represent systems for modeling cell-to-cell interactions and tissue function. The objective of this study was to develop an optimized nerve differentiation medium to incorporate into a 3D in vitro model to study innervation and cell targeting. A hybrid neuroblastoma cell line (NDC) was examined for its ability to differentiate into neurons, produce neurites, and functionally contact target cells. Neuronal differentiation of NDCs was optimized through a combinatorial approach which involved culturing cells in the presence of various extracellular matrices and soluble factors. A serum-free medium containing nerve growth factor (NGF), dimethyl sulfoxide (DMSO), or dexamethasone resulted in the greatest proportion of NDCs demonstrating a neuronal morphology. Similarly, with supplementation of cyclic AMP (cAMP) or NGF, neurite extension was optimized. Combining these factors generated an optimized differentiation and extension medium, relative to the individual components alone. In co-culture with epithelial cells, NDC neurites generated in the optimized medium formed contacts with epithelial targets and produced substance P. Similarly, NDCs seeded into a collagen matrix produced neurites that projected through the matrix to target epithelial cells, promoted epithelial stratification, and increased the rate of epithelial wound healing. As well, differentiated NDCs could target and alter acetylcholine receptor clustering in mouse C2C12 myotubes, demonstrating synaptic plasticity. Our data supports the use of NDCs, in combination with optimized medium, for generating an innervated in vitro model.

Bioactive hydrogel-filament scaffolds for nerve repair and regeneration.

The design of novel biomaterials is crucial for the advancement of tissue engineering in nerve regeneration. In this study we developed and evaluated novel biosynthetic scaffolds comprising collagen crosslinked with a terpolymer of poly(N-isopropylacrylamide) (PNiPAAm) as conduits for nerve growth. These collagen-terpolymer (collagen-TERP) scaffolds grafted with the laminin pentapeptide YIGSR were previously used as corneal substitutes in pigs and demonstrated enhanced nerve regeneration compared to allografts. The purpose of this project was to enhance neuronal growth on the collagen-TERP scaffolds through the incorporation of supporting fibers. Neuronal growth on these matrices was assessed in vitro using isolated dorsal root ganglia as a nerve source. Statistical significance was assessed using a one-way ANOVA. The incorporation of fibers into the collagen-TERP scaffolds produced a significant increase in neurite extension (p<0.05). The growth habit of the nerves varied with the type of fiber and included directional growth of the neurites along the surface of certain fiber types. Furthermore, the presence of fibers in the collagen-TERP scaffolds appeared to influence neurite morphology and function; neurites grown on fibers-incorporated collagen-TERP scaffolds expressed higher levels of Na channels compared to the scaffolds without fiber. Overall, our results suggest that incorporation of supporting fibers enhanced neurite outgrowth and that surface properties of the scaffold play an important role in promoting and guiding nerve regeneration. More importantly, this study demonstrates the potential value of tissue engineered collagen-TERP hybrid scaffolds as conduits in peripheral nerve repair.

Factors associated with assault-related firearm injuries in male adolescents.

PURPOSE: To identify factors associated with an increased prevalence of assault-related firearm injuries in male adolescents. METHODS: This study is a retrospective comparison of two samples of adolescent males from the same geographic localities regarding their involvement in the juvenile justice system (court involvement) and injury status (current or prior firearm injury at the time of the study). The subjects included adolescent male patients admitted to an urban, Level I trauma center for assault-related firearm injuries (court-involved and noncourt- involved, n = 65); and incarcerated juvenile offenders (prior firearm injury and no known firearm injury, n = 267). RESULTS: Two-thirds of the male assault-related pediatric firearm injury victims treated over a two-year period were involved in the juvenile justice system (court involved). Court-involved adolescents were almost 22 times more likely to have sustained an assault-related firearm injury, when compared to noncourt-involved patients with firearm injuries. Additional analysis documented recent substance use and/or involvement in criminal offenses in 82% of the victims. For most of the juvenile offenders (88%), court involvement preceded their injuries. Analysis of the injury patterns revealed an increased prevalence of truncal injuries (injuries to thorax or abdomen) in the court-involved victims, when compared to their noncourt-involved peers (40% and 14% for the court-involved and noncourt-involved samples, respectively; p <.05). Incarceration was associated with a 17-fold increase in the firearm injury prevalence over the court-involved, but not incarcerated, sample. CONCLUSIONS: These results suggest that involvement in substance use and/or the criminal justice system is associated with an increased risk of firearm injuries in male adolescents, and that an increased level of involvement in the juvenile justice system is associated with a concomitant increase in firearm injuries.

The relationship between substance use, drug selling, and lethal violence in 25 juvenile murderers.

The goal of the present study was to determine the relationship between substance use, drug selling, and lethal violence in adolescent male homicide offenders and their victims. The study employed a retrospective review of criminal justice databases and medical examiner records for murders committed by 25 adolescent males incarcerated in the Commonwealth of Virginia juvenile correctional centers from February 1992 to July 1996. The perpetrator sample was 84% African American and 16% white. The average age at the time of the offense was 15.0 years (range = 13.0 to 17.7 years). The victims were 84% male, 60% African American and 32% white. The median victim age was 28.0 years (mean = 34.8, range = 17 months to 75 years). The results indicated that 52% of the murders were committed by juveniles with identified involvement in drug selling, and 28% of the murders were drug-related. Toxicology results indicated recent drug or alcohol use in 27% of the victims; while 74% of the perpetrators reported substance use, 35% indicating daily use. Using discriminant analysis, it was possible to accurately classify 86% of the drug-related murders with the variables of recent victim drug use and perpetrator substance use history. The results indicated that adolescent males involved in the sale and distribution of illegal drugs comprised a significant percentage of those incarcerated for murder. Recent victim drug use and perpetrator substance use may be important variables in identifying drug-related juvenile homicides. These results underscore the link between substance use, drug selling, and lethal violence.

Factors associated with parenting among incarcerated juvenile offenders.

In regard to the injured offender, research indicates that violent victimization represents only one facet of a constellation of associated risks and consequences, including promiscuity and adolescent parenthood. A relationship between firearm injuries and self-reported promiscuity among incarcerated juvenile offenders has previously been noted. The present study was an attempt to gain additional insight into the larger consequences of violent injuries. Information pertaining to the fathering of children was collected from 258 incarcerated male adolescents from the Richmond, Virginia, metropolitan area during a two-year period. It was hypothesized that adolescent parenting would be associated with firearm injuries. The results indicated that 20% of the juvenile offenders fathered at least one child. Analyses revealed a significant relationship between firearm injuries and increased prevalence of adolescent parenting. Continued involvement in illegal activities, as indicated by a second commitment to a juvenile correctional center, also was associated with increased prevalence of adolescent parenting, while race and involvement in drug selling or violent offending were not. The social and economic implications of these findings, particularly in terms of the health care and social service delivery systems, are discussed.

Analysis of the antinociceptive actions of the kappa-opioid agonist enadoline (CI-977) in neonatal and adult rats: comparison to kappa-opioid receptor mRNA ontogeny.

Earlier reports indicate that kappa-opioid agonists may be especially potent in the formalin test of tonic nociception, and that neonatal rat pups are more sensitive to mu-agonists, when compared to adults. We have assessed the potency of enadoline (CI-977), a novel and selective kappa-opioid agonist, in the formalin and tail-flick nociceptive tests in 3-day-old rat pups and compared their responses to adults in the same tests. In addition, the recent cloning of the kappa-opioid receptor has allowed us to make the first evaluation of the ontogeny of kappa-opioid receptor mRNA in an effort to elucidate a possible mechanism for differences in sensitivity to kappa-opioid agonists. Enadoline was found to be a potent antinociceptive agent in the formalin test; the neonates were eight times more sensitive than the adults. kappa-Opioid receptor mRNA, however, is present in whole brain at adult levels as early as postnatal day 3. Previous studies have shown kappa-opioid receptor levels, as measured by radioligand binding and receptor autoradiography, to be present at postnatal day 3 as well. Consequently, it is unlikely that gross differences in receptor number subserved the modest increase in sensitivity to enadoline observed in the neonates in the formalin test. Enadoline was less potent and less effective in the tail-flick test in the neonates. The adults were similarly insensitive to the antinociceptive effects of enadoline in the tail-flick test. Additional studies indicated that enadoline significantly increased locomotor activity, as assessed by the open-field test, in neonates, while decreasing activity in the adults.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation and developmental expression of a rat cDNA encoding a cysteine-rich zinc finger protein.

A number of cysteine-rich proteins have recently been isolated by homology screening, differential library screens, and association with other proteins. In this report, we describe the isolation of the rat cysteine-rich protein from a rat brain library during a search for clones with homology to the delta-opioid receptor. One of the cDNAs isolated hybridized to a 1.8 kb mRNA abundantly expressed throughout the rat brain as well as in rat liver. In situ hybridization reveals a wide distribution in rat brain; in particular, abundant hybridization was detected in the hippocampus, cerebellum, habenula, reticular thalamic nucleus and interposed nucleus. Nucleotide sequence analysis of a 1403 bp cDNA clone indicated 77% identity with the cDNA for human cysteine-rich protein (hCRP), that translates into a 99% identity at the amino acid level. The predicted amino acid sequence suggests four zinc fingers, two of the C4 class and two of the C2HC class. This structural motif is characteristic of members of the LIM domain protein family. The mRNA is serum-inducible in Balb/c 3T3 cells. Additional study suggests that its expression is not induced by either NGF treatment of PC12h pheochromocytoma cells, or inflammation-induced injury in the spinal cord at up to 60 min after injury. It does appear to be developmentally expressed in rat brain, consistent with a potential role in neuronal development. The rat CRP clone will be useful for studying the function of CRPs in rodent models.

A comparison of the antinociceptive effects of opioid agonists in neonatal and adult rats in phasic and tonic nociceptive tests.

Changes in the attitudes about neonatal pain and pain management have recently resulted in increases in the administration of opioids to neonates. Little is known, however, about the relative potencies of the various opioid agonists employed, especially in comparison to adult responses. The first objective in the present study was to compare the antinociceptive potency of four clinically relevant opioids in neonatal and adult rats. The second objective was to compare and contrast these agents in two different types of nociceptive tests: tonic (formalin-induced inflammation) and phasic (tail flick and hot plate). Our results indicate that the opioid agonists morphine, meperidine, and fentanyl, and the mixed agonist buprenorphine were all effective antinociceptive agents in both neonates and adults in each of the three tests employed, and that the relative potencies of these agents appeared to be similar in neonates and adults. In general, the pups were more sensitive to the antinociceptive agents when tested in the phasic nociceptive tests, and the drugs were more potent in the tonic test than either of the phasic tests.

Cannabinoid receptors in developing rats: detection of mRNA and receptor binding.

Despite a large body of research directed at assessing the effects of perinatal cannabinoid exposure, little is known about the development of the cannabinoid receptor. Recent advances, including the cloning of the cannabinoid receptor, have afforded us the opportunity to plot the postnatal ontogeny of the cannabinoid receptor and its mRNA in whole brain using the methods of receptor binding and RNA blot hybridization, respectively. Our results indicate that cannabinoid receptor mRNA is present at adult levels as early as postnatal day 3. The Bmax, on the other hand, increases almost fifty percent with increasing postnatal age, while the affinity does not change. The Hill coefficients for all ages studied were approximately 1. These findings suggest the possibility of a developmental progression for cannabinoid receptor development with receptor mRNA appearing first, followed by a period of rapid proliferation of the receptors themselves.

Developmental expression of cannabinoid receptor mRNA.

Cloning of the cannabinoid receptor affords the opportunity to examine its developmental expression. Other G-protein-coupled receptor systems, those for the opioids for example, exhibit distinct ontogenies. For the initial study, therefore, cannabinoid receptor mRNA expression was assessed in rat pups postnatal days 3, 5, 8, 10, 12, 15, 18 and 21. The brains were grossly dissected into cerebellum/brainstem and forebrain, and total RNA was extracted by a modified acid-extraction method. Expression of the cannabinoid receptor was analyzed by two methods: polymerase chain reaction (PCR) and Northern blot analysis. Oligonucleotide primers based on bp 1-21 and bp 824-843 on the opposite strand were chosen for use in the PCR. The probe used in the Northern blot analysis was a full length cDNA corresponding to the rat cannabinoid receptor and was cloned in our lab based on published sequence information. Our results indicate that by postnatal day 3, cannabinoid receptor mRNA can be detected in the brain. Our results further indicate that cannabinoid mRNA expression steadily increases in the cerebellum/brainstem until postnatal days 18-21, while expression in the forebrain does not change. The findings from the present study indicate that cannabinoid receptor mRNA is present in very young rats. Our data also suggest, however, regional differences in the relative expression of message which may parallel cerebellar proliferation and organization.

Neonatal pain: a comprehensive survey of attitudes and practices.

We surveyed 352 physicians board certified in neonatal-perinatal medicine on their attitudes and practices in the area of pain and pain management in neonates and infants. In contrast to earlier surveys of this type, almost all respondents indicated that even the youngest and most premature infants are able to perceive pain, and most reported that they always advocated anesthesia during the intraoperative period. The use of analgesic agents in the postoperative period, however, was more variable. Respondents who indicated that neonates perceived less pain than adults reported seeing fewer signs of pain and using less analgesia in the postoperative period. They were also more likely to believe that analgesics are too dangerous to use in neonates and that physiologic factors such as incomplete myelination of the pain pathways and neural/physical immaturity (factors now known not to play a role) contribute to diminished pain sensitivity. Conversely, respondents who indicated that neonates do not perceive less pain than adults, the majority of respondents, reported seeing more signs of pain and using more medication in the postoperative period. These physicians also believed that the physiologic stress associated with pain can be more dangerous than the analgesics. We conclude that attitudes and reported practices have changed in the area of neonatal pain and pain management. Furthermore, our data indicate that these attitudes significantly predict reported postoperative medicating practices.

Tonic nociception in neonatal rats.

The issues of whether infants detect noxious stimuli and whether their nociceptive responses are suppressed by analgesics has been the focus of considerable controversy. Therefore, to more completely assess the nociceptive responses of neonatal rat pups to tonic pain, we tested 3-day-old rat pups using the formalin test. The responses of the young pups to formalin-produced injury were similar to those observed in adult rats, both behaviorally and in terms of their responsivity to morphine-induced antinociception. These results provide the first clear-cut evidence of integrated tonic pain responses in the neonate.

The ontogenesis of lithium-induced effects on suckling: inhibition and facilitation.

The ontogenesis of the effect of lithium on suckling behavior was assessed by administering lithium carbonate directly and acutely to 15-, 20-, 30-, and 35-day-old rat pups. Lithium significantly interfered with nipple attachment in 15-day-old rat pups in a dose-dependent pattern, but it facilitated attachment at some doses (40, 60, 80 mg/kg) in weanling-age rat pups. Furthermore, lithium pretreatment reversed quipazine-induced interference of attachment in weanling-age rats. These effects are similar to those previously reported with serotonergic antagonists, suggesting a similar mechanism, perhaps via the inositol phosphate second messenger system.

Pro-Leu-Gly-NH2 serves as a conditioned stimulus in the acquisition of conditioned tolerance.

The effect of Pro-Leu-Gly-NH2 (MIF) on the acquisition of tolerance to morphine-induced antinociception and its efficacy as a cue predictive of morphine administration was examined. Daily administration of MIF prior to morphine injection did not attenuate the acquisition of tolerance to the antinociceptive properties of morphine, as measured by the latency to hindpaw lick in a hot-plate test of analgesia. When the animals were tested 72 hr later without MIF pretreatment, they appeared to lose tolerance, as indicated by longer latencies to paw lick. These data suggest that in some situations MIF may interfere with the acquisition of tolerance by acting as a cue that reliably predicts the antinociceptive properties of morphine.

Acute administration of lithium carbonate interferes with suckling in neonatal rats.

These studies provide an animal model for the lithium-induced decrease in suckling reported in the clinical literature that allows for more precise determination of causal mechanisms. Nine-day-old rat pups were administered lithium carbonate via either intraperitoneal (IP) injections or intragastric (IG) gavage in doses approximating that which human infants might receive via breast milk. The pups were tested for their ability to locate and attach to the nipples of an anesthetized dam. Lithium significantly increased the pups' latency to attach to a nipple. Further tests of milk extraction using oxytocin-induced milk-letdowns indicate that lithium also interferes with milk withdrawal. Tests of motor and sensory deficits using an open-field and an olfactory choice test indicated that lithium did not similarly impair these behavioral facets of suckling. Alternative mechanisms for lithium-produced suppression of suckling are discussed.

Management uses of the Delphi.

In addition to its simplicity, the Delphi approach to planning and problem solving invites candor and uninhibited response from participants. It may also serve as a bridge to team building within the health care institution.