Development of the Nursing Community Apgar Questionnaire (NCAQ): a rural nurse recruitment and retention tool.

INTRODUCTION: Health professional shortages are a significant issue throughout the USA, particularly in rural communities. Filling nurse vacancies is a costly concern for many critical access hospitals (CAH), which serve as the primary source of health care for rural communities. CAHs and rural communities have strengths and weaknesses that affect their recruitment and retention of rural nurses. The purpose of this study was to develop a tool that rural communities and CAHs can utilize to assess their strengths and weaknesses related to nurse recruitment and retention. METHODS: The Nursing Community Apgar Questionnaire (NCAQ) was developed based on an extensive literature review, visits to multiple rural sites, and consultations with rural nurses, rural nurse administrators and content experts. RESULTS: A quantitative interview tool consisting of 50 factors that affect rural nurse recruitment and retention was developed. The tool allows participants to rate each factor in terms of advantage and importance level. The tool also includes three open-ended questions for qualitative analysis. CONCLUSIONS: The NCAQ was designed to identify rural communities' and CAHs' strengths and challenges related to rural nurse recruitment and retention. The NCAQ will be piloted and a database developed for CAHs to compare their results with those in the database. Furthermore, the NCAQ results may be utilized to prioritize resource allocation and tailor rural nurse recruitment and retention efforts to highlight a community's strengths. The NCAQ will function as a useful real-time tool for CAHs looking to assess and improve their rural nurse recruitment and retention practices and compare their results with those of their peers. Longitudinal results will allow CAHs and their communities to evaluate their progress over time. As the database grows in size, state, regional, and national results can be compared, trends may be discovered and best practices identified.

Direct evidence for an adhesive function in the noncholinergic role of acetylcholinesterase in neurite outgrowth.

Acetylcholinesterase (AChE) can promote neurite outgrowth through a mechanism that is independent of its role in hydrolyzing the neurotransmitter acetylcholine. It has been proposed that this neuritogenic capacity of AChE may result from its intrinsic capacity to function in adhesion. In this study we directly tested this hypothesis using neuroblastoma cell lines that have been engineered for altered cell-surface expression of AChE. Using a microtiter-plate adhesion assay and the electrical cell-substrate impedance-sensing (ECIS) method, we demonstrate that the level of cell-substratum adhesion of these cells directly correlates with their level of AChE expression. Furthermore, this adhesion is blocked by either an anti-AChE antibody or a highly specific AChE inhibitor (BW284c51), both of which have also been shown to block neurite outgrowth. In addition, cells that overexpress AChE showed enhanced neurite initiation. By employing cell lines with different levels of AChE expression in two types of cell-substratum adhesion assays, our current studies provide evidence for an adhesive function for AChE. These results, together with the fact that AChE shares sequence homology and structural similarities with several known cell adhesion molecules, support the hypothesis that AChE promotes neurite outgrowth, at least in part, through an adhesive function.

Evidence for the direct role of acetylcholinesterase in neurite outgrowth in primary dorsal root ganglion neurons.

Dorsal root ganglion (DRG) neurons show a transient peak expression of acetylcholinesterase (AChE) during periods of axonal outgrowth prior to synaptogenesis, suggesting that AChE has a non-enzymatic role during development. We have previously shown that perturbation of cell surface AChE in cultured embryonic rat DRG neurons results in decreased neurite outgrowth and neurite detachment. In this report, we demonstrate a direct correlation between endogenous AChE content and neurite outgrowth in primary DRG neurons. Adenoviral vectors were constructed using full-length rat AChE(T) cDNA in either the sense or antisense orientations to overexpress or knock down AChE expression, respectively. Treatment with the sense-expressing vector produced a 2.5-fold increase in AChE expression and a 2-fold increase in neurite length compared with either untreated or null virus-treated control cells. Conversely, treatment with the antisense-expressing vector reduced AChE expression by 40% and resulted in a reduction in neurite length of similar magnitude. We also observed that overexpression of AChE resulted in greater branching at the distal tips of each primary neurite as well as an increase in cell body size. These findings further indicate that AChE expressed on the axonal surface of developing DRG neurons may modulate their adhesive properties and thereby support axonal development.

Myelinated dorsal root ganglion cultures activate both the alternative and classical pathways of complement.

We used rat myelinated dorsal root ganglion (MDRG) cultures to study antibody and complement-mediated mechanisms of peripheral demyelinating diseases. Heat inactivated serum from a patient (LT) with peripheral neuropathy and a monoclonal IgM reactive with myelin-associated glycoprotein (anti-MAG) and sulfated glucuronosyl glycolipids (anti-SGGL) was used as an antibody source. Incubation of whole human serum (WHS) or WHS and anti-SGGL with MDRGs resulted in reduction of classical and alternative pathway hemolytic activities and the development of abnormal myelin sheaths. Incubation of MDRG cultures with C2-deficient serum showed activation of the alternative complement pathway. Classical pathway hemolytic activity was reduced when Factor B-depleted serum was incubated with MDRG cultures. The rat MDRG culture system provides a good model system of a peripheral nerve and has therefore been used by several investigators to study antibody and complement-mediated demyelination associated with peripheral neuropathies. However, our studies indicate a high degree of complement activation and membrane disruption of cultures incubated with WHS.

Morphogenic role for acetylcholinesterase in axonal outgrowth during neural development.

Acetylcholinesterase (AChE) is the enzyme that hydrolyzes the neurotransmitter acetylcholine at cholinergic synapses and neuromuscular junctions. However, results from our laboratory and others indicate that AChE has an extrasynaptic, noncholinergic role during neural development. This article is a review of our findings demonstrating the morphogenic role of AChE, using a neuronal cell culture model. We also discuss how these data suggest that AChE has a cell adhesive function during neural development. These results could have additional significance as AChE is the target enzyme of agricultural organophosphate and carbamate pesticides as well as the commonly used household organophosphate chlorpyrifos (Dursban). Prenatal exposure to these agents could have adverse effects on neural development by interfering with the morphogenic function of AChE.

Continuous adult development of multiple innervation in toadfish sonic muscle.

The sonic muscle of the oyster toadfish Opsanus tau produces unfused contractions at over 200 Hz for mating call production, requiring extreme muscle fiber synchronization. This multiply innervated muscle is sexually dimorphic and grows for life by fiber proliferation and hypertrophy. Previous descriptions of its multiple innervation did not consider fish size or sex. We examined neuromuscular junction (NMJ) development in adult fish of both sexes between 123 and 343 mm in total length (24.7790 g in mass). The NMJ was a tubelike trough that varied in length from 8 to 178 microm. Troughs were usually straight, although some consisted of consecutive ovals and some were branched. Median length of NMJs increased linearly with fish length (r2=.40; p=.002) from 58 to 75 microm. Modal lengths were mostly between 50 and 60 microm and did not increase ontogenetically, indicating that the median increase was caused by a greater number of large junctions in older fish. Median interval between NMJs (measured from the beginning of one junction to the next) ranged from 92 to 116 microm and did not vary with fish size (r2=.06; p=.285). Considering muscle fiber elongation, the data indicate an increase from 60 to 140 NMJs per fiber during fish growth. There were no sexual differences in NMJ length or spacing. In view of the slow conduction velocity of sonic muscle fibers, the addition of new NMJs and the relatively constant distance between them supports rapid and synchronized contraction necessary for sound production in both sexes.

Acetylcholinesterase antibody treatment results in neurite detachment and reduced outgrowth from cultured neurons: further evidence for a cell adhesive role for neuronal acetylcholinesterase.

Data from our laboratory and others demonstrate that acetylcholinesterase (AChE) is expressed transiently by neurons during periods of neurite outgrowth preceding synaptogenesis, suggesting an extrasynaptic function for this molecule. These findings, along with reports that AChE shares amino acid sequence homology and structural similarities with known cell adhesion molecules, have led to the theory that, during development, AChE may exert a morphogenic effect through cell adhesion. To further test this hypothesis, we have examined the effects of an AChE monoclonal antibody (MAB304) on neurite outgrowth in primary cultures of rat dorsal root ganglion (DRG) neurons. Short-term, high-concentration antibody treatment produced a rapid detachment of established DRG neurites, which was followed by regrowth upon removal of the antibody from the culture medium. This effect appeared to be site-specific, because other AChE antibodies that were able to detect AChE immunocytochemically failed to produce this disadhesion. Long-term, low-concentration antibody exposure produced a 50% reduction in total area of outgrowth, in which neurites were more densely packed and interlaced compared with the neurites in control cultures. These results extend our previous observations on the outgrowth perturbing effects of AChE inhibitor treatment and provide further evidence that AChE may support neurite outgrowth through a cell adhesive role.

Acetylcholinesterase inhibitor treatment delays recovery from axotomy in cultured dorsal root ganglion neurons.

We have previously reported that dorsal root ganglion neurons cultured in the presence of the highly specific, reversible acetylcholinesterase inhibitor 1,5-bis-(4-allyldimethylammoniumphenyl) pentan-3-one dibromide (BW284c51), showed significantly reduced neurite outgrowth and contained massive perikaryal inclusions of neurofilaments. In the present report we have more closely examined these changes in a time course study over a 21-day culture period using a combined morphological, immunocytochemical and enzymatic approach and additionally, describe, the effects of acetylcholinesterase inhibitor treatment on the state of neurofilament phosphorylation. Finally, we have examined the effects of co-administration of N6,2'-0-dibutyryladenosine 3':5'-cyclic monophosphate (dbcAMP) with BW284c51. At 1 day in culture, both control and treated cells displayed eccentrically located nuclei, numerous polysomes and perikaryal accumulations of neurofilaments which were immunoreactive with both phosphorylation- and nonphosphorylation-dependent neurofilament antibodies. These cytological changes, which are common features of the chromatolytic reaction following axotomy in vivo, rapidly resolved in the control neurons, where by 7 days in culture, the neurofilament accumulations had completely disappeared and neurite outgrowth was robust. In contrast, inhibitor-treated neurons retained the post-axotomy features up to 21 days and had significantly reduced neurite outgrowth. In addition, we have investigated a possible role of cyclic adenosine monophosphate (cAMP) in the recovery process since it has been shown to enhance neuritic outgrowth in cultured neurons. Our results demonstrate that the addition of dbcAMP, a membrane permeable analog of cAMP, significantly enhanced neuritic outgrowth and accelerated the recovery of BW284c51-treated dorsal root ganglion cells, as gauged by the disappearance of the axotomy-related cytological changes. Treatment with dbcAMP also increased acetylcholinesterase activity which has been positively correlated with neurite outgrowth both in vivo and in vitro. Together, these observations suggest that acetylcholinesterase has a non-cholinolytic, neurotrophic role in neuronal regeneration and development.

Generation and characterization of anti-sulfoglucuronosyl paragloboside monoclonal antibody NGR50 and its immunoreactivity with peripheral nerve.

Sulfoglucuronosyl paragloboside (SGPG) is a member of the sulfated glucuronic acid-containing glycolipid (SGGL) family found primarily in peripheral nerves. These glycolipids contain the HNK-1 carbohydrate epitope and are recognized by monoclonal IgM from patients with chronic demyelinating neuropathy and paraproteinemia. Recent studies indicate that SGGLs may serve as ligands for selectins, amphoterin, and laminin, suggesting that these glycolipids may play an important role in cellular adhesion. To elucidate the biological function of these glycolipids, we produced a murine monoclonal antibody (mAb) and studied its antigenic specificity. Using an enzyme-linked immunosorbent assay (ELISA), we found that the mAb designated as NGR50 belonged to the IgG2a subclass, and that the minimal titer (2 SD above the mean optical density value of control) of this mAb was 1:640, with 20 ng of purified SGPG as the antigen. Thin-layer chromatography (TLC) immunoblotting revealed that this mAb reacted specifically with SGPG and sulfoglucuronosyl lactosaminyl paragloboside (SGLPG), which is a structural analogue of the former, but not with other glycolipids. Desulfated derivates of SGPG and SGLPG did not react with mAb NGR50. Western blot analysis showed crossreactivity with human myelin-associated glycoprotein (MAG), but not with rat MAG or rat glycoprotein P0. Unlike anti-HNK-1 monoclonal antibody, however, NGR50 reacted only weakly with several proteins in the 20-30-kD regions, including human P0, suggesting that mAb50 has a different fine specificity as an anti-HNK-1 antibody. Immunocytochemical study of rat sciatic nerve using mAb NGR50 revealed positive staining at the outer surface of the myelin sheath and Schwann cells, as well as in the intervening connective tissues. Faint staining was also visible at the axolemmal-myelin interface; however, compact myelin was not stained.

Expression of a unique globo-series glycolipid in cultured rat dorsal root ganglion neurons: relationship with neuronal development.

Previous studied from the laboratory demonstrated the presence of a UDP-galactose:Gb3Cer alpha 1-3galactosyltansferase activity responsible for the synthesis of a unique glycosphingolipid (GSL), Gal alpha 1-3Gb3Cer, in cultured PC12 pheochromocytoma cells (21). In this investigation, we examined the presence of this enzyme activity in isolated rat embryonic dorsal root ganglion neurons (DRGN), which, like pheochromocytoma cells, originate from the neural crest cells. DRGN exhibited the alpha-galactosyltransferase activity and the activity was comparable to that of the PC12 cells while several other rat tissues, with the exception of kidney, showed minimal activity. In order to define the spatial and temporal expression of Gal alpha 1-3Gb3Cer in DRGN, we examined the expression of Gal alpha 1-3Gb3Cer in cultured DRGN derived from embryonic day 16 rat embryos. Using a polyclonal antibody raised against Gal alpha 1-3Gb3Cer, we examined the localization of this glycolipid in DRGN cells after 5, 8, 12, and 15 days in culture. Immunostaining was restricted to the neurons while Schwann cells were negative. At day 5, the immunostaining was weak and confined to the cell body of the DRGN, though neurites were present at this stage. The period between days 5 and 15 represented a period of rapid neuritic growth and continued enlargement of the cell bodies. Immunoreactivity in the cell bodies increased dramatically by day 8. By day 12, immunoreactivity was present in neurites, and by day 15, was strong in both cells bodies and neurites. The expression of Gal alpha 1-3Gb3Cer in vivo was confirmed by immunostaining of frozen sections of dorsal root ganglia. Our present studies which demonstrate neuron-specific expression of Gal alpha 1-3Gb3Cer during neurotigenesis combined with previous observations for its expression in PC12 cells, strongly implicates this GSL in neuronal development.

Inverse correlation of acetylcholinesterase (AChE) activity with the presence of neurofilament inclusions in dorsal root ganglion neurons cultured in the presence of a reversible inhibitor of AChE.

We have previously shown that treatment of cultured dorsal root ganglion neurons (DRGN) with a highly specific, reversible acetylcholinesterase (AChE) inhibitor, BW284c51, retards neuritic outgrowth in a dose dependent manner and is accompanied by the presence of abnormal, perikaryal neurofilament (NF) inclusions in approximately 40% of the cells. Since subpopulations of DRGN have been classified according to their levels of AChE activity, we have combined immunocytochemical and enzyme histochemical techniques to investigate a possible correlation between AChE activity and the presence of NF inclusion formation. Our results show that after inhibitor treatment, cells with low levels of AChE activity have a greater percentage of inclusions, with nearly 75% of cells with undetectable levels of AChE activity containing inclusions. In contrast, inclusions were present in only 3.2% of cells with high levels of AChE activity. This inverse relationship between AChE activity and the presence of NF inclusions supports our previous observations that this enzyme may have extra-synaptic functions which could affect neuronal development and regeneration.

Myelinogenic potential of an immortalized oligodendrocyte cell line.

The myelinogenic potential of an oligodendrocyte cell line (N20.1) immortalized by transformation with a temperature-sensitive retrovirus (Verity et al., J Neurochem 60:577-587, 1993) has been evaluated in a co-culture system utilizing dorsal root ganglion neurons. When N20.1 cells were placed in co-culture with dorsal root ganglion neurons at 39 degrees C, the temperature at which TAg expression is decreased relative to that in cells maintained at 34 degrees C, there was a dramatic decrease in the N20.1 proliferation rate compared to cells maintained in the absence of neurons at either temperature. This decrease in proliferation was observed within 3 days of co-culture and appeared to precede a further decrease in TAg expression that occurred with time in response to the neurons. In co-cultures the immunoreactivity of N20.1 cells for galactocerebroside increased with time, and the cells appeared to establish contact with neurites and initiate formation of membranous sheets. When the duration of co-culture was extended to 52 days, myelin-like figures were noted by electron microscopy. Thus, the extent of N20.1 differentiation is dependent on the presence of neurons and the duration of co-culture. This culture system represents a potentially powerful tool for the study of neuronal-glial interactions influencing myelinogenesis and remyelination.

Retardation of neuritic outgrowth and cytoskeletal changes accompany acetylcholinesterase inhibitor treatment in cultured rat dorsal root ganglion neurons.

Over the past two decades acetylcholinesterase (AChE) has been shown to be present in numerous non-cholinergic and non-cholinoceptive tissues. Interestingly, transient expression of AChE in developing nervous tissue corresponds temporally with neuronal migration and neuritic outgrowth. This observation has led our laboratory to investigate a possible novel, non-cholinergic role for AChE in the development of the nervous system. In a previous study, we demonstrated that the activity of AChE in cultured dorsal root ganglion neurons (DRGN) can be modulated by the substratum. In our current study, we have examined the effects of AChE inhibitor treatment on neuritic outgrowth on the highly permissive substratum Matrigel and the less permissive substratum Collagen Type I. DRGN received serial dilutions of the AChE-specific inhibitor 1,5-bis-(4-allyldimethylammoniumphenyl) pentan-3-one dibromide (BW284c51) ranging from 10(-4) to 10(-7) M. Results showed that neuritic outgrowth was significantly reduced in DRGN grown on Matrigel at 10(-5) and 10(-4) M BW284c51, while outgrowth on Collagen Type I was significantly reduced at 10(-6), 10(-5), and 10(-4) M concentrations of BW284c51. Inhibitor treatment did not affect cell survival and neuritic outgrowth from BW284c51-treated cells recovered to control levels after removal of the inhibitor from the medium. In addition, massive spiraling accumulations of 10 nm filaments were observed in the cell bodies of treated neurons, which resemble neurofibrillary inclusions observed in neuropathological diseases such as Pick's disease. This study demonstrates that AChE inhibitor treatment retards neuritic outgrowth and neuronal migration of cultured DRGN which is accompanied by cytoskeletal abnormalities in the cell body.(ABSTRACT TRUNCATED AT 250 WORDS)

cAMP-induced morphological changes in an immortalized Schwann cell line: a prelude to differentiation?

Schwann cells (SC), the myelinating cells of the peripheral nervous system, show a remarkable capacity to switch from a differentiated state to a proliferative state both during development and peripheral nerve regeneration. In order to better understand the regulatory mechanisms involved with this change we are studying a Schwann cell line transfected with the SV-40 large T gene (TSC). Serum-free medium combined with elevating intra-cellular cAMP levels produced a slower proliferating TSC whose morphology changed from pleiomorphic to process bearing, reminiscent of primary SC in culture. This change was abrogated by colcemid but was unaltered by cytochalasin D, indicating a major role for microtubules. Ultrastructural studies demonstrated numerous microtubules in the cellular extensions which correlated with strong immunocytochemical staining for tubulin in the processes. Analysis of cytoskeletal fractions from the treated cells revealed a greater proportion of tubulin in the polymerized state compared with untreated cells which closely resembled the distribution in primary SC. The cytoskeletal changes observed in the TSC as a result of elevating the intra-cellular cAMP levels may reflect the earliest cellular changes in the induction of myelination.

Release of membrane-associated growth factors during neural injury.

The release of membrane-associated growth factors after neural injury may influence the outcome of the recovery. For example, for remyelination to occur after neural injury it is critical for the glial cell to proliferate prior to remyelination in both the PNS and CNS. In the CNS, the relative response of the oligodendrocytes and astroglia to growth factors mobilized during neural injury may play a role in the cellular dynamics of repair of neural injury or scarring and subsequent failure to repair neural injury. In support of this view, we have studied the mitotic potential and cell cycle kinetics of cultured adult oligodendrocytes and found that these adult cells respond only weakly to factors such as FGF which are known to be potent mitogens for neonatal cells. However, given the same dose of FGF, adult astrocytes are mitotically stimulated to a much greater degree than are the adult oligodendrocytes (Vick and De Vries, unpublished observations). Given the pathways which may be operative in the release of growth factors after injury, it has not escaped our attention that, provided the released factors are in equilibrium with easily accessible and peripheral body fluids, these released factors may serve as new markers for neural injury. Further experiments are in progress to explore this possibility.

The uniqueness of rural nursing.

Rural nursing is a unique and challenging field of nursing that requires a "special breed" of nurse that is committed to high quality, comprehensive care at the individual, family, and community levels. The unique characteristics of rural nursing include close interaction with the community, a truly generalist approach, and increased autonomy, cohesiveness, and community visibility. This area of specialty practice requires nurses to be highly competent and well-prepared in all aspects of professional practice. Certainly, the demands of rural nursing are great, as are the benefits. Rural nursing can indeed be the essence of what nursing should be.

A nuclear matrix protein stabilized by lead exposure: current knowledge and future prospects.

p32/6.3, a low-abundance, highly conserved nuclear protein, is a target for lead. Very few low abundance nuclear proteins have been described and no others have been associated with lead. Its wide distribution and conservation indicate a fundamental nuclear role. Further, it increases many fold in grey matter of brain and spinal cord during the neonatal period; there are no other identified nuclear proteins which serve as markers for this period of nervous system development. There are several links between lead and p32/6.3. It is a major component of lead-induced intranuclear inclusion bodies from the kidney. Its accumulation in kidney is a relatively early event in the process of lead intoxication. Exposure to lead increases p32/6.3 in mouse neuroblastoma 2a cells within one day, blocking its degradation almost completely. These observations suggest that lead either structurally alters p32/6.3 or inhibits a protease for which p32/6.3 is a substrate. In these lead-treated cells nuclear envelope invaginations and small nuclear bodies increase. The possible involvement of lead and p32/6.3 with the formation and movement of nuclear bodies is discussed.

Frontier areas: opportunities for NPs' primary care services.

Demographers have recently designated frontier areas as distinct from other rural areas. Frontier counties are defined as those with less than six persons per square mile; as such, they constitute 45 percent of the U.S. land mass and include 2.2 million people. The health status among frontier residents is estimated to be lower than that of other rural or urban populations, and frontier health services, particularly primary care services, are scant. In this article, opportunities for nurse practitioners in frontier practice are explored, including specific suggestions for the development of new frontier NP practices.

Substratum-induced modulation of acetylcholinesterase activity in cultured dorsal root ganglion neurons.

Acetylcholinesterase (AChE) has been shown to be transiently expressed in the developing nervous system during periods of neuronal migration and axonal outgrowth. We are investigating the possible interaction of substratum with AChE activity in dorsal root ganglion neurons (DRGN) cultured on substrata with varying degrees of permissiveness for neurite outgrowth: (1) extracellular matrix substrata: reconstituted basal lamina Matrigel (MGEL), laminin (LAM) and type I collagen (COL), and (2) organotypic substrata: unfixed, frozen sections of sciatic nerve (SN) and spinal cord (SC). In group 1, histochemical staining for AChE in DRGN was lowest on MGEL where outgrowth was most vigorous, intermediate on LAM, and highest on COL where neurite outgrowth was reduced by 55% compared to Matrigel and highly fasciculated. A similar trend was seen when the cultures were assayed biochemically, 2.84 +/- 0.14 nmoles ACh hydrolyzed/ganglion/hr (MGEL), 4.42 +/- 0.19 (LAM), 5.79 +/- 0.37 (COL). In group 2, SN supported an expansive outgrowth with lower AChE activity than in DRGN grown on SC where outgrowth was minimal. These studies show that the levels of AChE activity can be modulated by substratum, perhaps in proportion to the permissiveness of the substratum to neuritic outgrowth. These results are discussed in relation to possible non-cholinergic roles of AChE.