Developmental expression of the TTX-resistant voltage-gated sodium channels Nav1.8 (SNS) and Nav1.9 (SNS2) in primary sensory neurons.

The development of neuronal excitability involves the coordinated expression of different voltage-gated ion channels. We have characterized the expression of two sensory neuron-specific tetrodotoxin-resistant sodium channel alpha subunits, Na(v)1. (SNS/PN3) and Na(v)1.9 (SNS2/NaN), in developing rat lumbar dorsal root ganglia (DRGs). Expression of both Na(v)1.8 and Na(v)1.9 increases with age, beginning at embryonic day (E) 15 and E17, respectively, and reaching adult levels by postnatal day 7. Their distribution is restricted mainly to those subpopulations of primary sensory neurons in developing and adult DRGs that give rise to unmyelinated C-fibers (neurofilament 200 negative). Na(v)1.8 is expressed in a higher proportion of neuronal profiles than Na(v)1.9 at all stages during development, as in the adult. At E17, almost all Na(v)1.8-expressing neurons also express the high-affinity NGF receptor TrkA, and only a small proportion bind to IB4, a marker for c-ret-expressing (glial-derived neurotrophic factor-responsive) neurons. Because IB4 binding neurons differentiate from TrkA neurons in the postnatal period, the proportion of Na(v)1.8 cells that bind to IB4 increases, in parallel with a decrease in the proportion of Na(v)1.8-TrkA co-expressing cells. In contrast, an equal number of Na(v)1.9 cells bind IB4 and TrkA in embryonic life. The differential expression of Na(v)1.8 and Na(v)1.9 in late embryonic development, with their distinctive kinetic properties, may contribute to the development of spontaneous and stimulus-evoked excitability in small diameter primary sensory neurons in the perinatal period and the activity-dependent changes in differentiation they produce.

Identification and characterization of a novel human vanilloid receptor-like protein, VRL-2.

Remarkable progress has been made recently in identifying a new gene family related to the capsaicin (vanilloid) receptor, VR1. Using a combination of in silico analysis of expressed sequence tag (EST) databases and conventional molecular cloning, we have isolated a novel vanilloid-like receptor, which we call VRL-2, from human kidney. The translated gene shares 46% and 43% identity with VR1 and VRL-1, respectively, and maps to chromosome 12q23-24.1, a locus associated with bipolar affective disorder. VRL-2 mRNA was most strongly expressed in the trachea, kidney, and salivary gland. An affinity-purified antibody against a peptide incorporating the COOH terminal of the receptor localized VRL-2 immunolabel in the distal tubules of the kidney, the epithelial linings of both trachea and lung airways, serous cells of submucosal glands, and mononuclear cells. Unlike VR1 and VRL-1, VRL-2 was not detected in cell bodies of dorsal root ganglia (DRG) or sensory nerve fibers. However, VRL-2 was found on sympathetic and parasympathetic nerve fibers, such as those innervating the arrector pili smooth muscle in skin, sweat glands, intestine, and blood vessels. At least four vanilloid receptor-like genes exist, the newest member, VRL-2 is found in airway and kidney epithelia and in the autonomic nervous system.

Diversity of expression of the sensory neuron-specific TTX-resistant voltage-gated sodium ion channels SNS and SNS2.

The differential distribution of two tetrodotoxin resistant (TTXr) voltage-gated sodium channels SNS (PN3) and SNS2 (NaN) in rat primary sensory neurons has been investigated. Both channels are sensory neuron specific with SNS2 restricted entirely to those small dorsal root ganglion (DRG) cells with unmyelinated axons (C-fibers). SNS, in contrast, is expressed both in small C-fiber DRG cells and in 10% of cells with myelinated axons (A-fibers). All SNS expressing A-fiber cells are Trk-A positive and many express the vanilloid-like receptor VRL1. About half of C-fiber DRG neurons express either SNS or SNS2, and in most, the channels are colocalized. SNS and SNS2 are found both in NGF-responsive and GDNF-responsive C-fibers and many of these cells also express the capsaicin receptor VR1. A very small proportion of small DRG cells express either only SNS or only SNS2. At least four different classes of A- and C-fiber DRG neurons exist, therefore, with respect to expression of these sodium channels.

Pain: molecular mechanisms.

Our understanding of chronic inflammatory and neuropathic pain at the molecular and cellular level has developed at an extraordinary rate in recent years. Inflammatory, or neuropathic, neuronal plasticity describes the process by which the neurons involved in pain transmission are converted from a state of normosensitivity to one in which they are hypersensitive. Here we summarize current theories on somatosensory neuroplasticity in a molecular context, highlighting key receptors, ion channels, and signal molecules involved. We also suggest new possibilities for drug design, based on the rational targeting of these molecular players.

A role for HSP27 in sensory neuron survival.

Peripheral nerve injury in neonatal rats results in the death of the majority of the axotomized sensory neurons by 7 d after injury. In adult animals, however, all sensory neurons survive for at least 4 months after axotomy. How sensory neurons acquire the capacity to survive axonal injury is not known. Here we describe how the expression of the small heat shock protein 27 (HSP27) is correlated with neuronal survival after axotomy in vivo and after NGF withdrawal in vitro. The number of HSP27-immunoreactive neurons in the L4 DRG is low at birth and does not change significantly for 21 d after postnatal day 0 (P0) sciatic nerve axotomy. In contrast, in the adult all axotomized neurons begin to express HSP27. One week after P0 sciatic nerve section the total number of neurons in the L4 DRG is dramatically reduced, but all surviving axotomized neurons, as identified by c-jun immunoreactivity, are immunoreactive for HSP27. In addition, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling reveals that very few HSP27-expressing neurons are dying 48 hr after neonatal axotomy. In vitro, a similar correlation exists between HSP27 expression and survival; in P0 DRG cultures, neurons that express HSP27 preferentially survive NGF withdrawal. Finally, overexpression of human HSP27 in neonatal rat sensory and sympathetic neurons significantly increases survival after NGF withdrawal, with nearly twice as many neurons surviving at 48 hr. Together these results suggest that HSP27 in sensory neurons plays a role in promoting survival after axotomy or neurotrophin withdrawal.

Neurotrophins: peripherally and centrally acting modulators of tactile stimulus-induced inflammatory pain hypersensitivity.

Brain-derived neurotrophic factor (BDNF) is expressed in nociceptive sensory neurons and transported anterogradely to the dorsal horn of the spinal cord where it is located in dense core vesicles in C-fiber terminals. Peripheral inflammation substantially up-regulates BDNF mRNA and protein in the dorsal root ganglion (DRG) in a nerve growth factor-dependent fashion and results in novel expression of BDNF by DRG neurons with myelinated axons. C-fiber electrical activity also increases BDNF expression in the DRG, and both inflammation and activity increase full-length TrkB receptor levels in the dorsal horn. Sequestration of endogenous BDNF/neurotrophin 4 by intraspinal TrkB-Fc fusion protein administration does not, in noninflamed animals, change basal pain sensitivity nor the mechanical hypersensitivity induced by peripheral capsaicin administration, a measure of C fiber-mediated central sensitization. TrkB-Fc administration also does not modify basal inflammatory pain hypersensitivity, but does block the progressive hypersensitivity elicited by low-intensity tactile stimulation of inflamed tissue. BDNF, by virtue of its nerve growth factor regulation in sensory neurons including novel expression in A fibers, has a role as a central modulator of tactile stimulus-induced inflammatory pain hypersensitivity.

Transcriptional and posttranslational plasticity and the generation of inflammatory pain.

Inflammatory pain manifests as spontaneous pain and pain hypersensitivity. Spontaneous pain reflects direct activation of specific receptors on nociceptor terminals by inflammatory mediators. Pain hypersensitivity is the consequence of early posttranslational changes, both in the peripheral terminals of the nociceptor and in dorsal horn neurons, as well as later transcription-dependent changes in effector genes, again in primary sensory and dorsal horn neurons. This inflammatory neuroplasticity is the consequence of a combination of activity-dependent changes in the neurons and specific signal molecules initiating particular signal-transduction pathways. These pathways phosphorylate membrane proteins, changing their function, and activate transcription factors, altering gene expression. Two distinct aspects of sensory neuron function are changed as a result of these processes, basal sensitivity, or the capacity of peripheral stimuli to evoke pain, and stimulus-evoked hypersensitivity, the capacity of certain inputs to generate prolonged alterations in the sensitivity of the system. Posttranslational changes largely alter basal sensitivity. Transcriptional changes both potentiate the system and alter neuronal phenotype. Potentiation occurs as a result of the up-regulation in the dorsal root ganglion of centrally acting neuromodulators and simultaneously in the dorsal horn of their receptors. This means that the response to subsequent inputs is augmented, particularly those that induce stimulus-induced hypersensitivity. Alterations in phenotype includes the acquisition by A fibers of neurochemical features typical of C fibers, enabling these fibers to induce stimulus-evoked hypersensitivity, something only C fiber inputs normally can do. Elucidation of the molecular mechanisms responsible provides new opportunities for therapeutic approaches to managing inflammatory pain.

Lateralized use of the mouth in production of vocalizations by marmosets.

We have found that the common marmoset, Callithrix jacchus, displays a larger left hemimouth during production of fear expressions, with or without vocalization, and a larger right hemimouth when producing a social contact call. Thus, marmosets have right hemisphere specialization for the production of negative emotional expressions and vocalizations and left hemisphere specialization for the production of social contact communication. These hemispheric specializations for social communication in marmosets are the same as those found in humans for speech production and for the control of emotional expressions. We suggest that hemispheric specializations for communication in humans may well have precursors in primate evolution.

Septic shock: an analysis of outcomes for patients with onset on hospital wards versus intensive care units.

OBJECTIVE: To determine if early interventions for septic shock were associated with reduced mortality. DESIGN: Retrospective cohort study. SETTING: University hospital intensive care unit (ICU) and general wards. PATIENTS: Forty-one consecutive patients prospectively identified with positive blood cultures and septic shock. Although all patients were eventually treated in an ICU, ten (24%) patients were on a general ward at the onset of septic shock, and 31 (76%) were in an ICU setting. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Over a period of 9 mos, a cohort of 41 patients who had positive blood cultures and septic shock was prospectively identified. The 28-day crude mortality was 46% (19 deaths). We compared the management of septic shock and outcome for patients on a general ward vs. those patients in an ICU setting. Of the ten patients on the ward at time of shock onset (median age 55.5 yrs; median Acute Physiology and Chronic Health Evaluation [APACHE] II score of 18.5), seven (70%) died. In contrast, the 31 patients receiving intensive care when shock developed were older and more ill (median age 66 yrs; median APACHE II 24), yet had a mortality of 39% (12 deaths). The odds ratio (OR) for death for ward patients compared with ICU patients was 3.57 (p=.17). In a multivariate logistic regression analysis, two risk factors for mortality were important: APACHE II score (p=.015) and ward status (p=.08). Candida species in the bloodstream is known to have a high attributable mortality. When type of bloodstream pathogen (Candida species vs. bacteria) was added to the model, APACHE II (OR 2.64 for 10-unit increase) remained significant (p=.014), but ward status (OR 3.97) became statistically nonsignificant (p=.222). The patients who were on a general ward when their shock developed had a median delay of 67 mins before transfer to an ICU setting. Ward patients received an intravenous fluid bolus after a median delay of 27 mins, whereas those in the ICU who received a fluid bolus did so after a median of 15 mins (p=.48). Ward patients also had a median delay of 310 mins to receive inotropic support compared with a median 22.5 mins (p=.037) for the patients in an ICU setting when shock started. CONCLUSIONS: The data suggest that for patients with septic shock on wards, there were clinically important delays in transfer of patients to the ICU, receipt of intravenous fluid boluses, and receipt of inotropic agents. However, the most powerful predictors of mortality were APACHE II scores and bloodstream infection with Candida species.

Tubular peptide hypermetabolism and urinary ammonia in chronic renal failure in man: a maladaptive response?

Excessive renal tubular peptide uptake and degradation reflecting hypercatabolism may be a maladaptive response in chronic renal failure (CRF). It may also offer an explanation for the increased ammoniagenesis, per surviving nephron, observed in CRF but as yet unexplained. Neither has been explored in man. We have shown in patients with normal renal function and heavy (>5.0 g/24 h) proteinuria that tubular catabolism of a technetium-labelled peptide marker, aprotinin, and urinary ammonia were increased compared to others with less proteinuria. We now measure tubular kinetics of aprotinin and urinary ammonia in 16 CRF patients with variable proteinuria. Metabolism and turnover of aprotinin and ammonia excretion were increased, corrected for glomerular filtration rate, to levels found in patients with normal function and heavy proteinuria.

Heat shock protein 27: developmental regulation and expression after peripheral nerve injury.

The heat shock protein (HSP) 27 is constitutively expressed at low levels in medium-sized lumbar dorsal root ganglion (DRG) cells in adult rats. Transection of the sciatic nerve results in a ninefold upregulation of HSP27 mRNA and protein in axotomized neurons in the ipsilateral DRG at 48 hr, without equivalent changes in the mRNAs encoding HSP56, HSP60, HSP70, and HSP90. Dorsal rhizotomy, injuring the central axon of the DRG neuron, does not upregulate HSP27 mRNA levels. After peripheral axotomy, HSP27 mRNA and protein are present in small, medium, and large DRG neurons, and HSP27 protein is transported anterogradely, accumulating in the dorsal horn and dorsal columns of the spinal cord, where it persists for several months. Axotomized motor neurons also upregulate HSP27. Only a minority of cultured adult DRG neurons are HSP27-immunoreactive soon after dissociation, but all express HSP27 after 24 hr in culture with prominent label throughout the neuron, including the growth cone. HSP27 differs from most axonal injury-regulated and growth-associated genes, which are typically present at high levels in early development and downregulated on innervation of their targets, in that its mRNA is first detectable in the DRG late in development and only approaches adult levels by postnatal day 21. In non-neuronal cells, HSP27 has been shown to be involved both in actin filament dynamics and in protection against necrotic and apoptotic cell death. Therefore, its upregulation after adult peripheral nerve injury may both promote survival of the injured neurons and contribute to alterations in the cytoskeleton associated with axonal growth.

Proximal renal tubular peptide catabolism, ammonia excretion and tubular injury in patients with proteinuria: before and after lisinopril.

1. Progression to renal failure may be linked to the degree of proteinuria through tubulo-interstitial mechanisms. However, there are no data in man on the kinetics of proximal renal tubular protein catabolism or markers of tubular injury before and after lisinopril. We developed a method to allow such studies, and found increased tubular catabolism of 99mTc-labelled aprotinin (Trasylol) in patients with nephrotic range proteinuria which was associated with increased ammonia excretion. 2. In this study, 10 patients with mild renal impairment (51Cr-EDTA clearance 63.7 +/- 8.3 ml.min-1.1.73 m-2) and heavy proteinuria (8.2 +/- 2.3 g/ 24 h) were given lisinopril (10-20 mg) for 6 weeks. Renal tubular catabolism of intravenous aprotinin was measured before and after lisinopril by renal imaging and urinary excretion of the free radiolabel over 26 h. Fractional degradation was calculated from these data. Fresh timed urine collections were also analysed for ammonia excretion every fortnight from 6 weeks before treatment. Total urinary N-acetyl-beta-D-glucosaminidase and the more tubulo-specific N-acetyl-beta-D-glucosaminidase 'A2' isoenzyme were also measured. 3. After lisinopril proteinuria fell significantly as expected (from 9.5 +/- 1.6 to 4.5 +/- 1.0 g/24 h, P < 0.01). This was associated with a reduction in metabolism over 26 h (from 1.7 +/- 0.1 to 1.2 +/- 0.1% dose/h, P < 0.01) and in fractional degradation of aprotinin (from 0.08 +/- 0.02 to 0.04 +/- 0.007/h, P < 0.04). Ammonia excretion also fell significantly (from 1.2 +/- 0.1 to 0.6 +/- 0.1 mmol/h, P < 0.0001), as did both total urinary N-acetyl-beta-D-glucosaminidase (P < 0.0001) and the N-acetyl-beta-D-glucosaminidase 'A2' isoenzyme (P < 0.015). These observations after lisinopril treatment have not been described previously. There was no significant change in blood pressure nor in glomerular haemodynamics.

Proteinuria and renal tubular damage: urinary N-acetyl-beta-D-glucosaminidase and isoenzymes in dissimilar renal disease.

Markers of renal tubular injury are difficult to interpret in patients with proteinura. The 24-hour urinary N-acetyl-beta-D-glucosaminidase (NAG) concentration was measured in 167 patients with dissimilar renal disease, function, and proteinuria. NAG isoenzymes were also separated in 69 patients, using a modified fast protein liquid chromatography technique. The 'A2' isoenzyme predominated at all levels of renal function and in all diagnostic groups. Urinary NAG and proteinuria were well correlated at all levels of renal function, as was NAG 'A2' isoenzyme. Proteinuria and urinary NAG were similarly correlated in patients with different glomerulonephritides, hypertensive nephrosclerosis, and chronic pyelonephritis, but not in those with diabetic nephropathy.

Oral sodium bicarbonate reduces proximal renal tubular peptide catabolism, ammoniogenesis, and tubular damage in renal patients.

Oral sodium bicarbonate (NaHCO3) is widely used to treat acidosis in patients with renal failure. However, no data are available in man on the effects on proximal renal tubular protein catabolism or markers of tubular injury. We have developed methods to allow such studies, and both increased tubular catabolism of 99mTc-labelled aprotinin (Apr*), as well as tubular damage were found in association with increased ammonia (NH3) excretion in patients with nephrotic range proteinuria. We now examine the effects of reducing renal ammoniogenesis, without altering proteinuria, using oral NaHCO3 in 11 patients with mild/moderate renal impairment and proteinuria. Renal tubular catabolism of Apr* was measured before and after NaHCO3 by renal imaging (Kidney uptake, K% of dose) and urinary excretion of free 99mTcO4- (metabolism, Met% of dose/h) over 26 h. Fractional degradation (Frac) was calculated from Met/K (/h). Fresh urine was also analyzed for NH3 excretion every fortnight from 6/52 before treatment. Total urinary N-acetyl-beta-D-glucose-aminidase (NAG) and the more tubulo-specific NAG "A2" were measured. 51CrEDTA clearance and 99mTc-MAG 3 TER were also assessed. After NaHCO3 Met over 26 h was significantly reduced (from 1.3 +/- 0.2% of dose/h to 0.9 +/- 0.1% dose/hr, p < 0.005), as was Frac of Apr* (from 0.06 +/- .006/h to 0.04 +/- 0.005/hr, p < 0.003). NH3 excretion also fell significantly (from 0.9 +/- 0.2 mmol/h to 0.2 +/- 0.05 mmol/h, p < 0.007), as did both total urinary NAG (from 169 mumol/24 h, 74-642 mumol/24 h to 79 mumol/ 24 h, 37-393 mumol/24 h, p < 0.01), and the NAG 'A2' isoenzyme (from 81.5 mumol/24 h, 20-472 mumol/24 h to 35.0 mumol/24 h, 6-388 mumol/24 h, p < 0.001). Proteinuria remained unaltered, and there was no change in blood pressure nor in glomerular haemodynamics. Oral NaHCO3 may thus pro-tect the proximal renal tubule and help delay renal disease progression.

Staphylococcus aureus bacteremia: the cost-effectiveness of long-term therapy associated with infectious diseases consultation.

OBJECTIVE: To investigate the cost-effectiveness of long-term therapy for Staphylococcus aureus bacteremia and to determine if an infectious diseases consultation affected the duration of therapy. METHODS: A decision analysis was performed based on data from the literature. To determine if consultation was related to therapy duration, a retrospective cohort study was performed using tightly matched pairs. RESULTS: The excess cost per life saved by long-term antibiotics was $500,000. The excess cost per life-year saved was $18,000. Nine pairs were matched. Patients who received consultation were more likely to receive long-term therapy than controls (median 41 days vs 15 days for controls, P = .04). CONCLUSIONS: The estimated cost per life-year saved by long-term therapy was similar to other accepted medical interventions. Infectious diseases consultation can encourage prolonged duration of antibiotic therapy for S aureus bacteremia.

Eye preferences in common marmosets (Callithrix jacchus): influence of age, stimulus, and hand preference.

Eye preferences of the common marmoset ( Callithrix jacchus ) were examined, taking into account age, arousal, and hand preference. Monocular eye use for looking through a small hole at a stimulus was recorded. Of 21 marmosets, 20 displayed right-eye preferences for viewing a piece of familiar food. In subjects tested at 3-8, 12, 15-18, and 22 months, eye preferences were consistent across age. A group bias, indicative of right-eyedness, was also found for viewing other stimuli. The stimuli included a watch, mirror, model of a beetle, and the experimenter's hand. However, when the marmosets viewed a threatening stimulus, a model resembling two rearing snakes, they displayed increased arousal (indicated by longer duration between monocular viewing events and increased incidence of aroused vocalisations) and the eye preferences shifted away from a preference for the right eye to either no preference or a left-eye preference. No relationship between eye preference and hand preference for holding food was found. Therefore, we suggest that eye preferences may reflect hemispheric specialisations for perceptual processing, according to the emotional valence of the stimulus.

Origin and significance of urinary N-acetyl-beta, D-glucosaminidase (NAG) in renal patients with proteinuria.

In patients with proteinuria, indices of tubular damage are unreliable since filtered plasma enzymes could contribute to tubular enzymuria. Previous work has suggested the existence of various forms of the 'A' isoenzyme of N-acetyl-beta, D-glucosaminidase (NAG), one of which could be kidney specific and thus a useful marker of renal tubular damage. By using fast protein liquid chromatography, two forms of the 'A' isoenzyme, 'A1' and 'A2' were separated in human urine, plasma and kidney tissue. The isoenzyme profile in pathological urine resembled that seen in kidney tissue, the 'A2' isoenzyme predominating. The ratio A2/A1 in the urine of renal patients was significantly greater than in the plasma of renal patients, end-stage renal failure patients and healthy volunteers. There was no difference in the plasma ratios of the three groups studied. The clearances of total NAG, 'A1' and 'A2' isoenzymes were all greater than that of the lower molecular weight protein transferrin. This indicates that the origin of urinary NAG in patients with proteinuria is from the kidney itself. Thus, analysis of urinary NAG and its isoenzymes may be of benefit as an early predictor of renal tubular damage and may also be useful as a non-invasive indicator of disease progression.