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Women laboring with epidural analgesia experience fever much more frequently than do women who chose other forms of analgesia, and maternal intrapartum fever is associated with numerous adverse consequences, including brain injury in the fetus. We developed a model of noninfectious inflammatory fever in the near-term pregnant rat to simulate the pathophysiology of epidural-associated fever and hypothesized that it would produce fetal brain inflammation.Twenty-four pregnant Sprague-Dawley rats were studied at 20 days gestation (term: 22 days). Dams were treated by injection of rat recombinant interleukin (IL)-6 or vehicle at 90-minute intervals, and temperature was monitored every 30 minutes. Eight hours after the first treatment, dams were delivered of fetuses and then killed. Maternal IL-6 was measured at delivery. Fetal brains (n = 24) were processed and stained for ED-1/CD68, a marker for activated microglia, and cell counts in the lateral septal and hippocampal brain regions were measured. Fetal brains were also stained for cyclooxygenase-2 (COX-2), a downstream marker of neuroinflammation. Eight fetal brains were further analyzed for quantitative forebrain COX-2 by Western blotting compared to a β-actin standard. Maternal temperature and IL-6 levels were compared between treatments, as were cell counts, COX-2 staining, and COX-2 levels by Mann-Whitney U test, repeated-measures analysis of variance, or Fisher exact test, as appropriate.Injection of rat IL-6 at 90-minute intervals produced an elevation of maternal temperature compared to vehicle (P.0001). IL-6 levels were elevated to clinically relevant levels at delivery in IL-6 compared to vehicle-treated animals (mean ± standard deviation: 923 ± 97 vs 143 ± 94 pg/mL, P = .0006). ED-1-stained cells were present in significantly higher numbers in fetal brains from IL-6 compared to saline-treated dams (median [interquartile range]: caudal hippocampus, 99 [94-104] and 64 [57-68], respectively, P = .002; lateral septum, 102 [96-111] and 68 [65-69], respectively, P = .002), as well as COX-2 immunostaining (lateral septum, 22 [20-26] and 17 [15-18], respectively, P = .005; dorsal hippocampus, 27 [22-32] and 16 [14-19], respectively, P = .013) and quantitative COX-2 Western blotting activity (mean ± standard error of the mean: vehicle, 0% of β-actin intensity versus IL-6, 41.5% ± 24%, P.001).Noninfectious inflammatory fever is inducible in the near-term pregnant rat by injection of IL-6 at levels comparable to those observed during human epidural labor analgesia. Maternal IL-6 injection causes neuroinflammation in the fetus.

Fever and increased maternal interleukin-6 (IL-6) plasma levels in labor are associated with an increased risk of adverse events in offspring, including neonatal seizures, cerebral palsy, and low intelligence scores at school age. However, the neural changes in the neonate that might mediate the adverse effects of maternal noninfectious fever are not fully characterized. This study was designed to test the hypothesis that induced maternal noninfectious fever alters neonatal neural progenitor cell proliferation and enhances microglial activation in the rat dentate gyrus of the hippocampus.Systemic vehicle or IL-6 was given 3 times to near-term pregnant rats (n = 7/group) every 90 minutes, and maternal core temperature was recorded. Neonatal brains were processed and analyzed for dentate gyrus cell proliferation (using Ki-67, n = 10/group, and glial fibrillary acidic protein, n = 6/group) and resident microglia activation (using ionized calcium-binding adaptor protein-1 [Iba-1], n = 6/group). In separate studies, the authors assessed microglia proliferation using Ki-67/Iba-1 costaining (n = 5/group).Compared to controls, exposure to IL-6 resulted in significant maternal temperature increase [mean temperature difference 0.558°C (95% CI, 0.417-0.698; P.0001)]. Following maternal IL-6, Ki-67 cell proliferation in the dentate gyrus was 55 % higher in neonates whose mother received IL-6 (38.8 ± 9.2) compared with those that received vehicle (25.1 ± 7.8); mean difference 13.7 (95% CI, 5.68-21.71); (P = .0021). Glial fibrillary acidic protein cell proliferation was 40% higher in the neonatal dentate gyrus whose mother received IL-6 when compared to controls (713 ± 85.52 vs 500 ± 115); mean difference 212 (95% CI, 82.2-343.4); (P = .004). Resident microglial activation was 90% higher in the dentate gyrus of neonates whose mother received IL-6 when compared to controls (71.8 ± 9.3 vs 37.8 ± 5.95); mean Iba-1 in stained cells was significantly different between IL-6 and vehicle groups 34 (95% CI, 23.94-44.05); (P.0001). Proliferating microglia, determined by the colocalization of Ki-67 and Iba-1, were not different in the vehicle (8.8 % ± 3.19 %) and the IL-6 (5.6% ± 2.3%) groups (mean difference 3.2% (95% CI, -0.8-7.25) (P = .1063).IL-6 is sufficient to induce maternal systemic temperature increases in near-term pregnant rats as well as neuronal, glial, and neuroinflammatory changes in the dentate gyrus of the neonatal hippocampus. These alterations might disrupt fetal neurodevelopment during a vulnerable period.

Early exposure to common anesthetic and sedative agents causes widespread brain cell degeneration and apoptosis in the developing rat brain, associated with persistent learning deficits in rats. This study was designed to determine whether the α2 adrenergic receptor agonist, dexmedetomidine, produces brain cell degeneration and apoptosis in postnatal day-7 rats in the same brain areas when compared to ketamine.Systemic saline, ketamine 20 mg/kg, or dexmedetomidine at 30 or 45 μg/kg were given six times to postnatal day 7 rats (n = 6/group) every 90 min. Twenty-four hours after the initial injection, brain regions were processed and analyzed for cell degeneration using the silver stain and for apoptosis using activated caspase-3 immunohistochemistry.Exposure to ketamine resulted in significant cellular degeneration and apoptosis in limbic brain regions, but nonsignificant changes in primary sensory brain regions. In contrast, dexmedetomidine produced significant cellular degeneration and apoptosis in primary sensory brain regions, but nonsignificant changes in limbic regions.These data show that ketamine and dexmedetomidine result in anatomically distinct patterns of cell degeneration and apoptosis in the brains of 7-day-old rat pups. The meaning and the clinical significance of these findings remain to be established.

Summary A 38-year-old woman presented with a pronounced increase in symptoms and proliferation of urticaria pigmentosa (UP) after acute psychological stress, which was quantified using the Spielberger’s State–Trait Anxiety Inventory. Immunohistochemical examination of a skin biopsy from a new UP lesion showed a large number of activated mast cells expressing corticotrophin-releasing factor receptor-1 (CRF-R1) and there was high serum CRF. This is the first documented report to our knowledge of UP worsening associated with acute stress, possibly through activation of skin mast-cell CRF-R1.

Two different RNAi methods were used to inhibit the expression of prohormone convertase 1 (PC1) in At-T20 cells. Transient transfection of double stranded RNA and stable expression of a vector expressing hairpin-loop RNA targeting PC1 reduced cholecystokinin (CCK) secretion from At-T20 cells. PC1 mRNA and protein were also decreased in the vector transfected cells. This treatment caused a shift in the forms of cholecystokinin (CCK) secreted, decreasing CCK 22 and increasing CCK 8. Stable expression of RNAi effectively decreased PC1 expression. The observed decrease in CCK seen with these RNAi treatments further supports a role for PC1 in CCK processing in these cells.

Background. The pathophysiology of neurocognitive dysfunction and developmental delay after cardiopulmonary bypass (CPB) in infants is not known. It is known that head trauma, stroke, and seizures cause dysfunction of the blood brain barrier (BBB) that is associated with increased inducible transcription factor gene expression in the cells of the barrier. The purpose of this study was to determine the effects of CPB and hypothermic circulatory arrest on expression of the transcription factor FOS and the function of the BBB in an infant animal model. Methods. Infant lambs (n 36; 10 –12 days) were exposed to 0, 15, 30, 60, or 120 minutes of normothermic (38°C) CPB or 2 hours of hypothermic circulatory arrest at 16°C. After terminating bypass 15 animals had their brains perfusion-fixed and removed for immunohistochemical analysis of expression of the transcription factor FOS. The other animals were perfused with fluorescent albumin to visualize the brain microvasculature. Brain sections were analyzed with a laser scanning confocal microscope. Results. Control animals (n 6, sham operated and cannulated) exhibited normal vasculature with negligible leakage and no FOS protein expression in neurons or endothelial cells anywhere in the brain. Significant FOS expression in barrier-associated structures including the blood vessels, choroid plexus, and ependyma but not neurons occurred at all times on bypass. CPB caused leakage of fluorescent albumin from blood vessels in all animals. Two hours of normothermic CPB (n 4) caused significant (p < 0.01) leakage in the cerebellum, cortex, hippocampus, and corpus callosum. Animals exposed to circulatory arrest experienced severe leakage throughout the brain (p < 0.001) and FOS expression in all cells. Conclusions. These experiments indicate that the BBB is dysfunctional after all time points on normothermic CPB, BBB dysfunction is worsened by hypothermic circulatory arrest, and BBB dysfunction is associated with intense molecular activity within the barrier-forming cells. Dysfunction of the BBB may contribute to neurologic complications after heart surgery. (Ann Thorac Surg 2004;78:1418 –25) © 2004 by The Society of Thoracic Surgeons

The use of “multimodal” combination analgesic therapies or novel single molecules possessing multiple analgesic targets is becoming increasingly attractive. In previous experiments we showed that a substance P antagonist injected intrathecally potentiated the antinociceptive effects of potent opioid receptor agonist, biphalin. Based on examination of the biphalin structure–activity relationship, we designed and synthesized a novel chimeric peptide, termed AA501 (N′(Tyr-D-Ala-Gly-Phe), Nʺ(Z-Trp) hydrazide, Z=benzyloxycarbonyl). AA501 consists of an opioid receptor agonist pharmacophore related to biphalin and a substance P receptor antagonist pharmacophore, both linked by a hydrazide bridge. The present study evaluates the ability of a novel chimeric peptide, AA501, to bind to opioid and substance P receptors and to produce antinociception in tail-flick and formalin tests, and in a neuropathic pain model when administered intrathecally to rats.

Physiological studies of odor-elicited responses from the olfactory epithelium and bulb in the tiger salamander, Ambystoma tigrinum, have elucidated a number of features of olfactory coding that appear to be conserved across several vertebrate species. This animal model has provided an accessible in vivo system for observing individual and ensemble olfactory responses to odorant stimulation using biochemical, neurophysiological, and behavioral assays. In this paper we have complemented these studies by characterizing 35 candidate odorant receptor genes. These receptor sequences are similar to those of the large families of olfactory receptors found in mammals and fish. In situ hybridization, using RNA probes to 20 of these sequences, demonstrates differential distributions of labeled cells across the extent and within the depth of the olfactory epithelium. The distributions of cells labeled with probes to different receptors show spatially restricted patterns that are generally localized to different degrees in medial-lateral and anterior-posterior directions. The patterns of receptor expression in the ventral olfactory epithelium (OE) are mirrored in the dorsal OE. We present a hypothesis as to how the sensory neuron populations expressing different receptor types responding to a particular odorant may relate to the distribution patterns of epithelial and bulbar responses previously characterized using single-unit and voltage-sensitive dye recording methods.

Recent animal models of experimental nerve injury have proven useful in evaluating potential sympathetic involvement in neuropathic pain syndromes. We have employed a widely adopted unilateral L 5 /L 6 spinal nerve ligation model to compare the development of mechanical allodynia with neurochemical changes both at the site of peripheral nerve injury and in the dorsal root ganglia (DRG). We have focused on the expression of neuropeptide Y (NPY), a well-studied regulatory peptide and phenotypic marker of sympathetic neurons, and functionally related Y-receptor binding sites following nerve injury. In sympathetic neurons, NPY is colocalized and coreleased with norepinephrine (NE) at peripheral sites of action. Furthermore, NPY gene expression is induced within the population of medium- and large-diameter DRG neurons of the A β -fiber class after experimental nerve injury. We therefore hypothesized that concurrent alterations in NPY and NE expression by sympathetic and sensory neurons may be a contributing factor to sympathetically-maintained neuropathic conditions. Animals with unilateral L 5 /L 6 spinal nerve ligation developed mechanical allodynia of the hind paw ipsilateral to the site of injury that persisted until sacrifice at postoperative day 10. A significant induction of preproneuropeptide Y-encoding (PPNPY) mRNA, as detected by in situ hybridization histochemistry (ISHH), occurred in populations of medium- and large-diameter DRG neurons ipsilateral to the site of injury. Immunohistochemical analysis indicated a marked decline in the number of labeled sympathetic axons positive for tyrosine hydroxylase-like and NPY-like immunoreactivities (TH-Ll and NPY-LI, respectively) proximal to the site of nerve injury and almost complete elimination of immunopositive fibers distal to the site of ligation. Whereas, the extent of colocalization of NPY-LI to TH-LI-positive sympathetic axons in unaffected L 4 or L 5 nerve segments exceeded 80%, this figure declined to approximately 50% in regenerating axons of ligated spinal nerve L 5 . The portion of NPY-LI that was not colocalized to sympathetic TH-LI-positive fibers was most likely contributed by regenerating sensory axons, consistent with marked de novo synthesis of NPY by DRG neurons. In end bulb axon terminals, i.e. morphological profiles characteristic of neuromas, NPY-LI-positive elements that were not colocalized to TH-LI-positive sympathetic elements appeared to be spatially segregated from those of sympathetic origin with colocalized TH-LI and NPY-LI. Receptor autoradiography indicated that small- and medium-diameter DRG somata of the C-fiber class normally express both Y 1 and Y 2 receptor subtypes. The pattern of the distribution of Y-receptor binding sites appeared to be relatively unaffected by spinal nerve ligation. In contrast, there was a marked increase in the density of Y 2 receptor binding sites in the proximal segment of ligated spinal nerve L 5 , consistent with previously published data indicating differential transport of the Y 2 autoregulatory receptor subtype to nerve terminals. Induction of NPY gene expression in injured DRG neurons is consistent with appearance of NPY-LI-positive end bulbs derived from regenerating sensory axons that are found in developing neuromas containing a relatively high density of transported prejunctional Y 2 receptors. Newly established functional interactions of spatially segregated sensory- and sympathetically-derived end bulbs in developing neuromas may enhance neuronal hyperexcitability engendered by aberrant electrical activity at the site of injury. Injury-related alterations in the regulatory activities of NPY released within the DRG at somally-distributed Y-receptors may also contribute to the development and/or persistence of symptoms characteristic of sympathetically-maintained pain. Finally, at later times NPY-mediated modulation of NE release from invading sympathetic axon terminals within the DRG may affect the extent of α 2 receptor-mediated neuronal hyperexcitability associated with neuropathic pain.

The objective of the present study was to determine how c-fos gene expression in brainstem structures after a brief episode of social defeat stress is related to behavioral sensitization to cocaine challenge. Social stress was defined as defeat in a brief confrontation with an aggressive resident mouse and subsequent 20-min exposure to the resident's threats behind a protective screen. Mice were treated with cocaine (40 mg/kg, i.p.) immediately or 1 week after social defeat stress. Fos-like immunoreactive (Fos-LI) cell nuclei were analyzed in the ventral tegmental area (VTA), dorsal raphe nucleus (DR), periaqueductal grey area (PAG) and locus coeruleus (LC). One episode of social stress induced behavioral sensitization to cocaine as indicated by an augmented locomotor response to a challenge injection 7 days after a single defeat. In naive mice, social stress markedly increased the number of Fos-LI nuclei in the DR, PAG and LC, but not in the VTA. Similarly, cocaine administration resulted in a significantly increased number of Fos-LI nuclei in the same areas. Administration of cocaine immediately following social defeat significantly reduced the number of Fos-LI nuclei in the DR, PAG and LC. Cocaine-induced Fos expression returned in the PAG and DR, but not in the LC, 1 week after social stress. In conclusion, the present results suggest that the presence of brainstem Fos be related to the ability to express stress-induced behavioral sensitization to cocaine.

Altered gene expression occurs in the brain after global ischemia. We have developed a model to examine the effects of cardiopulmonary bypass and hypothermic circulatory arrest (HCA) on the induction of the immediate-early gene c-fos in the brains of neonatal lambs. We then tested the effects of the noncompetitive N-methyl-D-aspartate antagonist, aptiganel hydrochloride (Cerestat), on c-fos expression and neuronal injury.Neonatal lambs (weight, 4 to 6 kg) anesthetized with isoflurane were supported by cardiopulmonary bypass, subjected to 90 or 120 minutes of HCA at 15 degrees C, and rewarmed on bypass to 38 degrees C. One hour after cardiopulmonary bypass was terminated, the brains were perfusion fixed and removed for in situ hybridization and immunohistochemical analysis. Some animals survived 3 days before their brains were removed to examine for neuronal necrosis. One group of lambs (n = 20) received aptiganel (2.5 mg/kg). A second group (n = 25) received saline vehicle only.Increasing duration of HCA induced a corresponding increase in c-fos messenger RNA expression throughout the hippocampal formation and cortex. However, Fos protein synthesis peaked after 90 minutes of HCA and decreased significantly (p0.01) after 120 minutes of HCA. Aptiganel administration caused a significant decrease in (p0.001) c-fos messenger RNA expression and Fos protein synthesis after 90 minutes of HCA and preserved Fos protein synthesis after 120 minutes of HCA. Neuronal necrosis was observed in the brains of vehicle-treated lambs after 120 minutes of HCA but was significantly decreased (p0.05) in the lambs given aptiganel.These experiments indicate that the transcriptional processes of immediate-early genes remain intact, whereas translational processes are impaired after prolonged HCA. The inability to synthesize Fos proteins after 120 minutes of HCA was associated with neuronal degeneration. Aptiganel preserved translational processes and caused a significant improvement in the neurologic outcome.

Prior studies in rodents have shown significant depletion of reduced glutathione (GSH) in peripheral organs following acute systemic or central administration of opioids. However, little information exists on whether opioid administration affects concentrations of brain GSH. Recently, clinical observations have indicated acute declines of GSH concentrations in the cerebrospinal fluid of cancer patients after acute intracerebroventricular (ICV) morphine which may contribute to the development of organic behavioral brain syndromes associated with central opioid analgesia. Collectively these data led us to investigate the affect of acute systemic and central morphine on regional concentrations of GSH in rat brain. Systemic morphine had no effect on GSH concentrations in selected brain areas. In contrast, ICV morphine resulted in selective GSH depletion in the caudate nucleus, consistent with concurrent excitatory locomotive behavior. This change may have reflected morphine-induced oxidative stress together with increased metabolic activity within the extrapyramidal system.

Background Cardiopulmonary bypass (CPB) and hypothermic circulatory arrest (HCA) are associated with neurological injury. Altered immediate-early gene expression occurs rapidly in the brain in response to ischemia, hypoxia, and severe metabolic stress, which results in long-term changes in the molecular phenotype of neurons. This study determined the effects of CPB and HCA on the expression of the immediate-early gene c-fos. Methods Neonatal lambs were subjected to 2 h of CPB at 38 degrees C (n = 4) or 60 min (n = 6), 90 min (n = 7), and 120 min (n = 6) of HCA at 15 degrees C. One hour after terminating CPB at 38 degrees C, the brains were analyzed for FOS-encoding mRNA and FOS-like immunoreactivity in the hippocampal formation. Other animals (n = 15), subjected to the same CPB and HCA protocol, were allowed to survive 3-5 days before their brains were examined for dead neurons. Results Minimal c-fos mRNA and FOS proteins were observed in neurons of animals subjected to normothermic bypass and of those that served as controls. Non-neuronal FOS proteins were observed in the choroid plexus, ependyma, and blood vessels at all times, including normothermic CPB, but not in the control animals without CPB. The magnitude of c-fos mRNA expression in hippocampal neurons increased directly with the duration of HCA. In contrast, expression of FOS proteins peaked after 90 min of HCA and declined significantly thereafter. Dead neurons were seen in surviving animals after 2 h of HCA only. Conclusions Cardiopulmonary bypass and HCA alter immediate-early gene expression in the brain. Translational processes are impaired after 120 min of HCA and correlate with neuron death in the hippocampus.

Major complications arising from diabetes mellitus include neuropathic pain and altered peripheral inflammatory responses. Somatostatin (SOM), calcitenin gene-related peptide (CGRP), and substance P (SP) are neuropeptides that modulate pain responses transmitted by primary sensory afferents, the cell bodies of which are located in the dorsal root ganglion (DRG). Thus, the goal of the present study was to determine whether the diabetic condition is associated with altered neuropeptide gene expression in lumbar DRG of the rat. We employed an established animal model in which streptozotocin (STZ, 55 mg/kg) is administered to 6 week-old rats. The hallmark symptoms of hyperglycemia (blood glucose400 mg/dl), polydipsia, polyuria, and severe weight loss were maximal at 6 weeks postadministration, at which time animals were sacrificed. For determination of peptide encoding mRNAs distributed in DRG neurons, in situ hybridization histochemistry utilizing S-end-labeled oligonucleotides complimentary to sequences of preprosomatostatin (PPSOM), preprocalcitonin gene related peptide (PPCGRP), preprotachykinin (PPT), or preproneuropeptide Y (PPNPY) mRNA was performed. Silver grains were detected overlying DRG cells by autoradiography on sections of tissue counterstained with thionin. Semiquantitative analysis of differences in silver grain signal were made using an image analysis system, which expressed signals as fCi/microns2. In diabetic rats there was a significant decrease in DRG PPSOM (54%, p0.01), and PPCGRP (33%. p0.05) mRNA hybridization from the normal values PPT mRNA hybridization signal and SP-like immunoreactivity were not significantly changed in diabetic rat DRGs compared to control. In contrast, there was an increase in the number of cells labeled with PPNPY hybridization in DRG from diabetic rats. These data suggest that CGRP and SOM synthesis in primary sensory neurons is reduced in STZ-induced diabetic rats. These changes could contribute to the painful neuropathies and altered inflammatory responses seen in diabetes mellitus.

Steady-state levels of the prototypic tachykinin neuropeptide substance P (SP) and its major precursor form substance P-glycine (SP-G) were detected and authenticated in guinea pig vagal and respiratory tissues by radioimmunoassay (RIA), combined high-performance liquid chromatography (HPLC)/RIA analyses, and immunohistochemistry. Four antisera were employed: anti-SP that recognizes the amidated COOH-terminal of SP and is specific for the mature peptide, anti-SP4-10 that recognizes the midportion 4-10 amino acid sequence of SP and is highly specific for both mature SP and extended precursor forms of SP, anti-SP-G that is highly specific for the unamidated COOH-terminal of SP-G, and affinity-purified anti-SP-G-K that is capable of detecting SP-G and minor forms of SP precursor in immunohistochemical analyses. In all examined areas, the content of substance P4-10-like immunoreactivity (SP4-10-LI) quantified by RIA with the use of anti-SP4-10 was greater than that quantified by RIA with the use of anti-SP serum, thereby providing biochemical evidence of steady-state expression of extended precursor forms of SP. Immunohistochemical analyses demonstrated labeled axonal profiles indicating the presence of immunoreactive SP as well as immunoreactive forms of SP precursor within lung hilum and in small fibers in the parenchyma, with no evidence of labeled neuronal cell bodies in these same areas.

This report describes the generation and biochemical characterization of a high-affinity antiserum that recognizes an epitope contained in the midportion sequence of substance P, i.e., substance P4-10. Designated A47, this reagent bound a variety of related peptide species containing the substance P4-10 sequence with apparent equipotency. A double radioimmunoassay procedure was developed that utilized A47, in combination with a traditional high-affinity COOH-terminally directed anti-substance P serum, to provide quantification of mature and immature forms of substance P in CNS tissues. Across most rat CNS areas, levels of substance P-like immunoreactivity were consistently 15% higher when monitored by analyses using A47 versus anti-substance P serum. In the dorsal root ganglia, an apparent enhancement in levels of substance P-like immunoreactivity of approximately 40%, when quantified by analyses using A47 versus anti-substance P serum, was observed; this most likely reflected the presence of an active biosynthetic pool of intermediate processing forms of substance P in this tissue. Coordinated HPLC/radioimmunoassay analyses of extracted dorsal root ganglia tissues demonstrated multiple peaks of immunoreactivity corresponding to mature substance P and to several of its precursor forms found in the normal biosynthetic pathway. Of the total recovered HPLC-fractionated immunoreactivities, that corresponding to the putative immediate precursor to substance P, i.e., substance P-glycine, was the predominant peak. In an additional series of HPLC/radioimmunoassay analyses, selective decreases in immunoreactive peaks corresponding to precursor forms of substance P were observed in dorsal root ganglia tissues from rats treated with the neurotoxic agent capsaicin. These results indicated decreased turnover of substance P as a consequence of drug treatment. Finally, initial immunohistochemical analyses employing affinity-purified A47 produced an unusual pattern of labeling characterized by well defined punctate terminal elements within the superficial aspects of the dorsal horn of the spinal cord.

SUMMARY: Although mesenchymal stem cells (MSC) from different tissue sources share many characteristics and generally fulfill accepted criteria for MSC (plastic adherence, certain surface marker expression, and ability to differentiate into mesenchymal tissues), we are increasingly learning that they can be distinguished at the level of cytokine production and gene expression profiles. Their ability to differentiate into different tissues including endodermal and ectodermal lineages, also varies according to tissue origin. Importantly, MSC from fetal sources can undergo more cell divisions before they reach senescence than MSC from adult tissue such as bone marrow or adipose tissue. As we learn more about the differentiation and plasticity of MSC from different sources, health care providers in the future will use them tailored to different medical indications.

The biosynthetic enzyme peptidylglycine alpha-amidating monooxygenase catalyzes the formation of a variety of biologically active alpha-amidated peptides from respective COOH-terminal glycine-extended peptide precursors. Peptidylglycine alpha-amidating monooxygenase activity is dependent on copper, ascorbate, and molecular oxygen and is inhibited by the relatively selective copper chelator N,N-diethyldithiocarbamate or its disulfide dimer disulfiram (Antabuse). In the present study, chronic disulfiram treatment (100 mg/kg/day, for 12-25 days) resulted in significant changes in several neurochemical parameters in the mouse central nervous system, including levels of substance P-like, unamidated substance P-Gly-like, and protease-generated substance P-Gly-Lys-like immunoreactivities (SP-LI, SP-G-LI, and SP-G-K-LI, respectively). Combined high performance liquid chromatography/radioimmunoassay analyses of the extracted SP-LI, SP-G-LI, and SP-G-K-LI species indicated very similar chromatographic and immunochemical behavior as demonstrated for chemically authentic peptide standards. Additionally, changes in levels of monoamines and their metabolites were observed after drug administration. Complementary immunohistochemical analyses using affinity-purified anti-SP-G sera localized these drug-induced changes in levels of immunoreactive unamidated precursor to neural elements that normally express SP. As a functional corollary to alterations in neurochemical parameters, we observed significant disulfiram-induced increases in pain thresholds, potentiated by capsaicin treatment. Overall, our results indicate that the observed changes in steady state levels of immunoreactive SP and of the immature COOH-terminal extended forms of SP may reflect compensatory biosynthetic and posttranslational processing events in SP-containing neural systems after pharmacological challenge.

Current evidence from clinical and laboratory studies confirms that mast cells play a central role in the pathogenesis and pathophysiology of interstitial cystitis (IC). In this article, we focus on the role of the mast cell in IC and examine the ways in which mast cells and other pathophysiologic mechanisms are interrelated in this disease. Identifying the patients with IC who have mast cell proliferation and activation will enable us to address this aspect of disease pathophysiology in these individuals with targeted pharmacotherapy to inhibit mast cell activation and mediator release.

Prohormone convertase 2 is widely co-localized with cholecystokinin in rodent brain. To examine its role in cholecystokinin processing, cholecystokinin levels were measured in dissected brain regions from prohormone convertase 2 knock-out mice. Cholecystokinin levels were lower in hippocampus, septum, thalamus, mesencephalon, and pons in knock-out mice than wild-type mice. In cerebral cortex, cortex-related structures and olfactory bulb, cholecystokinin levels were higher than wild type. Female mice were more affected by the loss of prohormone convertase 2 than male mice. The decrease in cholecystokinin levels in these brain regions shows that prohormone convertase 2 is important for cholecystokinin processing. Quantitative polymerase chain reaction measurements were performed to examine the relationship between peptide levels and cholecystokinin and enzyme expression. They revealed that cholecystokinin and prohormone convertase 1 mRNA levels in cerebral cortex and olfactory bulb were actually lower in knock-out than wild type, whereas their expression in other brain regions of knock-out mouse brain was the same as wild type. Female mice frequently had higher expression of cholecystokinin and prohormone convertase 1, 2, and 5 mRNA than male mice. The loss of prohormone convertase 2 alters CCK processing in specific brain regions. This loss also appears to trigger compensatory mechanisms in cerebral cortex and olfactory bulb that produce elevated levels of cholecystokinin but do not involve increased expression of cholecystokinin, prohormone convertase 1 or 5 mRNA.

Prohormone convertase (PC1) is found in endocrine cell lines that express cholecystokinin (CCK) mRNA and process pro CCK to biologically active products. Other studies have demonstrated that PC1 may be a one of the enzymes responsible for the endoproteolytic cleavages that occur in pro CCK during its biosynthesis and processing. Prohormone convertase 1 (PC1) has a distribution that is similar to cholecystokinin (CCK) in rat brain. A moderate to high percentage of CCK mRNA-positive neurons express PC1 mRNA. CCK levels were measured in PC1 knockout and control mice to assess the degree to which loss of PC1 changed CCK content. CCK levels were decreased 62% in hippocampus, 53% in amygdala and 57% in pons-medulla in PC1 knockout mice as compared to controls. These results are highly correlated with the colocalization of CCK and PC1. The majority of CCK mRNA-positive neurons in the pyramidal cell layer of the hippocampus express PC1 mRNA and greater than 50% of CCK mRNA-positive neurons in several nuclei of the amygdala also express PC1. These results demonstrate that PC1 is important for CCK processing. PC2 and PC5 are also widely colocalized with CCK. It may be that PC2, PC5 or another non-PC enzyme are able to substitute for PC1 and sustain production of some amidated CCK. Together these enzymes may represent a redundant system to insure the production of CCK.

We investigated the effect of epidural local anesthetic blockade on urinary substance P levels in five patients suffering from painful flare-ups of interstitial cystitis. Urine was collected in 24-h intervals commencing at the onset of an epidural bolus of 0.25% bupivacaine followed by maintenance epidural infusions of 0.05% bupivacaine. Substance P was measured by radioimmunoassay. After initiation of the epidural infusion, urinary substance P levels increased and then declined in all patients. All patients reported a decrease in pain intensity. We hypothesize that acute release, followed by depletion, of substance P from bladder sensory nerve endings accounts for the transient increase of peptide levels in urine and may contribute to the decrease in pain intensity during a 3-day epidural infusion.Substance P levels in urine initially increased and then declined in a series of 5 patients who achieved pain control by epidural local anesthetic infusion during a flare-up of interstitial cystitis.

During posttranslational processing to generate CCK 8, pro-cholecystokinin (CCK) undergoes endoproteolytic cleavage at three sites. Several studies using endocrine and neuronal tumor cells in culture and recombinant enzymes and synthetic substrates in vitro have pointed to the subtilisin/kexin-like enzymes prohormone convertase (PC) 1, PC2, and PC5 as potential candidates for these endoproteolytic cleavages. In these experimental models, they all appear to be able to cleave pro-CCK to make the correct products. One rodent model has provided information about the role of PC2. PC2 knockout mouse brains had less CCK 8 than wild-type, although a substantial amount of CCK was still present. The degree to which CCK levels were reduced in these mice was regionally specific. These data indicated that PC2 is important for normal production of CCK but that it is not the only endoprotease that is involved in CCK processing. To evaluate whether PC1 and PC5 are possible candidates for the other enzymes involved in CCK processing, the distribution of PC1, PC2, and PC5 mRNA was studied in rat brain. Their colocalization with CCK mRNA was examined using double-label in situ hybridization. PC2 was the most abundant of these enzymes in terms of the intensity and number of cells labeled. It was widely colocalized with CCK. PC1 and PC5 mRNA-positive cells were less abundant, but they were also widely distributed and strongly colocalized with CCK in the cerebral cortex, hippocampus, amygdala, ventral tegmental area, and substantia nigra zona compacta. The degree of colocalization of the enzymes with CCK was regionally specific. It is clear that PC1 and PC5 are extensively colocalized with CCK and could be participating in CCK processing in the rat brain and may be able to substitute for PC2 in its absence. These three enzymes may represent a redundant system to ensure production of biologically active CCK.

The present study characterized the response of P-glycoprotein (P-gp) and multidrug resistance-associated protein (MRP1) to chronic ritonavir (RIT) exposure by assessing increases in P-gp and MRP1 protein expression and activity. LS-180V intestinal carcinoma cells were exposed for 3 days to 1–100 μM RIT concurrently with controls. P-gp and MRP1 protein was quantified by Western blot analysis. Cell accumulation assays, using the P-gp substrate rhodamine 123 (RH123), the P-gp/MRP1 substrate doxorubicin (DOX), and the MRP substrate carboxyfluorescein (CBF), were performed as a measure of transporter activity. RIT strongly induced P-gp and MRP1 expression (maximum 6-fold and 3-fold increases, respectively) in a concentration-dependent fashion. Following extended exposure to RIT (> 10 μM), cells accumulated

To elucidate mechanisms of acute and chronic pain, it is important to understand how spinal excitatory systems influence opioid analgesia. The tachykinin substance P (SP) represents the prototypic spinal excitatory peptide neurotransmitter/neuromodulator, acting in concert with endogenous opioid systems to regulate analgesic responses to nociceptive stimuli. We have synthesized and pharmacologically characterized a chimeric peptide containing overlapping NH 2 - and COOH-terminal functional domains of the endogenous opioid endomorphin-2 (EM-2) and the tachykinin SP, respectively. Repeated administration of the chimeric molecule YPFFGLM-NH 2 , designated ESP7, into the rat spinal cord produces opioid-dependent analgesia without loss of potency over 5 days. In contrast, repeated administration of ESP7 with concurrent SP receptor (SPR) blockade results in a progressive loss of analgesic potency, consistent with the development of tolerance. Furthermore, tolerant animals completely regain opioid sensitivity after post hoc administration of ESP7 alone, suggesting that coactivation of SPRs is essential to maintaining opioid responsiveness. Radioligand binding and signaling assays, using recombinant receptors, confirm that ESP7 can coactivate μ-opioid receptors (MOR) and SPRs in vitro . We hypothesize that coincidental activation of the MOR- and SPR-expressing systems in the spinal cord mimics an ongoing state of reciprocal excitation and inhibition, which is normally encountered in nociceptive processing. Due to the ability of ESP7 to interact with both MOR and SPRs, it represents a unique prototypic, anti-tolerance-forming analgesic with future therapeutic potential.

Background This study determined the induction profiles of immediate-early genes in the ovine brain after cardiopulmonary bypass (CPB) and hypothermic circulatory arrest (HCA), and the effects of the noncompetitive N-methyl-D-aspartate antagonist, aptiganel, on immediate-early gene expression, neuronal necrosis, and functional outcome. Methods Cannulas were inserted into isoflurane-anesthetized neonatal lambs undergoing CPB. One group received 2.5 mg/kg intravenous aptiganel. Animals underwent 90 or 120 min of HCA at 16 degrees C, were rewarmed to 38 degrees C, and were weaned from CPB. One hour after CPB was discontinued, brain perfusion was fixed and removed for immunohistochemical analysis in one half of the animals. The other half survived 2 or 3 days before their brains were evaluated for neuronal degeneration. Data were analyzed using analysis of variance; P < 0.05 was considered significant. Results Cardiopulmonary bypass and HCA differentially induced c-Jun and Fos proteins in the hippocampal formation, with c-Jun expression increasing with the duration of HCA, whereas Fos protein expressions were greatest after 90 min of HCA. The c-Jun protein was expressed in all neurons except the dentate gyrus. The Fos proteins were expressed in all neurons, including the dentate gyrus. Neuronal necrosis was observed in CA1 (73%) and CA3 (29%) neurons but not in the dentate gyrus after 120 min of HCA. Aptiganel completely inhibited c-Jun expression (P < 0.001) but not Fos, improved functional outcome, and attenuated neuronal necrosis (P < 0.05). Conclusions The c-Jun and c-Fos proteins are expressed differentially in hippocampal neurons after CPB and HCA. Expression of c-Jun is associated with neuronal necrosis, whereas Fos protein expression is associated with survival. Aptiganel inhibits c-Jun expression, attenuates neuronal necrosis, and improves outcome.

Complications arising from diabetes mellitus include hormonal dysfunctions such as impairment in the regulation of gonadatroph and corticotroph secretion. Preprotachykinin (PPT) mRNA encoding the peptide substance P (SP), has been localized in the anterior pituitary. The goal of this study was to determine if streptozotocin (STZ)-induced diabetes affects the SP content or PPT mRNA level in the pituitary of male rats. We injected STZ (55 mg/kg) to 6-week-old rats which developed hyperglycemia (blood glucose400 mg/dl) by 6 weeks post-injection. SP-like immunoreactivity in the pituitary dropped 54%. In situ hybridization was performed using a PPT-specific oligonucleotide with signal intensity differences semi-quantified using an image analysis system. Normal pituitary had a regional distribution of PPT mRNA, with no detectable signal in the posterior or intermediate lobes, while the anterior lobe displayed a distinctive pattern of labeled cells arranged in clusters. In diabetic rats there was a 23% decrease in the PPT-mRNA hybridization signal compared to controls (P0.05). The changes observed in PPT gene expression and SP content may be additional factors participating in the hormonal complications seen in diabetes mellitus.

The experiments described in the present study approached nerve injury from both a biochemical and anatomical perspective by monitoring changes in expression of preprotachykinin (PPT) mRNA encoding the prototypic tachykinin substance P and related peptide species in neurons of the rat dorsal root ganglia (DRG) following unilateral chronic constriction injury of the sciatic nerve. In situ hybridization histochemistry (ISHH) analyses in conjunction with computer-assisted image processing were employed to quantify levels of PPT mRNA distributed in DRG neurons. Injury-induced changes in PPT mRNA expression by affected DRG neurons included: 1. (1) at early postoperative times, generally increased levels of PPT mRNA associated with small and intermediate-size B cells exhibiting normal morphology, 2. (2) at late postoperative times, markedly decreased levels of PPT mRNA associated with degenerating B cells, and 3. (3) induction of PPT gene expression by large A cells which is highly correlated with degenerative morphological changes. The significant aspects of these changes are discussed with special emphasis on the contribution of altered transmitter expression by DRG neurons to the pathophysiology of causalgia. In particular, the induction of PPT gene expression by many of the large neurons undergoing degenerative changes may represent an important biochemical parameter which is associated with the development and persistence of experimental allodynia.

We examined the effects of chronic diethyldithiocarbamate (DDC) treatment on the concentrations of methionine-enkephalin, mature and unamidated forms (-Gly) of luteinizing hormone releasing hormone (LHRH) and substance P (SP) in various regions of the central nervous system (CNS). Chronic DDC treatment resulted in elevations of LHRH-Gly like immunoreactivity in the preoptic area (POA) and the medial basal hypothalamus (MBH), as well as elevations in SP-Gly like immunoreactivity in all areas of the CNS examined. Castration altered the ratios of SP-G-like/SP-like immunoreactivity in the pons, and LHRH-Gly like immunoreactivity in the MBH. Met-enkephalin concentrations were significantly elevated in the pons and medulla of intact DDC-treated animals, and in the POA of both intact- and castrated DDC-treated animals. Results demonstrate that it is possible to detect basal levels of unamidated LHRH and SP in many areas of the CNS, with ratios of unamidated/amidated peptides representing a unique and sensitive method for determining altered posttranslational processing of these transmitters, especially under altered endocrine states such as castration. Pharmacological blockade of terminal enzymatic processing of these peptides may be useful in studying upstream regulatory events in peptidergic neurons.

The undecapeptide substance P and the alkaloid morphine sulfate are two agents previously thought to have opposite roles in the mediation of spinal nociceptive processes. The present report, however, demonstrates that low doses of substance P when coadministered with marginally effective doses of morphine sulfate into the rat subarachnoid space produce a markedly enhanced analgesic response, as monitored by the tail-flick test. This pharmacological effect is blocked by prior treatment with the opioid antagonist naloxone, indicating that the potentiated analgesic response is mediated by opioid-responsive neurons. In addition, the putative immediate precursor form of substance P (i.e., substance P-glycine) may substitute for the mature compound in the potentiated pharmacological effect. Moreover, the described synergism is unaffected by transection of the spinal cord, demonstrating the lack of supraspinal modulation of the observed phenomenon. Based on these observations, we are now able to dissociate opioid-potentiating and analgesic properties of substance P from traditional hyperalgesic effects realized at significantly higher concentrations. Consistent with previous biochemical data, a likely mechanism underlying the peptide-mediated enhancement of opioid analgesia may center on the ability of substance P to release endogenous opioid peptides within the local spinal cord environment. Finally, the pharmacological relationship of coadministered substance P and morphine sulfate established here supports the hypothesis that spinal tachykinin and opioid systems have a direct functional interaction in the modulation of local nociceptive responses.

Previous work from this laboratory has provided biochemical characterization of several posttranslational processing intermediates of the neuropeptide substance P (SP) in central nervous system (CNS) tissues, including the COOH-terminal glycine-extended dodecapeptide Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-Gly (SP-G). SP-G is a major species of unprocessed SP found in rodent CNS tissues, and is the likely immediate precursor form of SP in the biosynthetic scheme. Here we present extensive characterization of the normal regional distribution of SP-G, as compared to SP, throughout the rat CNS via coordinated biochemical and morphological analyses. By radioimmunoassay (RIA), an approximate 10-fold variation in regional levels of SP-G-like immunoreactivity (SP-G-LI) was observed, ranging from 0.30 pmol/g in the amygdala, to 6.49 pmol/g in the medulla. On a normalized basis, the regional variation of unamidated precursor relative to mature peptide (SP-G-LI/SP-LI molar ratio) ranged from 0.30% in the amygdala to 5.15% in the dorsal root ganglia (DRG). Overall, the highest SP-G-LI/SP-LI ratios were found in DRG, medulla, and spinal cord, i.e. CNS areas associated with primary sensory afferent innervation via capsaicin-sensitive unmyelinated small diameter fibers. In addition, chromatographic and RIA analyses of extracted brain tissues indicated that the quantified immunoreactivities corresponding to SP, SP-G, as well as an additional COOH-terminal Gly-Lys-extended precursor, i.e., SP-G-K, displayed very similar chromatographic behavior as demonstrated for chemically authentic standards. These biochemical data were complemented by immunohistochemical analyses demonstrating a pattern of immunohistochemical staining for the presence of SP-G-LI as a defined subset of SP-LI-containing neural elements. Here, reaction product was localized to dendritic, axonal, and terminal neuronal elements in representative CNS regions of the rat, with relatively high levels of SP-G-LI found within anatomical areas containing a high density of sensory terminal structures. In an attempt to provide correlative functional anatomy, a group of rats was treated with colchicine, in order to differentially localize SP-LI- and SP-G-LI-containing somata after inhibition of axoplasmic transport. Most prominently, colchicine administration engendered immunohistochemical visualization of both SP-LI- and SP-G-LI-positive cells in mesencephalic and brainstem regions associated with stress, pain responses, and central control of autonomic function. Within this context, the coordinate expression of both SP-LI- and of SP-G-LI-positive somata in discrete brain areas is probably indicative of high ongoing rates of tachykinin synthesis coupled to utilization.

Biochemical studies on the hippocampus of acutely prepared rabbits revealed more than two fold increases in cyclic GMP levels following tetanic potentiation of the pathway from the medial septal region to CA1 pyramidal cells. Diazepam, administered intravenously, prevented the elevation in cyclic GMP levels in this region and also attenuated the post-tetanic potentiation seen following the presentation of trains at frequencies within theta rhythm. The results imply a modulatory role for cyclic nucleotides in the enhancement of pyramidal cell excitability and suggest that the biochemical mechanism for the psychoactive benzodiazepines may well include the suppression of cyclic GMP levels.

The effects of muscimol on rabbit hippocampal pyramidal cell firing were studied and compared after iontophoretic, topical, and intravenous administration of the drug. All modes of application resulted in a bicuculline-sensitive, strychnine-insensitive, depression of the monosynaptically activated population spike evoked by micro-stimulation of the contralateral hippocampal field. These findings indicate that systemically administered muscimol selectively activates hippocampal GABA receptors suggesting that this compound may be useful for studying limbic system physiology.

In rats, arginine-vasopressin (AVP) iontophoretically applied to lateral septal neurons activated by fimbria stimulation produced a decrease in the amplitude of the fimbria-evoked field response and a decrease in the frequency of fimbria-evoked action potentials. A second group of rats, treated with α-methyl-p-tyrosine, showed a significantly smaller decrease in responses after AVP iontophoresis, suggesting an interaction between catecholamines and AVP at the synaptic level.

Field and extracellular unitary potentials were recorded in the ventromedial nucleus of the hypothalamus (VMH) of urethane-anesthetized rabbits after stimulation of the fimbria and stria terminalis. Stimulation of the lateral portion of the fimbria, which carries fibers from the ventral subiculum of the hippocampal formation, evoked a two-component response. An early excitatory response, with an average latency of 10 msec, predominated along the lateral margins of the VMH. A later inhibitory potential with an average latency of 15 msec was seen predominantly within the central portions of the VMH. Stimulation of the dorsal component of the stria terminalis produced two similar response patterns: an early excitatory response with an average latency of 16 msec, followed by an inhibitory potential with an average latency of 25 msec. The topographical distribution of these two components of the response was nearly identical to that produced by lateral fimbria stimulation. In contrast, stimulation of the ventral component of the stria terminalis evoked a simple excitatory response with an average latency of 10 msec which was maximal within the core of the VMH. Extracellular unitary recordings showed that the early negativity associated with stimulation of each of these three pathways reflects a monosynaptic excitation of VMH cells and that there was convergence of the three excitatory inputs at the single cell level.

Halothane was found to have suppression effects on hippocampal tetanic and post-tetanic potentiation, even when no suppressive effects were seen with respect to pretetanic control responses. At the same time, halothane stimulated an elevation of cyclic AMP in hippocampal tissue. An initial halothane-related excitatory effect on pyramidal cells was also noted. It is suggested that in the hippocampus, halothane can modulate the potentiation phenomena by altering cyclic AMP concentrations.

Norepinephrine (NE) and dopamine (DA) are present in the lateral septum and presumably modulate activity of neurons in this region. Iontophoretic techniques were used to study the actions of NE and DA on stimulation-evoked activity of neurons in the lateral septum. Stimuli were applied to the fimbria, and the effects of NE and DA on field and unitary responses were recorded. Norepinephrine strongly inhibited the monosynaptically driven negative field potential and associated unitary responses. Dopamine produced an inhibitory, excitatory, or bimodal pattern of changes in the monosynaptic responses. Ventrally in the lateral septum, where a later polysynaptic excitatory response to fimbria stimulation was also seen, NE and DA augmented the polysynaptic responses, while producing inhibitory (NE) or inhibitory-excitatory (DA) effects on the monosynaptic response. Effects of NE and DA may play a role in modulation of the relay of descending hippocampal input to the medial septum, which is relayed by way of neurons in the lateral septum. In this manner catecholamines may indirectly produce modulatory effects in the theta pace-making cells in the medial septum.