The management of endoscopic retrograde cholangio- pancreatography-related infections risk: results of an italian survey at regional level.

Background and aim: Among the Endoscopic retrograde cholangiopancreatography (ERCP) adverse events, an increasingly arising problem is the transmission of Multi Drug Resistant (MDR) Bacteria through duodenoscopes. The aim of this survey was to evaluate the current clinical practice of management of ERCP associated infections in Emilia-Romagna, Italy. Methods: An online survey was developed including 12 questions on management of ERCP associated infections risk. The survey was proposed to all 12 endoscopy centers in Emilia Romagna that perform at least > 200 ERCPs per year. Results: 11 centers completed the survey (92%). Among all risk factors of ERCP infections, hospitalization in intensive care units, immunosuppressant therapies, and previous MDR infections have achieved a 80 % minimum of concurrence by our respondents. The majority of them did not have a formalized document in their hospital describing categories and risk factors helpful in the detection of patients undergoing ERCP with an high-level infective risk (9/11, 82%). Most centers (8/11, 72%) do not perform screening in patients at risk of ERCP infections. Post procedural monitoring is performed by 6 of 11 centers (55%). Conclusion: Our survey showed that, at least at regional level, there is a lack of procedures and protocols related to the management of patients at risk of ERCP infections.

Myeloperoxidase-positive cell infiltration of normal colorectal mucosa is related to body fatness and is predictive of adenoma occurrence.

Body fatness is a risk factor for colorectal cancer, and promotes an inflammatory environment. Indeed, inflammation in normal colorectal mucosa may be a factor linking body fatness to colorectal carcinogenesis. In this study, we evaluated myeloperoxidase (MPO)-positive cells infiltration of normal colorectal mucosa as a marker of cancer-promoting inflammation in overweight and obese subjects. One hundred and three subjects with normal colonoscopy entered the study. Waist circumference (WC) and body mass index (BMI) were measured, and MPO-positive cells on histological sections of biopsies of normal colorectal mucosa were counted under a light microscope. The occurrence of adenomas was then evaluated on follow-up colonoscopies. Mean MPO-positive cell count (±s.e.m.) was higher in subject with a WC equal or above the obesity cutoff values according to gender (2.63±0.20 vs 2.06±0.18, P=0.03), and in subjects with BMI equal or above 25 kg m-2 (2.54±0.18 vs 1.97±0.20, P=0.03). A Cox proportional hazard model showed that mean MPO-positive cell count in normal colorectal mucosa was the only factor independently related to occurrence of adenomas in follow-up colonoscopies. Though preliminary, these results show that MPO-positive cell infiltration in normal colorectal mucosa is related with body fatness, as evaluated by WC and BMI, and it may be considered a useful and simple marker to estimate adenoma occurrence risk.

Biochemical markers in vascular cognitive impairment associated with subcortical small vessel disease - A consensus report.

BACKGROUND: Vascular cognitive impairment (VCI) is a heterogeneous entity with multiple aetiologies, all linked to underlying vascular disease. Among these, VCI related to subcortical small vessel disease (SSVD) is emerging as a major homogeneous subtype. Its progressive course raises the need for biomarker identification and/or development for adequate therapeutic interventions to be tested. In order to shed light in the current status on biochemical markers for VCI-SSVD, experts in field reviewed the recent evidence and literature data. METHOD: The group conducted a comprehensive search on Medline, PubMed and Embase databases for studies published until 15.01.2017. The proposal on current status of biochemical markers in VCI-SSVD was reviewed by all co-authors and the draft was repeatedly circulated and discussed before it was finalized. RESULTS: This review identifies a large number of biochemical markers derived from CSF and blood. There is a considerable overlap of VCI-SSVD clinical symptoms with those of Alzheimer's disease (AD). Although most of the published studies are small and their findings remain to be replicated in larger cohorts, several biomarkers have shown promise in separating VCI-SSVD from AD. These promising biomarkers are closely linked to underlying SSVD pathophysiology, namely disruption of blood-CSF and blood-brain barriers (BCB-BBB) and breakdown of white matter myelinated fibres and extracellular matrix, as well as blood and brain inflammation. The leading biomarker candidates are: elevated CSF/blood albumin ratio, which reflects BCB/BBB disruption; altered CSF matrix metalloproteinases, reflecting extracellular matrix breakdown; CSF neurofilment as a marker of axonal damage, and possibly blood inflammatory cytokines and adhesion molecules. The suggested SSVD biomarker deviations contrasts the characteristic CSF profile in AD, i.e. depletion of amyloid beta peptide and increased phosphorylated and total tau. CONCLUSIONS: Combining SSVD and AD biomarkers may provide a powerful tool to identify with greater precision appropriate patients for clinical trials of more homogeneous dementia populations. Thereby, biomarkers might promote therapeutic progress not only in VCI-SSVD, but also in AD.

Fixed combination of hyaluronic acid and chondroitin-sulphate oral formulation in a randomized double blind, placebo controlled study for the treatment of symptoms in patients with non-erosive gastroesophageal reflux.

BACKGROUND: Proton pump inhibitors (PPIs) are a major breakthrough in the medical management of gastroesophageal reflux disease (GERD). In several patients with non erosive reflux disease symptoms (NERD) the response to PPIs is partial or limited and symptoms relief needs the administration of additional medications. AIM: The aim of this study was to evaluate the effect of a new medical device, based on an oral fixed combination of hyaluronic acid and chondroitin-sulphate (HA+CS), in a bioadhesive carrier, in adults with symptoms of non erosive gastroesophageal reflux and with a low response to PPIs. PATIENTS AND METHODS: Twenty patients who had experienced heartburn and/or acid regurgitation for at least 3 days during a 7 day run-in period, without endoscopic mucosal breaks, were randomized in a double blind crossover study to receive four daily doses of a fixed oral combination of HA+CS and placebo for 14 days. Relief of cardinal symptoms of GERD was evaluated at the end of each period. RESULTS: A significant greater Sum of Symptoms Intensity Difference, compared to placebo, was observed after HA+CS treatment (-2.7 vs 0.5 - p < 0.01), being both heartburn (-1.6 vs 0.5 - p < 0.03) and acid regurgitation (-1.1 vs 0.1 - p < 0.03) significantly improved by the medical device. A speed of action ≤ 30 min was significantly more frequently reported by patients during HA+CS administration than with placebo (60% vs 30% - p = 0.05). Total disappearance of symptoms was observed in 50% of the patients compared to 10% during placebo administration (p = 0.01 between group comparison). CONCLUSIONS: A fixed combination of HA+CS has demonstrated to be effective in gastroesophageal reflux control, with a rapid onset of action.

Ghrelin in central neurons.

Ghrelin, an orexigenic peptide synthesized by endocrine cells of the gastric mucosa, is released in the bloodstream in response to a negative energetic status. Since discovery, the hypothalamus was identified as the main source of ghrelin in the CNS, and effects of the peptide have been mainly observed in this area of the brain. In recent years, an increasing number of studies have reported ghrelin synthesis and effects in specific populations of neurons also outside the hypothalamus. Thus, ghrelin activity has been described in midbrain, hindbrain, hippocampus, and spinal cord. The spectrum of functions and biological effects produced by the peptide on central neurons is remarkably wide and complex. It ranges from modulation of membrane excitability, to control of neurotransmitter release, neuronal gene expression, and neuronal survival and proliferation. There is not at present a general consensus concerning the source of ghrelin acting on central neurons. Whereas it is widely accepted that the hypothalamus represents the most important endogenous source of the hormone in CNS, the existence of extra-hypothalamic ghrelin-synthesizing neurons is still controversial. In addition, circulating ghrelin can theoretically be another natural ligand for central ghrelin receptors. This paper gives an overview on the distribution of ghrelin and its receptor across the CNS and critically analyses the data available so far as regarding the effects of ghrelin on central neurotransmission.

Diagnostic value of endoscopic markers for celiac disease in adults: a multicentre prospective Italian study.

AIM: Some endoscopic features of duodenal mucosa are marker of mucosal injury, the most common cause being celiac disease (CD). The aim of this study was to prospectively assess the diagnostic value of the endoscopic markers for the diagnosis of CD in the adult population undergoing routine upper endoscopy. METHODS: This was a prospective multicenter study conducted at 37 Italian endoscopic centers. A total of 509 consecutive patients submitted to routine upper endoscopy who presented one or more of following endoscopic markers were included: 1) mucosal mosaic pattern in the bulb and/or descending duodenum (DD); 2) nodularity in the bulb and/or DD; 3) scalloping of Kerkring's folds; 4) reduction in the number or absence of folds in the DD. 4 biopsies samples were taken from descending duodenum. In patients with histological findings consistent with CD, according to Oberhuber classification, sierologic test (EMA, tTGA) were performed for confirm the diagnosis. RESULTS: At endoscopy, 249 patients showed an isolated marker; 260 subjects showed a coexistence of more than one marker; 369 patients (72.5%) presented mucosal lesions at histological examination and in 347 of these patients the diagnosis of CD was confirmed by serologic markers (94.0%). For 10 patients the diagnosis remained uncertain because of negative sierology and exclusion of other other cause of mucosal lesions. The diagnosis of CD was made in 61.3% patients who showed the mosaic pattern, in 65.7% of patients with nodular mucosa, in 64.4% of patients with scalloping of folds, in 40.2% of patients with reduction of folds, and in 61.5% of patients with loss of folds and in 83.6% of patients who showed the coexistence of more than one marker. The endoscopic markers overall had a PPV of 68% for the diagnosis of CD; the markers that singularly have demonstrated a higher correlation with CD are: mosaic mucosa of DD (PPV 65.0%), nodular mucosa of the bulb and DD (PPV 75.5%), and scalloping of folds (PPV 64.4%). CONCLUSION: The study confirms the important role of endoscopy in the diagnostic process of CD not only for the bioptic sampling in patients with clinical suspicion of CD, but especially for the opportunity to evaluate alterations of the duodenal mucosa suggestive of CD in the general population and, consequently, to identify those patients who should undergo a duodenal biopsy.

Costorage and coexistence of neuropeptides in the mammalian CNS.

The term neuropeptides commonly refers to a relatively large number of biologically active molecules that have been localized to discrete cell populations of central and peripheral neurons. I review here the most important histological and functional findings on neuropeptide distribution in the central nervous system (CNS), in relation to their role in the exchange of information between the nerve cells. Under this perspective, peptide costorage (presence of two or more peptides within the same subcellular compartment) and coexistence (concurrent presence of peptides and other messenger molecules within single nerve cells) are discussed in detail. In particular, the subcellular site(s) of storage and sorting mechanisms within neurons are thoroughly examined in the view of the mode of release and action of neuropeptides as neuronal messengers. Moreover, the relationship of neuropeptides and other molecules implicated in neural transmission is discussed in functional terms, also referring to the interactions with novel unconventional transmitters and trophic factors. Finally, a brief account is given on the presence of neuropeptides in glial cells.

In vivo cellular and molecular mechanisms of neuronal apoptosis in the mammalian CNS.

Apoptosis has been recognized to be an essential process during neural development. It is generally assumed that about half of the neurons produced during neurogenesis die before completion of the central nervous system (CNS) maturation, and this process affects nearly all classes of neurons. In this review, we discuss the experimental data in vivo on naturally occurring neuronal death in normal, transgenic and mutant animals, with special attention to the cerebellum as a study model. The emerging picture is that of a dual wave of apoptotic cell death affecting central neurons at different stages of their life. The first wave consists of an early neuronal death of proliferating precursors and young postmitotic neuroblasts, and appears to be closely linked to cell cycle regulation. The second wave affects postmitotic neurons at later stages, and is much better understood in functional terms, mainly on the basis of the neurotrophic concept in its broader definition. The molecular machinery of late apoptotic death of postmitotic neurons more commonly follows the mitochondrial pathway of intracellular signal transduction, but the death receptor pathway may also be involved.Undoubtedly, analysis of naturally occurring neuronal death (NOND) in vivo will offer a basis for parallel and future studies aiming to elucidate the mechanisms of pathologic neuronal loss occurring as the result of conditions such as neurodegenerative disorders, trauma or ischemia.

Variant estrogen receptor messenger RNA species detected in human primary hepatocellular carcinoma.

The development of hepatocellular carcinoma (HCC) in addition to cirrhosis affects males in a significantly higher proportion than females. Liver estrogen receptors increase when HCC develops in males; however, these tumors usually respond poorly to antiestrogens. We have, therefore, hypothesized that, similar to breast cancer, estrogen receptors in males with HCC may be mutated. Variant estrogen receptor transcripts (lacking exon 5 of the hormone binding domain) were investigated by reverse transcription-PCR in 14 patients (7 males and 7 females) with HCC. While females mostly displayed the wild-type transcript (both in peritumoral and in tumor liver tissue), males showed both transcripts in the cirrhotic tissue and almost only the variant in the tumor. As the variant ER transcripts when translated could give rise to truncated receptors still able to constitutively activate transcription, they may be key factors in favoring deregulated proliferation in the male liver.

Selection of more pathogenic hepatitis C virus genotype II during long-term follow-up of interferon-treated patients.

The behavior of hepatitis C virus (HCV) infection with regards to type and number of HCV genotypes (tested with genotype-specific nested polymerase chain reaction) was evaluated in 60 patients with anti-HCV-positive chronic active hepatitis without cirrhosis [17 untreated and 43 subjects undergoing single or repeat courses of interferon (IFN) therapy] during a mean follow-up period of 76 +/- 18 months. In untreated patients (2 genotype I, 6 genotype II, 9 mixed infections) 4 out of 9 mixed infections selected for genotype II at the end of follow-up. Of the 43 treated patients 10 were long-term responders with histological remission, 6 were short-term responders, and 22 did not respond. Fifteen of the latter patients received another course of IFN therapy, and only 3 patients responded. Eight of the 10 responders had infection with a single genotype (4 gt I, 3 gt II, 3 gt III). After IFN therapy, all but 2 patients cleared the HCV infection. The responders to the second IFN course (1 gt I, 1 gt II, 1 gt III) remained viremic. Of the short-term responders, 2/6 patients had genotype II and 4 had a mixed infection (3 gt II +/- I and 1 gt II +/- III); gt III became prevalent in the latter in all but one patient. Of the nonresponders 18/24 had more than one genotype, 5 were genotype II at baseline and one had genotype I. At the end of the follow-up period 15/18 with mixed infection had selected for gt II (P < 0.01 vs. untreated patient).(ABSTRACT TRUNCATED AT 250 WORDS)

Apoptosis in the mammalian CNS: Lessons from animal models.

It is generally assumed that about half of the neurons produced during neurogenesis die before completion of maturation of the central nervous system (CNS). Neural cell death is also relevant in aging and several neurodegenerative diseases. Among the modalities by which neurons die, apoptosis has very much attracted the interest of investigators because in this type of cell death neurons are actively responsible for their own demise by switching on a number of genes and activating a series of specific intracellular pathways. This review focuses on the cellular and molecular mechanisms of apoptosis in normal and transgenic animal models related to naturally occurring neuronal death within the CNS. We will also consider some examples of apoptotic cell death in canine neuropathologies. A thorough analysis of naturally occurring neuronal death in vivo will offer a basis for parallel and future studies involving secondary neuronal loss such as those in neurodegenerative disorders, trauma or ischaemia.

[Outcome of isolated small bowel transplantation in adults: experience from a single Italian center]. / Risultati del trapianto di intestino isolato nell'adulto: esperienza di un singolo centro italiano.

AIM: Isolated small bowel transplantation is becoming the treatment of choice for adult patients with serious parenteral nutrition (PN) related complications: we report our three-year experience (December 2000-December 2003) from a single Italian center (Modena-Italy), with one of the larger European series. METHODS: We transplanted 14 patients, with a previous mean PN course of 27 months and a mean 21-month post-transplantation follow-up (range 3-36 months), obtaining a one-year actuarial survival rate of 92.3% with no intraoperative deaths. RESULTS: We lost 1 patient (7.2%), died for post-transplantation overwhelming sepsis following Cytomegalovirus (CMV) enteritis. Thirteen patients are alive, with one-year actuarial graft survival rate of 85.1%: 1 patient underwent graft removal (7.2%) for intractable severe acute rejection. Our immunosuppressive regimen was based on tacrolimus and 3 induction protocols: daclizumab (8 patients) with steroids, alemtuzumab (4 patients) and thymoglobulin (2 patients) without steroids. In 9 cases, we added sirolimus. Nine recipients experienced 22 episodes of acute cellular rejection (ACR), treated successfully in all cases but one. One patient (7.2%) was treated successfully for Post Transplant Lymphoproliferative Disease (PTLD) and is disease-free after 8 months. CONCLUSIONS: Small bowel transplantation can achieve optimal results depending on appropriate immunosuppressive management and candidate selection, added to shorter ischemia time and careful donor and graft selection.

The early intracellular signaling pathway for the insulin/insulin-like growth factor receptor family in the mammalian central nervous system.

Several studies support the idea that the polypeptides belonging to the family of insulin and insulin-like growth factors (IGFs) play an important role in brain development and continue to be produced in discrete areas of the adult brain. In numerous neuronal populations within the olfactory bulb, the cerebral and cerebellar cortex, the hippocampus, some diencephalic and brainstem nuclei, the spinal cord and the retina, specific insulin and IGF receptors, as well as crucial components of the intracellular receptor signaling pathway have been demonstrated. Thus, mature neurons are endowed with the cellular machinery to respond to insulin and IGF stimulation. Studies in vitro and in vivo, using normal and transgenic animals, have led to the hypothesis that, in the adult brain, IGF-I not only acts as a trophic factor, but also as a neuromodulator of some higher brain functions, such as long-term potentiation and depression. Furthermore, a trophic effect on certain neuronal populations becomes clearly evident in the ischemic brain or neurodegenerative disorders. Thus, the analysis of the early intracellular signaling pathway for the insulin/IGF receptor family in the brain is providing us with new intriguing findings on the way the mammalian brain is sculpted and operates.

Connections of two types of flat cone bipolars in the rabbit retina.

The synaptic connections of two types of cone bipolar cells in the rabbit retina were studied with the electron microscope after labeling in vitro with 4',6-diamidino-2-phenylindole (DAPI), intracellular injection with Lucifer Yellow, and photooxidation (Mills and Massey [1992] J. Comp. Neurol. 321:133). Both types of bipolars belong to the flat variety, because they make basal junctions with a group of four to ten neighboring cone pedicles. One cell type has an axonal arborization that occupies strata 1 through 3 of the inner plexiform layer (IPL). At ribbon synaptic junctions, it is presynaptic to ganglion cell dendrites and to reciprocal dendrites belonging to narrow-field bistratified (AII) amacrine cells. In addition, it contacts and is contacted by other amacrine cell processes of unknown origin. The other cell type has an axonal arborization entirely confined to stratum 2 of the IPL; it is pre- or postsynaptic to a pleomorphic population of amacrine cell processes, and, in particular, it receives input from the lobular appendages of AII. Thus, these two bipolar types probably belong to the off-variety because they make basal junctions with cone photoreceptors and send their axon to sublamina a of the IPL, which is occupied by the dendrites of off-ganglion cells. They are also part of the rod pathway because they receive input from AII amacrine cells.

Apoptosis of undifferentiated progenitors and granule cell precursors in the postnatal human cerebellar cortex correlates with expression of BCL-2, ICE, and CPP32 proteins.

Naturally occurring apoptotic cells have been demonstrated in the postnatal cerebellum of rodents (Wood et al. [1993] Neuron 11:621-632; Krueger et al. [1995] J. Neurosci. 15:3366-3374). The nature of these cells differs among species: they are considered to be granule cells in mouse and astrocytes in rat. We labeled proliferating and apoptotic cells in the postnatal human cerebellar cortex by using antibodies against the Ki-67/proliferating cell nuclear antigen and the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling method for fragmented DNA. We also immunocytochemically detected some proteins encoded by genes modulating apoptosis and specific markers of neuronal/glial differentiation. Proliferating cells were observed from birth to 4 months, representing 31-35% of cells within the external granular layer (EGL). Apoptotic cells were detected during the first 3 months and corresponded to 5-7% of EGL cells. Much lower percentages were calculated in other cortical layers and white matter. The balance between proliferation and apoptosis was quantitatively favorable to the latter during the first postnatal week. Expression of BCL-2, CPP32, and interleukin-1beta-converting enzyme (ICE) proteins was spatially and developmentally regulated in parallel with apoptosis. Apoptotic cells were often CPP32/ICE immunoreactive but negative for BCL-2. Some apoptotic cells were positive for vimentin and, less frequently, for alpha-internexin or type-III beta tubulin, but never expressed the glial fibrillary acidic protein. This study demonstrates that apoptosis is a significant phenomenon in early postnatal development of human cerebellar cortex and shares some of the regulatory mechanisms described in other vertebrates.

Distribution of protein gene product 9.5 (PGP 9.5) in the vertebrate retina: evidence that immunoreactivity is restricted to mammalian horizontal and ganglion cells.

Using light microscopic immunocytochemistry, we have studied the distribution of protein gene product 9.5 (PGP 9.5), a neuron-specific protein first extracted from human brain (Doran et al., '83:J. Neurochem. 40:1542-1547), in the vertebrate retina. Retinas were obtained from frog, chicken, rat, rabbit, cow, cat, dog, and human. No immunoreactivity was observed in frog and only a faint staining was present in chicken. In mammalian retinas, a strong positive reaction was restricted to horizontal and ganglion cells, with minor interspecies variations. Immunostaining was present throughout the cell body and the dendritic tree in horizontal cells. At the level of retinal ganglion cells, immunolabel was particularly abundant in cell bodies and axons forming the optic nerve. Only the main dendrites were stained, the remainder of the dendritic tree giving rise to a diffuse punctate reaction in the inner plexiform layer. In rats, displaced amacrine cells, which are known to contribute largely (40-50%) to the total neuronal population within the ganglion cell layer (Perry, '81: Neuroscience 6:931-944) were not immunoreactive, as demonstrated from (i) analysis of the morphology, cell size and cell density of immunoreactive neurons in wholemounts; (ii) colocalization of retrograde label and PGP 9.5 immunoreactivity in about 80% of ganglion cells after injection of peroxidase into the optic nerve; and (iii) reduction of immunoreactivity in the inner plexiform and ganglion cell layers following optic nerve transection. Western blot analysis of extracts from rabbit retinas indicated that the immunoreactive species is PGP 9.5 or a closely related molecule. Recent studies have demonstrated that PGP 9.5 is a ubiquitin carboxyl-terminal hydrolase (Wilkinson et al., '89:Science 246:670-673). The present results, therefore, suggest that differences in the ubiquitination process exist between retinal neurons.

Synapse-independent and synapse-dependent apoptosis of cerebellar granule cells in postnatal rabbits occur at two subsequent but partly overlapping developmental stages.

It has long been known that cells in the external granular layer die during postnatal development of the cerebellum. More recent findings indicate that at certain developmental stages, cell death occurs upon activation of an apoptotic program. We show that cerebellar granule cells in rabbits undergo programmed cell death at two different stages of maturation. At postnatal day 5 (P5), granule cell precursors and pre-migratory granule cells in the external granular layer incorporate the S-phase markers 5-bromo-2'-deoxyuridine and 5-iodo-2'-deoxyuridine with a pattern that is dependent upon the interval between the administration of the two tracers. Within 12-24 h after proliferation a significant number of labeled cells show typical ultrastructural alterations of apoptosis. DNA electrophoresis and cleavage of poly-ADP-ribose polymerase confirm the activation of the apoptotic machinery. After Southern blotting and immunodetection, incorporated 5-bromo-2'-deoxyuridine is present at the level of low size DNA oligomers as soon as 12 h after cell division. Therefore, this apoptotic phase is intrinsic to external granular layer neurons and independent of synaptic interactions with targets.Apoptotic cells, although fewer in number, are detected also in the internal granular layer and tend to increase from P5 to P10. It seems unlikely that these cells undergo DNA fragmentation in the external granular layer and subsequently migrate to their final destination, considering the data on cell cycle kinetics and the rapid tissue clearance by the glia. Parallel fiber-Purkinje spine synapses are already present in the molecular layer at P5. Therefore, the post-migratory granule cells likely undergo apoptosis as a failure to make proper synaptic contacts in the forming molecular layer. We conclude that the massive apoptosis of pre-migratory cells likely has a role in regulating the size of this rapidly expanding population of pre-mitotic neurons. The less tumultuous cell death of post-mitotic granule cells in the internal granular layer appears to be linked to the formation of the mature synaptic circuitry of the developing cerebellar cortex.

Ultrastructural visualization of glutamate and aspartate immunoreactivities in the rat dorsal horn, with special reference to the co-localization of glutamate, substance P and calcitonin-gene related peptide.

Antisera raised against the fixation products of L-glutamate and L-aspartate were used, singly or in combination, to study the ultrastructural localization of the amino acids in the rat dorsal horn, with post-embedding immunogold techniques. Immunostaining for each of the amino acids was also combined with immunolocalization of GABA, an important inhibitory neurotransmitter in the spinal cord, or synaptophysin, a synaptic vesicle glycoprotein. In addition, we examined the localization of glutamate immunoreactivity in relation to that of calcitonin-gene related peptide and substance P, two neuropeptides present in high concentrations in the dorsal horn. Glutamate- and aspartate-immunoreactive neuronal cell bodies, dendrites, axons and terminals were apparent in the first three laminae of the dorsal horn. In somatic and dendritic profiles, the immunolabel was present over the general cytoplasm and mitochondria; in the terminals, it was found over small, agranular vesicles, mitochondria and, at times, synaptic densities. Quantitative estimation indicated that the colloidal gold density in the glutamate-immunoreactive terminals was five-fold more than in any other neuronal profile. Both glutamate- and aspartate-immunopositive terminals made asymmetric synaptic contacts onto unlabelled dendrites; glutamate-positive terminals often formed the core of type I and II glomeruli. After double labelling of the same sections, glutamate and aspartate immunoreactivities consistently occurred in different axonal and terminal profiles. In these preparations, it was clearly seen that glutamate-immunoreactive terminals were far more numerous than (more than 10-fold) those immunoreactive for aspartate. Double labelling for glutamate or aspartate and GABA also revealed distinct staining of different terminals. Simultaneous immunolocalization of each of the amino acids and synaptophysin showed the amino acid and glycoprotein immunoreactivities co-localized in small, agranular vesicles in immunoreactive terminals. Finally, triple labelling of the same sections for glutamate, calcitonin gene-related peptide and substance P revealed that glutamate was often co-localized with either of the two neuropeptides in the same axonal boutons; terminals that showed simultaneous labelling for glutamate, calcitonin gene-related peptide and substance P were also noted. In all cases, the glutamate immunoreactivity was restricted to small, clear vesicles whereas the neuropeptide immunoreactivities were present in larger, dense-cored vesicles. Our observations demonstrate that there is an abundant glutamate immunoreactivity in the superficial layers of the rat dorsal horn, localized in neuronal profiles distinct from those containing aspartate or GABA.(ABSTRACT TRUNCATED AT 400 WORDS)

Newly-generated cells from the rostral migratory stream in the accessory olfactory bulb of the adult rat.

Cell proliferation in the accessory olfactory bulb of the adult rat was analysed after systemic injection of 5-bromo-2'-deoxyuridine, detected immunocytochemically at different survival times and compared with proliferating cell nuclear antigen immunostaining. As previously described in the main olfactory bulb, local cell proliferation was absent or very limited. By contrast, starting from 15 days after bromodeoxyuridine administration, many immunoreactive nuclei were present in the granular layer, and to a lesser extent, in other layers of the accessory olfactory bulb. This suggests that the newly-generated cells are migrating elements of the rostral migratory stream which are known to reach the olfactory bulb in 15 days. By immunocytochemical detection of the polysialylated isoform of the neural cell adhesion molecule, a weakly-adhesive cell-surface molecule expressed by newly-generated/migrating cells of the rostral migratory stream, we found a high number of immunoreactive cells in the different layers of the accessory olfactory bulb. Most of these cells were observed in the granular layer and showed the morphology of migrating neuroblasts. Some immunoreactive cells displaying neuronal morphology were also detected in the external plexiform and glomerular layers. Double labelling experiments demonstrated that these cells are newly-generated cells. These results demonstrate the occurrence of newly-added cells in the accessory olfactory bulb of the adult rat, which likely correspond to the neuronal precursors originating from the rostral migratory stream. This could be relevant since the accessory olfactory bulb of rodents plays an important role in the hard wiring of a simple olfactory memory system for sexual pheromones.

Dorsal rhizotomy induces transient expression of the highly sialylated isoform of the neural cell adhesion molecule in neurons and astrocytes of the adult rat spinal cord.

Expression of the weakly adhesive, highly sialylated isoform of the neural cell adhesion molecule is a feature common to cell capable of migration and conformation changes. 11,18,19 Polysialylated neural cell adhesion molecule also intervenes in axonal outgrowth and synaptogenesis during development and after lesion. 11,13 High levels of polysialylated neural cell adhesion molecule immunoreactivity are normally visible in laminae I,II and X of the adult rat spinal cord. 2,15 We how here that unilateral cervical dorsal rhizotomy induced no detectable changes in immunoreactivity in these areas. However, 24 h after lesion, polysialylated neural cell adhesion molecule immunoreactivity appeared in neurons scattered in laminae III-IX, ipsi-and contralateral to lesion. This reaction increased particularly on the contralateral side, became maximal at four days and disappeared eight days later. At this time, there was immunolabelling of astrocytes with an activated morphology. The astrocytic labelling, predominant on the side ipsilateral to the lesion, was strongest 12 days after rhizotomy, then diminished progressively. Deafferentation thus causes a transient expression of polysialylated neural cell adhesion molecule within areas of the spinal cord distinct from those which permanently express this adhesion molecule. Such expression occurs both in neurons and glial cells, with a temporal pattern specific to each type of cell.