Irina I. Stoyanova

Mast cells and inflammatory mediators in chronic ulcerative colitis

Chronic ulcerative colitis (CUC) is an inflammatory destructive disease of the large intestine characterized by motility and secretion disorders. In the past decade, attention has been paid to the role of neuronal structures and mast cells in regulating inflammatory and immune responses in inflammatory bowel disease (IBD). The present study was performed to demonstrate neuronal fibres (NF) and cells containing substance P (SP), tryptase and serotonin (SER) in the colonic wall of patients with CUC in remission. Biopsy specimens of 6 patients with CUC were investigated with immunocytochemical methods. Normal colon tissue obtained from 6 patients with rectal carcinoma was used as a control. An increased number of SP- and SER-positive NF was found in all the layers of the intestinal wall. The number of SER-containing endocrine cells in the mucosal glands was also increased per crypt. Tryptase-, SP- and SER-immunopositive mast cells were found in higher amounts than in control specimens in close apposition to the basal lamina of the glands among the epithelial cells and in other layers of the gut wall. Two types of mast cells were found: mast cells containing both tryptase and SP, and mast cells containing tryptase only. It is concluded that interactions between neuronal elements and mast cells play a significant role in the progress and maintenance of inflammatory processes in CUC.

Localization of nitric oxide synthase in rat trigeminal primary afferent neurons using NADPH-diaphorase histochemistry

SummaryNitric oxide (NO) is a ubiquitous gaseous neurotransmitter that has been ascribed to a large number of physiological roles in sensory neurons. It is produced by the enzyme nitric oxide synthase (NOS). To identify the NOS-containing structures of rat trigeminal primary afferent neurons, located in the trigeminal ganglion (TrG) and mesencephalic trigeminal nucleus (MTN), histochemistry to its selective marker nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) was applied in this study. In the TrG approximately half of the neuronal population was NADPH-d reactive. Strongly positive were neurons mainly of small-to-medium size. Neuronal profiles of large diameter were less intensely stained. In addition, NADPH-d-positive nerve fibers were dispersed throughout the ganglion. Nitrergic neurons were located in the caudal part and mesencephalic-pontine junction of the MTN. Most of them were large-sized pseudounipolar cells. In a more rostral aspect, the reactive psedounipolar MTN profiles gradually decreased in number and intensity of staining. There, only a fine meshwork of stained thin fibers and perisomatic terminal arborizations, and also some isolated perikarya of NADPH-d stained multipolar MTN neurons, were observed. The predominant NADPH-d localization in smaller in size TrG neurons, which are considered nociceptive, suggests that NO may play a role in the pain transmission in the rat trigeminal afferent pathways. In addition, the wide distribution of NADPH-d activity in large pseudounipolar and certain multipolar MTN neurons provides substantial evidence that NO may also participate in mediating proprioceptive information from the orofacial region. The differential expression patterns of nitrergic fibers in the TrG and MTN suggest that trigeminal sensory information processing is controlled by nitrergic input through different mechanisms.

Localization of orexin-A-immunoreactive fibers in the mesencephalic trigeminal nucleus of the rat

Orexin A is a neuropeptide located exclusively in neurons in the hypothalamic nuclei involved in the central regulation of many brain functions, related to motor activity and state-dependent processes. Orexins modulate behavioral state via actions across multiple terminal fields. In order to determine whether the mesencephalic trigeminal neurons may receive a direct hypothalamic orexinergic input, the distribution of orexin A immunoreactivity was examined in the rat mesencephalic trigeminal nucleus (MTN), using orexin A immunohistochemistry. Orexin-A-immunostained nerve fibers and terminals were found in a close apposition to the perikarya of primary afferent neurons in the MTN with a marked rostrocaudal gradient in their density. In the caudal pontine MTN, only scattered orexin-A-immunoreactive fibers were found, while more rostrally in the pons, and in the midbrain-pontine junction part of the nucleus, orexin-A-immunopositive varicosities were relatively more abundant, located in close proximity to or often surrounding the neuronal profiles. At the level of the inferior or superior colliculi, a large number of orexin-A-containing neuronal processes and terminal arborizations were observed traveling toward and contacting mesencephalic trigeminal neurons, some of which were multipolar. The results of this study show that MTN neurons receive orexin A hypothalamic innervation with a somatotopic arrangement of the projections in the nucleus. The central orexinergic system may exert direct influence upon jaw movements at the level of the MTN and thus to participate in the control of feeding behavior.

Orexin-A and Orexin-B during the postnatal development of the rat brain

Orexin-A and orexin-B are hypothalamic neuropeptides isolated from a small group of neurons in the hypothalamus, which project their axons to all major parts of the central nervous system. Despite the extensive information about orexin expression and function at different parts of the nervous system in adults, data about the development and maturation of the orexin system in the brain are a bit contradictory and insufficient. A previous study has found expression of orexins in the hypothalamus after postnatal day 15 only, while others report orexins detection at embryonic stages of brain formation. In the present study, we investigated the distribution of orexin-A and orexin-B neuronal cell bodies and fibers in the brain at three different postnatal stages: 1-week-, 2-week-old and adult rats. By means of immunohistochemical techniques, we demonstrated that a small subset of cells in the lateral hypothalamus, and the perifornical and periventricular areas were orexin-A and orexin-B positive not only in 2-week-old and adult rats but also in 1-week-old animals. In addition, orexin-A and orexin-B expressing neuronal varicosities were found in many other brain regions. These results suggest that orexin-A and orexin-B play an important role in the early postnatal brain development. The widespread distribution of orexinergic projections through all these stages may imply an involvement of the two neurotransmitters in a large variety of physiological and behavioral processes also including higher brain functions like learning and memory.

Immunocytochemical study on the liver innervation in patients with cirrhosis

In the liver, the autonomic nervous system plays an important role in degenerative and inflammatory changes. The aim of the present study was to investigate the distribution of neuronal fibres containing neuropeptides in livers of 5 patients with cirrhosis by immunocytochemical localization at the light and electron microscopical level of substance P (SP), neuropeptide Y (NPY), somatostatin (SOM), and calcitonin gene-related peptide (CGRP). In patients with alcoholic cirrhosis, a decreased number of neuronal fibres was found in the portal tract and fibrous septa as well as in the sinusoids of regenerative nodules. NPY- and SP-immunoreactive neuronal fibres were more numerous than CGRP-containing fibres. They were located mainly in portal tracts. These findings led to the conclusion that peptidergic innervation plays a role in inflammatory and fibrotic changes in cirrhotic liver.

Peptidergic nerve fibres in the human liver

The autonomic nervous system plays a significant role in liver physiology and pathology. The aim of the present study was to investigate peptidergic nerve fibres in the liver of patients with malignant gastrointestinal tumors that are not metastasizing in this organ. Using light and electron microscopic immunohistochemistry, somatostatin (SOM)-, neuropeptide Y (NPY)-, substance P (SP)- and calcitonin gene-related peptide (CGRP)-immunoreactive (IR) nerve fibres (NF) were detected in the portal tract and perisinusoidally. Histologically, the liver showed dilated sinusoids, filled with lymphoid cells, and scarcely marked perisinusoidal fibrosis. Neuropeptide-IR NF were found in close contact with hepatic sinusoids. Numerous IR varicosities were detected in the sinusoidal wall. We discuss the origin and role of these NF in the liver. Probable quantitative changes in peptidergic NF ensue the inflammatory reaction in sinusoids in malignant gastrointestinal tumors. This could also reflect the increased exposure of the liver to toxic substances in the portal blood flow.

Ghrelin stimulates synaptic formation in cultured cortical networks in a dose-dependent manner

Ghrelin was initially related to appetite stimulation and growth hormone secretion. However, it also has a neuroprotective effect in neurodegenerative diseases and regulates cognitive function. The cellular basis of these processes is related to synaptic efficacy and plasticity. Previous studies indicated that ghrelin has an excitatory effect on neuronal activity, and stimulates synaptic plasticity in vivo. Plasticity in the adult brain occurs in many different ways, including changes in synapse morphology and number. Therefore, we used in vitro neuronal cultures to investigate how ghrelin affects synaptogenesis. We used dissociated cortical cultures of newborn rats, chronically treated with different doses of ghrelin (0.5, 1, 1.5 and 2μM). After one-, two-, three- or four weeks cultures were immunostained for the presynaptic marker synaptophysin. In parallel, additional groups of non-treated cultures were immunostained for detection of ghrelin receptor (GHSR1). During development, GHSR1was increasingly expressed in all type of neurons, as well as the synaptophysin. Synaptic density depended on ghrelin concentration, and was much higher than in controls in all age groups. In conclusion, ghrelin leads to earlier network formation in dissociated cortical networks and an increase in number of synapses. The effect is probably mediated by GHSR1. These findings suggest that ghrelin may provide a novel therapeutic strategy for the treatment of disorders related to synaptic impairment.

In vivo testing of a 3D bifurcating microchannel scaffold inducing separation of regenerating axon bundles in peripheral nerves

Artificial nerve guidance channels enhance the regenerative effectiveness in an injured peripheral nerve but the existing design so far has been limited to basic straight tubes simply guiding the growth to bridge the gap. Hence, one of the goals in development of more effective neuroprostheses is to create bidirectional highly selective neuro-electronic interface between a prosthetic device and the severed nerve. A step towards improving selectivity for both recording and stimulation have been made with some recent in vitro studies which showed that three-dimensional (3D) bifurcating microchannels can separate neurites growing on a planar surface and bring them into contact with individual electrodes. Since the growing axons in vivo have the innate tendency to group in bundles surrounded by connective tissue, one of the big challenges in neuro-prosthetic interface design is how to overcome it. Therefore, we performed experiments with 3D bifurcating guidance scaffolds implanted in the sciatic nerve of rats to test if this new channel architecture could trigger separation pattern of ingrowth also in vivo. Our results showed that this new method enabled the re-growth of neurites into channels with gradually diminished width (80, 40 and 20 µm) and facilitated the separation of the axonal bundles with 91% success. It seems that the 3D bifurcating scaffold might contribute towards conveying detailed neural control and sensory feedback to users of prosthetic devices, and thus could improve the quality of their daily life.

Time-dependent changes in ghrelin-immunoreactivity in dissociated neuronal cultures of the newborn rat neocortex

Ghrelin is a hormone, initially described as a gastric peptide stimulating appetite and growth hormone secretion, which also has an important role in the regulation of many other processes, including higher brain functions. Ghrelin has been described in situ in different parts of the brain, but so far there has been no data about its expression in cell cultures. Therefore, we aimed in this study to investigate the levels of ghrelin in dissociated cortical neurons at various times in culture. We applied the ABC immunocytochemical method for the detection of ghrelin in one-day-, one-week-, and two-week-old cultures. Our results clearly show that at the early stages after plating the cultures 86.2% (+/-8.93) of the neurons are ghrelin-positive and their number decreases during the culturing period. As ghrelin is present in the majority of cultured newborn neurons, when the neuronal differentiation and network formation take place, it may also influence the early synaptic formation and cell-to-cell interactions, which are both very important for network functions like learning and memory.

Immunohistochemical detection of collagen type III and IV in relation with transformation of Ito cells in liver sinusoids of patients with reactive biliary hepatitis.

Reactive biliary hepatitis is a defined morphological entity, which is a result of chronic diseases of the gall bladder, biliary ducts or pancreas. The aim of the present study was to describe the morphology of reactive biliary hepatitis and its significance for progression of liver fibrosis, and in particular Ito cell (fat storing cell) transformation and occurrence of collagen type III and IV in the liver. Liver tissue from 19 patients with reactive biliary hepatitis was investigated light microscopically and immunohistochemically. Histologically, the liver showed features of mild to severe portal and lobular inflammation. The number of Ito cells increased periportally and pericentrally. Deposition of collagen type III and IV was increased in portal tracts, septa and perisinusoidal spaces, mainly in periportal zones of the lobules. Ultrastructurally, collagen type III immunoreactive fibrillar networks were found to be increased in the space of Disse around transitional cells. Collagen type IV immunoreactive deposits were detected around newly proliferating bile ducts in portal stroma and in the space of Disse. Ito cells were mainly transformed into transitional and myofibroblast-like cells. We discuss here the role of Ito cells and certain cytokines in the process of fibrosis of the liver in the course of reactive biliary hepatitis. It is proposed that bile acid retention in bile ducts during non-specific reactive inflammation or a gut endotoxin may cause transformation of Ito cells and increased collagen type III and IV in this type of hepatitis.