Liliana Rytel

Co-expression of PACAP with VIP, SP and CGRP in the Porcine Nodose Ganglion Sensory Neurons

Our previous study revealed the expression of substance P (SP) and calcitonin gene‐related peptide (CGRP) in sensory distal ganglion of the vagus (nodose ganglion) neurons in the pig. As these neuropeptides may be involved in nociception, the goal of these investigations was to determine possible expression of vasoactive intestinal polypeptide (VIP), SP and CGRP in the pituitary adenylate cyclase‐activating polypeptide‐immunoreactive (PACAP‐IR) porcine nodose perikarya. Co‐expression of these substances was examined using a double‐labelling immunofluorescence technique. To reveal the ganglionic cell bodies, the pan‐neuronal marker protein gene product 9.5 (PGP 9.5) was used. Quantitative analysis of the neurons revealed that 67.25% of the PGP 9.5+ somata in the right‐side ganglion and 66.5% in the left side, respectively, co‐expressed PACAP‐IR. Moreover, 60.6% of the PACAP‐IR cells in the right‐side ganglion and 62.1% in the left, respectively, co‐expressed VIP. SP‐IR was observed in 52.2 and 39.9% of the right and left ganglia, respectively. CGRP was found in 27.7 and 34.1% of the right and left distal ganglion of the vagus, respectively. High level of co‐expression of PACAP with VIP, SP and CGRP in the distal ganglia of the vagus sensory perikarya directly implicates studied peptides in their functional interaction during nociceptive vagal transduction.

Co-localization of zinc transporter 3 (ZnT3) with sensory neuromediators and/or neuromodulators in the enteric nervous system of the porcine esophagus

Zinc transporter 3 (ZnT3) is one of the zinc transporters family. It is closely connected to the nervous system, where enables the transport of zinc ions from the cytoplasm to synaptic vesicles. This substance has been described within the central and peripheral nervous system, especially in the enteric nervous system (ENS). The aim of the present study was to describe the co-localization of ZnT3 with selected neuromediators and/or neuromodulators participating in sensory stimuli conduction in neurons of the ENS within the porcine esophagus. Co-localization of ZnT3 with substance P (SP), leucine enkephalin (LENK) and calcitonin gene-related peptide (CGRP) was studied using standard double-immunofluorescence technique. The obtained results show that ZnT3, SP and/or LENK may occur in the same enteric neurons, and the degree of co-localization of these substances clearly depends on the fragment of esophagus studied and the type of enteric ganglia. In contrast, the co-localization of ZnT3 with CGRP was not observed during the present investigation. The obtained results suggest that ZnT3 in the ENS may be involved in the conduction of sensory and/or pain stimuli.

Reduction of the number of neurones in the caudal mesenteric ganglion innervating the ovary in sexually mature gilts following testosterone administration.

The effect of testosterone on the morphological and chemical plasticity of the porcine caudal mesenteric ganglion (CaMG) ovary‐projecting neurones was investigated. To identify the neurones on day 3 of the oestrous cycle, the ovaries of both the control and experimental gilts were injected with Fast Blue retrograde neuronal tracer. From next day until day 20 of the anticipated second studied cycle, experimental gilts were injected with testosterone, whereas control gilts received oil. Testosterone injections increased testosterone (by approximately 3.5‐fold) and 17β‐oestradiol (by approximately 1.6‐fold) levels in the peripheral blood and decreased the following in the CaMG: the total number of Fast Blue‐positive perikarya (including small ones); the population of small perikarya in the caudal, ventral and dorsal ganglional regions; the numbers of dopamine‐β‐hydroxylase (DβH) and/or neuropeptide Y (NPY), somatostatin (SOM), galanin (GAL) small and large perikarya; the numbers of small perikarya containing DβH (but not NPY, SOM, GAL); and the density of DβH and/or NPY, SOM nerve fibres. A disappearance of small and large non‐noradrenergic perikarya and an increase in the total number of androgen receptor‐immunoreactive perikarya was noted. Our results suggest that elevated androgen levels occurring during pathological states may regulate ovary function(s) by affecting the CaMG gonad‐supplying neurones.

Morphological and neurochemical characterization of the ovarian sympathetic chain ganglia perikarya in testosterone-treated sexually matured pigs.

We studied the effect of testosterone overdose on the number, distribution and chemical coding of ovarian neurons in the sympathetic chain ganglia (SChGs) in pigs. On day 3 of the estrous cycle the ovaries of both the control and experimental gilts were injected with retrograde neuronal tracer Fast Blue to identify the neurons innervating gonads. From the following day to the expected day 20 of the second studied cycle the experimental pigs were injected with testosterone, while the control pigs received oil, and subsequently the SChG Th16-S2 were collected. Testosterone injections increased testosterone (∼3.5 fold) and estradiol-17β (∼1.6 fold) levels in the peripheral blood, and reduced the following in the SChGs: the total number of Fast Blue-positive neurons, the numbers of perikarya in the L3-L5 ganglia, the numbers of perikarya in the ventral, dorsal and central regions of the SChGs, and the numbers of DβH(+)/NPY(+), DβH(+)/GAL(+), DβH(+)/NPY(-), DβH(+)/SOM(-) and DβH(+)/GAL(-) perikarya. In the testosterone-affected SChGs, the perikarya DβH(-)/SOM(+), DβH(-)/GAL(+) and DβH(-)/NPY(-) were absent. In these ganglia, the population of androgen receptor-positive perikarya was increased, while the population of estrogen receptor-expressing perikarya was lowered. Our data indicate that in the pig SChGs elevated androgen levels occurring during pathological states may affect the morphology and chemical coding of ovarian neurons.

Bisphenol A Causes Liver Damage and Selectively Alters the Neurochemical Coding of Intrahepatic Parasympathetic Nerves in Juvenile Porcine Models under Physiological Conditions

Bisphenol A (BPA) is an extremely common polymer that is used in typical everyday products throughout the world, especially in food and beverage containers. Within the last ten years, it has been found that the BPA monomer tends to leach into foodstuffs, and nanogram concentrations of it may cause a variety of deleterious health effects. These health problems are very evident in developing children and in young adults. The aim of this study was to expose developing pigs to dietary BPA at both legally acceptable and ten-fold higher levels. Livers that had been exposed to BPA showed vacuolar degeneration, sinusoidal dilatation, vascular congestion and glycogen depletion that increased with exposure levels. Furthermore, the livers of these models were then examined for irregularities and double-labeled immunofluorescence was used to check the innervated hepatic samples for varying neuronal expression of selected neuronal markers in the parasympathetic nervous system (PSNS). It was found that both the PSNS and all of the neuronal markers showed increased expression, with some of them being significant even at recommended safe exposure levels. The implications are quite serious since these effects have been observed at recommended safe levels with expression increasing in-line with exposure levels. The increased neuronal markers studied here have been previously correlated with behavioral/psychological disorders of children and young adults, as well as with childhood obesity and diabetes. However, further research must be performed in order to develop a mechanism for the above-mentioned correlations.

Immunohistochemical characteristics of porcine intrahepatic nerves under physiological conditions and after Bisphenol A administration

BACKGROUND The neurochemistry of hepatic nerve fibres was investigated in large animal models after dietary exposure to the endocrine disrupting compound known as bisphenol A (BPA). MATERIALS AND METHODS Antibodies against neuronal peptides were used to study changes in hepatic nerve fibres after exposure to BPA at varying concentrations using standard immunofluorescence techniques. The neuropeptides investigated were substance P (SP), galanin (GAL), pituitary adenylate cyclase activating polypeptide (PACAP), calcitonin gene regulated peptide (CGRP) and cocaine and amphetamine regulated transcript (CART). Immunoreactive nerve fibres were counted in multiple sections of the liver and among multiple animals at varying exposure levels. The data was pooled and presented as mean ± standard error of the mean. RESULTS It was found that all of the nerve fibres investigated showed upregulation of these neural markers after BPA exposure, even at exposure levels currently considered to be safe. These results show very dramatic increases in nerve fibres containing the above-mentioned neuropeptides and the altered neurochemical levels may be causing a range of pathophysiological states if the trend of over-expression is extrapolated to developing humans. CONCLUSIONS This may have serious implications for children and young adults who are exposed to this very common plastic polymer, if the same trends are occurring in humans.

Levels of zearalenone and its metabolites in sun-dried kapenta fish and water of Lake Kariba in Zambia — A preliminary study

Contamination of food with mycotoxins and the associated possibilities of human intoxication is a serious problem in Africa. One of the most widespread mycotoxins is zearalenone (ZEN), which usually occurs in food of vegetable origin. On the other hand, information about ZEN in products of animal origin in African countries is extremely scanty. During the present study, levels of ZEN and its analogs: α-zearalenol (α-ZEL) and β-zearalenol (β-ZEL) were measured by high performance liquid chromatography (HPLC) with fluorescence detection in sun-dried kapenta fish - traditional Zambian food, as well as in the water of Lake Kariba - the main source of kapenta fish in Zambia. This study revealed that levels of ZEN in sun-dried kapenta fish fluctuated from 27.2 μg·kg-1 to 53.9 μg·kg-1, whereas the contamination of water from Lake Kariba with ZEN is rather minimally similar to the content of ZEN analogs in both kapenta fish and water. The obtained results have shown that sun-dried kapenta fish of Lake Kariba contain ZEN and may contribute to the exposure of consumers to this substance.

Adam Opalski (1897–1963)

Adam Opalski was born on November 26 (some sources say 28), 1897 in Olkusz, a town located about 40 km from Cracow in Poland. He attended elementary school in Kielce and middle school in Warsaw [1]. In 1917, Opalski started to study at the Medical Faculty of Warsaw University. He got a medical degree in 1924 and took a job in the internal diseases department at Baby Jesus Clinical Hospital, and a year later at the Warsaw Neurological Clinic [2]. Initially, Opalski worked as a volunteer and assistant. In the years 1928/29 and 1932/33, he went to Munich for an internship founded by the National Fund of Science and the Rockefeller Foundation [1]. In Germany, he worked at the laboratory of Professor Walther Spielmayer. During the internship Opalski studied the anatomy of the trigeminal ganglion under physiological conditions and during pathological states. However, his most important observations concerned altered glial cells occurring in the brain in Wilson’s disease. Opalski called these cells “outgrown giant glial cells” [3]. Interestingly, Opalski found these cells in tissues which were previously studied by Professors Alzheimer and Spielmayer, who did not see them [4]. Many years later, in 1965, professor Mossakowski, a student and co-worker of Opalski, called these “Opalski cells” in honour of their discoverer [5]. At present, this term is found in many textbooks of neurology and neuropathology. During his stay in Munich, Opalski also wrote an assistant professorial dissertation entitled “Morphology and pathogenesis of inflammations within ependyma and subependymal glial cells” [4]. The defence of this dissertation took place in Warsaw in 1935. Opalski became the head of the Laboratory of Brain Histopathology at the Neurological Clinic in Warsaw, where he introduced new methods of brain labelling imported from Germany [4]. He was also a docent at the Department of Neurology of Warsaw University. In 1939, when the Second World War broke out, Adam Opalski served in the Polish army as a medical officer, was taken prisoner and interned in Radom [1]. When Opalski was released from the prison camp in 1941, he worked in various Warsaw hospitals. In 1942, he became the head of Neurological Clinic and started the underground teaching of Polish medical students. During the Warsaw Uprising in 1944 the city was completely destroyed. Opalski saved the equipment of the Clinic, driving it away from Warsaw at risk to his life [4]. After the liberation of Warsaw, in February 1945 Opalski became head of Department of Neurology with Clinic at Warsaw University. Despite difficult post-war conditions, he organized student education and developed the activities of his department. He participated actively in the Polish Neurological Society and edited the journal “Polish Neurology and Psychiatry” [4]. At this time Opalski also worked as a scientist. In 1946, based on two clinical cases, he described the submedullary syndrome, which is now also known as Opalski’s sub-bulbar syndrome [6]. This disease is a rare neurological syndrome considered to be a variation of lateral medullary syndrome (Wallenberg’s syndrome) [7]. Since 1946 only a few cases of Opalski’s sub-bulbar syndrome have been described in the scientific literature [8]. This syndrome is caused by post-stroke ischemia in the area of branches of the posterior medullary artery and may be a result of various reasons including differences in the diameter of the vertebral arteries, dissection of the vertebral artery, or hypotonia secondary to spinocerebellar tract injury [7, 8]. Nevertheless, the most common cause of the syndrome is atherosclerosis [9]. The principal clinical features of Opalski’s sub-bulbar syndrome are as follows [7, 9]: ipsilateral hemiparesis and ataxia, Horner’s syndrome, hypoesthesia within the face, as well as alternate hypoesthesia for pain and temperature within * Liliana Rytel liliana_rytel@wp.pl

The Influence of Bisphenol A (BPA) on Neuregulin 1-Like Immunoreactive Nerve Fibers in the Wall of Porcine Uterus

Bisphenol A (BPA), a substance commonly used in the manufacture of plastics, shows multidirectional negative effects on humans and animals. Due to similarities to estrogens, BPA initially leads to disorders in the reproductive system. On the other hand, it is known that neuregulin 1 (NRG-1) is an active substance which enhances the survivability of cells, inhibits apoptosis, and protects tissues against damaging factors. Because the influence of BPA on the nervous system has also been described, the aim of the present study was to investigate for the first time the influence of various doses of BPA on neuregulin 1-like immunoreactive (NRG-1-LI) nerves located in the porcine uterus using the routine single- and double-immunofluorescence technique. The obtained results have shown that BPA increases the number and affects the neurochemical characterization of NRG-1-LI in the uterus, and changes are visible even under the impact of small doses of this toxin. The character of observed changes depended on the dose of BPA and the part of the uterus studied. These observations suggest that NRG-1 in nerves supplying the uterus may play roles in adaptive and protective mechanisms under the impact of BPA.

Cocaine- and amphetamine-regulated transcript (CART) peptide in the enteric nervous system of the porcine esophagus

Cocaine- and amphetamine-regulated transcript peptide (CART) is widely distributed within the central and peripheral nervous system. In the brain, CART is considered as the main anorectic peptide involved in the regulation of food intake. Contrary to the central nervous system, a lot of aspects connected with the distribution and functions of CART within the enteric nervous system (ENS) still remain unknown. The aim of the present study was to investigate, for the first time, the population of CART-like immunoreactive (CART-LI) neurons within the porcine esophagus and the denotation of their neurochemical coding. During this experiment, the distribution of CART-LI neurons and the colocalization of CART with other neuronal active substances were examined using standard double- and triple-immunofluorescence techniques in enteric plexuses of cervical, thoracic, and abdominal esophagus fragments. The obtained results showed that CART is present in a relatively high percentage of esophageal neurons (values fluctuated from 45.2±0.9% in the submucous plexus of the thoracic esophagus to 58.1±5.0% in the myenteric plexus of the same fragment of the esophagus). Moreover, CART colocalized with a wide range of other active neuronal substances, mainly with the vesicular acetylcholine transporter (VAChT, a marker of cholinergic neurons), neuronal isoform of nitric oxide synthase (nNOS, a marker of nitrergic neurons), vasoactive intestinal polypeptide (VIP) and galanin (GAL). The number of CART-positive neuronal cells and their neurochemical coding clearly depended on the fragment of esophagus studied and the type of enteric plexus. The obtained results suggest that CART may play important and multidirectional roles in the neuronal regulation of esophageal functions.