Anita Mikołajczyk

Distribution and chemical coding of sensory neurons innervating the skin of the porcine hindlimb

The aim of the present study was to establish the origin and chemical phenotyping of neurons involved in skin innervation of the porcine hind leg. The dorsal root ganglia (DRGs) of the lumbar (L4-L6) and sacral (S1-S3) spinal nerves were visualized using the fluorescent tracer Fast Blue (FB). The morphometric analysis of FB-positive (FB+)neurons showed that in the L4, L5, S1 and S2 DRGs, the small-sized perikarya constituted the major population, whereas in the L6 and S3 DRGs the medium-sized cells made up the major population. In all these ganglia, large-sized FB+ perikarya constituted only a small percentage of all FB+ neurons. Immunohistochemistry revealed that small- and medium-sized FB+ perikarya contained sensory markers such as: substance P (SP), calcitonin gene related peptide (CGRP) and galanin (GAL); as well as various other factors such as somatostatin (SOM), calbindin-D28k (CB), pituitary adenylate cyclase-activating polypeptide (PACAP) and neuronal nitric oxide synthase (nNOS). Meanwhile large-sized FB+ perikarya usually expressed SP, CGRP or PACAP. In the lumbar DRGs, some large cells also contained SOM and CB. Double-labeling immunohistochemistry showed that SP-positive neurons co-expressed CGRP, GAL or PACAP; while PACAP-positive cells co-expressed GAL or nNOS. Neurons stained for SOM were also immunoreactive for CB or GAL, while neurons stained for nNOS were also immunoreactive for GAL. In conclusion, the present data has indicated that the distribution and chemical phenotyping of the porcine skin-projecting neurons are different within DRGs of the lumbar (forming a femoral nerve) and sacral (forming a sciatic nerve) spinal nerves.

Distribution and Neurochemistry of the Porcine Ileocaecal Valve Projecting Sensory Neurons in the Dorsal Root Ganglia and the Influence of Lipopolysaccharide from Different Serotypes of Salmonella spp. on the Chemical Coding of DRG Neurons in the Cell Cultures

The ileocecal valve (ICV)—a sphincter muscle between small and large intestine—plays important roles in the physiology of the gastrointestinal (GI) tract, but many aspects connected with the innervation of the ICV remain unknown. Thus, the aim of this study was to investigate the localization and neurochemical characterization of neurons located in the dorsal root ganglia and supplying the ICV of the domestic pig. The results have shown that such neurons mainly located in the dorsal root ganglia (DRG) of thoracic and lumbar neuromers show the presence of substance P (SP), calcitonin gene-related peptide (CGRP), and galanin (GAL). The second part of the experiment consisted of a study on the influence of a low dose of lipopolysaccharide (LPS) from Salmonella serotypes Enteritidis Minnesota and Typhimurium on DRG neurons. It has been shown that the LPS of these serotypes in studied doses does not change the number of DRG neurons in the cell cultures, but influences the immunoreactivity to SP and GAL. The observed changes in neurochemical characterization depend on the bacterial serotype. The results show that DRG neurons take part in the innervation of the ICV and may change their neurochemical characterization under the impact of LPS, which is probably connected with direct actions of this substance on the nervous tissue and/or its pro-inflammatory activity.

Distribution and neurochemistry of porcine urinary bladder-projecting sensory neurons in subdomains of the dorsal root ganglia: a quantitative analysis

The aim of the present study has been to verify the inter- and intraganglionic distribution pattern of porcine urinary bladder-projecting (UBP) neurons localized in the sacral dorsal root ganglia (DRGs). The morphology and chemical phenotype of these cells have also been investigated. These neurons were visualized using the fluorescent tracer Fast Blue (FB) which was injected bilaterally into the urinary bladder wall of five juvenile female pigs. The intraganglionic distribution showed that small- and medium-sized FB+ perikarya were mainly located in the central (S3-S4) and periphero-central (S2) region of the ganglia, while large cells were heterogeneously distributed. Immunohistochemistry revealed that the most frequently observed markers in small and medium-sized UBP perikarya were: neurofilament 200, lectin from Bandeiraea simplicifolia (Griffonia simplicifolia) isolectin B4, substance P, calcitonin gene-related peptide, pituitary adenylate cyclase-activating polypeptide and transient receptor potential vanilloid 1. Moreover, UBP neurons containing these substances were also mainly observed in the central and periphero-central region of the ganglion. Differences in the percentage of traced cells and their neuropeptide content were observed between the S2, S3 and S4 DRGs. In conclusion, the present study, for the first time, describes the arrangement of UBP DRGs neurons within particular subdomains of sacral ganglia, taking into account their size and chemical phenotype.

Neurochemical difference between somato- and viscero-projecting sensory neurons in the pig

The chemical coding of porcine somato (skin)- and viscero (urinary bladder)-projecting sensory neurons have been studied and compared using immunohistochemistry. Cell bodies of skin and bladder afferents were identified following Fast Blue injections into the skin of the hind leg as well as into wall of the urinary bladder, respectively. Immunohistochemistry revealed that small and medium-sized neurons projecting to both skin and bladder contained all of the studied substances i.e. substance P (SP), calcitonin gene-related pepide (CGRP), transient receptor potential vanilloid (TRPV1), lectin from Bandeiraea simplicifolia - Griffonia simplicifolia isolectin B4 (IB4) and galanin (GAL). Moreover, small-sized sensory neurons projecting to the bladder and skin of hind leg showed predominantly immunoreactivity to SP and TRPV1 and CGRP, as well as to CGRP and TRPV1 and IB4. It is worth stressing that the subset of sensory neurons innervating the skin exhibited these substances more often than bladder-projecting neurons. In addition, medium-sized skin-projecting neurons contained SP/GAL; SP/CGRP and CGRP/IB4 much more often than their bladder counterparts. On the other hand, small-sized perikarya that innervate the skin were less frequently expressed TRPV1, CGRP and GAL than the bladder-projecting neurons. In conclusion, the present report describes, for the first time, significant differences in the chemical coding between somato- and viscero-projecting sensory neurons in dorsal root ganglia. Moreover, these results provide morphological basis for further functional studies, which may explain the exact roles played by various subpopulations of somato- and viscero-projecting sensory neurons.

Neuroimmunological Implications of Subclinical Lipopolysaccharide from Salmonella Enteritidis

Mounting evidence has indicated that lipopolysaccharide (LPS) is implicated in neuroimmunological responses, but the body’s response to subclinical doses of bacterial endotoxin remains poorly understood. The influence of a low single dose of LPS from Salmonella Enteritidis, which does not result in any clinical symptoms of intoxication (subclinical lipopolysaccharide), on selected cells and signal molecules of the neuroimmune system was tested. Five juvenile crossbred female pigs were intravenously injected with LPS from S. Enteritidis (5 μg/kg body weight (b.w.)), while five pigs from the control group received sodium chloride in the same way. Our data demonstrated that subclinical LPS from S. Enteritidis increased levels of dopamine in the brain and neuropeptides such as substance P (SP), galanin (GAL), neuropeptide Y (NPY), and active intestinal peptide (VIP) in the cervical lymph nodes with serum hyperhaptoglobinaemia and reduction of plasma CD4 and CD8 T-lymphocytes seven days after lipopolysaccharide administration. CD4 and CD8 T-lymphocytes from the cervical lymph node and serum interleukin-6 and tumour necrosis factor α showed no significant differences between the control and lipopolysaccharide groups. Subclinical lipopolysaccharide from S. Enteritidis can affect cells and signal molecules of the neuroimmune system. The presence of subclinical lipopolysaccharide from S. Enteritidis is associated with unknown prolonged consequences and may require eradication and a deeper search into the asymptomatic carrier state of Salmonella spp.

Detailed Characterization of Sympathetic Chain Ganglia (SChG) Neurons Supplying the Skin of the Porcine Hindlimb

It is generally known that in the skin sympathetic fibers innervate various dermal structures, including sweat glands, blood vessels, arrectores pilorum muscles and hair follicles. However, there is a lack of data about the distribution and chemical phenotyping of the sympathetic chain ganglia (SChG) neurons projecting to the skin of the pig, a model that is physiologically and anatomically very representative for humans. Thus, the present study was designed to establish the origin of the sympathetic fibers supplying the porcine skin of the hind leg, and the pattern(s) of putative co-incidence of dopamine-β-hydroxylase (DβH) with pituitary adenylate cyclase-activating polypeptide (PACAP), somatostatin (SOM), neuronal nitric oxide synthase, substance P, vasoactive intestinal peptide, neuropeptide Y (NPY), leu5-enkephalin and galanin (GAL) using combined retrograde tracing and double-labeling immunohistochemistry. The Fast Blue-positive neurons were found in the L2–S2 ganglia. Most of them were small-sized and contained DβH with PACAP, SOM, NPY or GAL. The findings of the present study provide a detailed description of the distribution and chemical coding of the SChG neurons projecting to the skin of the porcine hind leg. Such data may be the basis for further studies concerning the plasticity of these ganglia under experimental or pathological conditions.