Paolo Clavenzani

Anatomical evidence for ileal Peyer’s patches innervation by enteric nervous system: a potential route for prion neuroinvasion?

We have examined the innervation of the gut-associated lymphoid system of the sheep ileum, with a view to identifying potential sites for neuroinvasion by pathogens, such as prions (PrPSc). Special attention has been paid to the follicles of Peyer’s patches (PPs), which are major sites of PrPSc accumulation during infection. Evidence exists that the enteric nervous system, together with the parasympathetic and sympathetic pathways projecting to the intestine, are important for PrPSc entry into the central nervous system. Thus, PrPSc might move from PPs to the neurons and nerve fibres that innervate them. We investigated, by immunohistochemistry and retrograde tracing (DiI) from the follicles, the distribution and phenotype of enteric neurons innervating the follicles. Antibodies against protein gene product 9.5, tyrosine hydroxylase, dopamine β hydroxylase, choline acetyltransferase, calbindin (CALB), calcitonin gene-related peptide (CGRP), and nitric oxide synthase were used to characterise the neurons. Immunoreactivity for each of these was observed in fibres around and inside PP follicles. CGRP-immunoreactive fibres were mainly seen at the follicular dome. Retrograde tracing revealed submucosal neurons that contributed to the innervation of PPs, including Dogiel type II neurons and neurons immunoreactive for CALB and CGRP. The major source of the adrenergic fibres are the sympathetic ganglia. Our results thus suggest that enteric and sympathetic neurons are involved during the first stage of neuroinvasion, with neurons connecting to them acting as potential carriers of PrPSc to the central nervous system.

Characterisation of neurons expressing calbindin immunoreactivity in the ileum of the unweaned and mature sheep.

We have identified the enteric neuron types expressing immunoreactivity for the calcium-binding protein calbindin D28k (CALB) in cryostat sections and whole-mount preparations of myenteric (MP) and submucosal (SMP) plexuses of sheep ileum. We wished to determine whether CALB-IR in the sheep enteric nervous system was expressed in Dogiel type II cells, as in guinea-pig and rat ileum, and could therefore be used as a marker for intrinsic primary afferent neurons. The neurochemical coding of CALB-containing myenteric and submucosal neurons in ileum of unweaned lamb and mature sheep and its co-localisation with various neural markers was studied immunohistochemically. An antiserum against neuronal nuclear protein (NeuN) failed to detect the entire neuronal population; it was expressed only in 48% of neuron-specific enolase (NSE)-immunoreactive (NSE-IR) neurons. Human neuronal protein appeared to occur in the large majority or all neurons. Almost all CALB-IR neurons were: (1) radially multidendritic; (2) eccentric multidendritic; (3) Dogiel type II. CALB-IR occurred in 20–25% of myenteric and 65–75% of submucosal neurons in lamb and mature sheep, with higher values in mature sheep. Nearly all CALB-IR neurons were common choline acetyltransferase (cChAT)-IR, whereas only about 20% of cChAT-IR somata were CALB-IR. In lamb and mature sheep, 90% of MP CALB-IR neurons were peripheral choline acetyltransferase (pChAT)-IR. In lamb SMP, 80±13% of CALB-IR cells were also pChAT-IR, whereas all those in mature SMP were pChAT-IR. Fewer myenteric CALB-IR neurons exhibited tachykinin (TK) in mature sheep (49%) than in lamb (88%). This was also the case for submucosal ganglia (mature sheep, 63%; lamb, 89%). In lamb MP, 77±7% of CALB-IR cells were NeuN-positive. In mature sheep, 73±10% of CALB-IR somata were NeuN-IR, but NeuN failed to stain SMP neurons. In the MP of suckling and mature sheep, Dogiel type II CALB-IR neurons were calcitonin gene-related peptide (CGRP)-IR. In the SMP at both stages, Dogiel type II CALB-IR somata (about 50% of CALB-IR neurons) were also CGRP-IR. Only small proportions of CALB-IR neurons showed immunoreactivity for calretinin or nitric oxide synthase (NOS), although large populations of CALB and NOS neurons occurred in the ganglia. Thus, CALB is a marker of most Dogiel type II neurons in the sheep but is not confined to Dogiel II neurons. CGRP is a more selective marker of Dogiel type II neurons, being only found in this neuron type.

Functional and neurochemical changes of the gastrointestinal tract in a rodent model of Parkinson's disease.

Patients with Parkinsons disease develop motor disturbances often accompanied by peripheral autonomic dysfunctions, including gastrointestinal disorders, such as dysphagia, gastric stasis and constipation. While the mechanisms subserving enteric autonomic dysfunctions are not clearly understood, they may involve the enteric dopaminergic and/or nitrergic systems. In the present study, we demonstrate that rats with unilateral 6-hydroxydopamine lesion of nigrostriatal dopaminergic neurons develop a marked inhibition of propulsive activity compared to sham-operated controls, as indicated by a 60% reduction of daily fecal output at the 4th week of observation. Immunohistochemical data revealed that 6-hydroxydopamine treatment did not affect the total number of HuC/D-positive myenteric neurons in both the proximal and distal segments of ileum and colon. Conversely, in the distal ileum and proximal colon the number of nitrergic neurons was significantly reduced. These results suggest that a disturbed distal gut transit, reminiscent of constipation in the clinical setting, may occur as a consequence of a reduced propulsive motility, likely due to an impairment of a nitric oxide-mediated descending inhibition during peristalsis.

Relationship between pectoralis major muscle histology and quality traits of chicken meat

A trial was conducted to evaluate the influence of myodegeneration of pectoralis major muscle on quality traits and chemical composition of breast meat of heavy-size male broilers. For this purpose, a total of 72 pectoralis major muscles were randomly collected from broilers farmed under homogeneous conditions and graded into three categories (mild, n=22; moderate, n=33; and severe, n=17) based on the presence of abnormal fibers (giant fibers, fibers with hyaline degeneration, and damaged and/or necrotic fibers) evaluated by histological and immunohistochemical analysis. Color, pH, drip loss, Allo-Kramer shear values, and chemical composition (moisture, proteins, total lipids, ashes, and collagen) were determined on nonmarinated breast meat. Purge loss and cook loss, total yield, and Allo-Kramer shear values were measured on vacuum-tumbled samples. Samples showing moderate myodegeneration had the highest mean cross-sectional area of the fibers, while samples with severe myodegeneration had myofibers of different diameter and without the characteristic polygonal shape, multifocal degeneration and necrosis, as well as infiltration of CD3-immunoreactive cells. Cooking losses of nonmarinated meat were lower in the mild group with respect to moderate and severe groups (21.4 vs. 24.7 and 24.7%; P<0.001). Breast muscles with severe damage, in comparison with mild degenerated samples, showed higher moisture (75.4 vs. 74.4%; P<0.05) and lower protein percentages (21.1 vs. 22.6%; P<0.001). The lipid percentage of severely degenerated samples was higher than that from moderate group (2.94 vs. 2.36; P<0.05), while collagen content was not modified by histological lesion levels. Marinated meat from the mild group had higher uptake and total marinade yield after cooking. In conclusion, almost all breast fillets of heavy broiler chickens produced under intensive farming systems had histological lesions, which reflected on the chemical composition of the meat and the impaired water holding/binding capacities of the meat.

Central projections and entries of capsaicin-sensitive muscle afferents.

The entry pathway and central distribution of A delta and C muscle afferents within the central nervous system (CNS) were investigated by combining electron microscopy and electrophysiological analysis after intramuscular injection of capsaicin. The drug was injected into the rat lateral gastrocnemius (LG) and extraocular (EO) muscles. The compound action potentials of LG nerve and the evoked field potentials recorded in semilunar ganglion showed an immediate and permanent reduction in A delta and C components. The morphological data revealed degenerating unmyelinated axons and terminals in the inner sublamina II and in the border of laminae I-II of the dorsal horn at L4-L5 and C1-C2 (subnucleus caudalis trigemini) spinal cord segments. Most degenerating terminals were the central bouton (C) of type I and II synaptic glomeruli. Furthermore, degenerating peripheral axonal endings (V2) presynaptic to normal C were found. Since V2 were previously found degenerated after cutting the oculomotor nerve (ON) or L4 ventral root, we conclude that some A delta and C afferents from LG and EO muscles entering the CNS by ON or ventral roots make axoaxonic synapses on other primary afferents to promote an afferent control of sensory input.

Neurochemical features of boar lumbosacral dorsal root ganglion neurons and characterization of sensory neurons innervating the urinary bladder trigone

Porcine lumbosacral dorsal root ganglion (DRG) neurons were neurochemically characterized by using six neuronal markers: calcitonin gene‐related peptide (CGRP), substance P (SP), neuronal nitric oxide synthase (nNOS), neurofilament 200kDa (NF200), transient receptor potential vanilloid 1 (TRPV1), and isolectin B4 (IB4) from Griffonia simplicifolia. In addition, the phenotype and cross‐sectional area of DRG neurons innervating the urinary bladder trigone (UBT) were evaluated by coupling retrograde tracer technique and immunohistochemistry. Lumbar and sacral DRG neuronal subpopulations were immunoreactive (IR) for CGRP (30 ± 3% and 29 ± 3%, respectively), SP (26 ± 8% and 27 ± 12%, respectively), nNOS (21 ± 4% and 26 ± 7%, respectively), NF200 (75 ± 14% and 81 ± 7%, respectively), and TRPV1 (48 ± 13% and 43 ± 6%, respectively), and labeled for IB4 (56 ± 6% and 43 ± 10%, respectively). UBT sensory neurons, which were distributed from L2 to Ca1 DRG, had a segmental localization, showing their highest density in L4–L5 and S2–S4 DRG. Lumbar and sacral UBT sensory neurons expressed similar percentages of NF200 immunoreactivity (64 ± 33% and 58 ± 12%, respectively) but showed a significantly different immunoreactivity for CGRP, SP, nNOS, and TRPV1 (56 ± 9%, 39 ± 15%, 17 ± 13%, 62 ± 10% vs. 16 ± 6%, 16 ± 11%, 6 ± 1%, 45 ± 24%, respectively). Lumbar and sacral UBT sensory neurons also showed different IB4 labeling (67 ± 19% and 48 ± 16, respectively). Taken together, these data indicate that the lumbar and sacral pathways probably play different roles in sensory transmission from the UBT. The findings related to cell size also reinforced this hypothesis, because lumbar UBT sensory neurons were significantly larger than sacral ones (1,112 ± 624 μm2 vs. 716 ± 421 μm2). J. Comp. Neurol. 521:342–366, 2013.

Nitric oxide synthase immunoreactivity and NADPH-d histochemistry in the enteric nervous system of Sarda breed sheep with different PrP genotypes in whole-mount and cryostat preparations.

Until now, significant differences in the neurochemical pattern of enteric neurons have been demonstrated in all species studied; however, some strong similarities also occur across species, such as the occurrence of nitric oxide synthase immunoreactivity (NOS-IR) in inhibitory motor neurons to muscle. In consideration of the insufficient data regarding the enteric nervous system (ENS) of sheep, we investigated the myenteric plexus and submucosal plexus of the ovine ileum. Since the pivotal role of the ENS in the early pathogenesis of sheep scrapie, the “prototype” of prion diseases, has been suggested, we have focused our observations also on the hosts PrP genotype. We have studied the morphology and distribution of NOS-IR neurons and their relationships with the enteric glia in whole-mount preparations and in cryostat sections. NOS-IR neurons, always encircled by glial processes, were located in both plexuses. Many NOS-IR fibers were seen in the circular muscle layer, in the submucosa, and in the mucosa. In the submucosa they were close to the lymphoid tissue. No differences in the distribution and percentage of NOS-IR fibers and neurons were observed among sheep carrying different PrP genotype, thus making unlikely their contribution in the determinism of susceptibility/resistance to scrapie infection.

Morphology and neurochemistry of descending and ascending myenteric plexus neurons of sheep ileum.

The specific patterns of gastrointestinal motility in large herbivores may relate to differences in the organization of enteric nerve circuits, compared with other mammals. To investigate this possibility, we characterized the morphologies, chemical phenotypes, and projections of myenteric plexus (MP) neurons of the sheep ileum. Morphologies and projections were investigated after application of the carbocyanine dye (1,1′, di‐octadecyl‐3,3,3′,3′,‐tetramethylindo‐carbocyanine perchlorate, DiI) to fixed tissues. To study chemical phenotypes, the fluorescent tracer Fast Blue (FB) was injected into the wall of the ileum, in vivo, 12–14 cm oral to the ileo‐caecal junction. Over 80% of the descending and ascending DiI‐labeled neurons had typical Dogiel type I morphology, whereas only a few Dogiel type II neurons were observed. Nevertheless, there were long projections (up to 10 cm) of Dogiel type II neurons in both directions. Both type II and type I neurons were neurofilament immunoreactive (IR). We observed long projections of descending (up to 18 cm) and ascending (up to 12–14 cm) FB‐labeled MP neurons. Nitric oxide synthase (NOS)‐IR, peripheral choline acetyltransferase (pChAT)‐IR, and substance P (SP)‐IR occurred in both descending and ascending myenteric neurons. NOS‐IR was in approximately 60% of FB‐labeled descending and ascending neurons, whereas those expressing pChAT‐IR were 67 ± 15% and 60 ± 14%, respectively. Descending neurons expressing SP‐IR were 48 ± 15% and ascending were 56 ± 12%. NOS‐IR and pChAT‐IR, and SP‐IR and pChAT‐IR were commonly colocalized in both ascending and descending pathways. In descending pathways, almost all SP‐IR neurons were also pChAT‐IR (98 ± 3%) and NOS‐IR (99 ± 2 NOS+/SP+/pChAT−). Many FB‐labeled descending neurons showed both NOS‐ and pChAT‐IR. Descending neurons may represent inhibitory motor neurons (NOS+/SP+/pChAT−) and two classes of interneurons (pChAT+/NOS−, and pChAT+/NOS+/SP+). In ascending pathways, most neurons are pChAT+/NOS+/SP+. Thus, in sheep, ascending interneurons and ascending excitatory motor neurons both have the same phenotype, and other markers are needed to distinguish them. Anat Rec, 2007.

Localization of peripheral autonomic neurons innervating the boar urinary bladder trigone and neurochemical features of the sympathetic component

The urinary bladder trigone (UBT) is a limited area through which the majority of vessels and nerve fibers penetrate into the urinary bladder and where nerve fibers and intramural neurons are more concentrated. We localized the extramural post-ganglionic autonomic neurons supplying the porcine UBT by means of retrograde tracing (Fast Blue, FB). Moreover, we investigated the phenotype of sympathetic trunk ganglia (STG) and caudal mesenteric ganglia (CMG) neurons positive to FB (FB+) by coupling retrograde tracing and double-labeling immunofluorescence methods. A mean number of 1845.1±259.3 FB+ neurons were localized bilaterally in the L1-S3 STG, which appeared as small pericarya (465.6±82.7 µm2) mainly localized along an edge of the ganglion. A large number (4287.5±1450.6) of small (476.1±103.9 µm2) FB+ neurons were localized mainly along a border of both CMG. The largest number (4793.3±1990.8) of FB+ neurons was observed in the pelvic plexus (PP), where labeled neurons were often clustered within different microganglia and had smaller soma cross-sectional area (374.9±85.4 µm2). STG and CMG FB+ neurons were immunoreactive (IR) for tyrosine hydroxylase (TH) (66±10.1% and 52.7±8.2%, respectively), dopamine beta-hydroxylase (DβH) (62±6.2% and 52±6.2%, respectively), neuropeptide Y (NPY) (59±8.2% and 65.8±7.3%, respectively), calcitonin-gene-related peptide (CGRP) (24.1±3.3% and 22.1±3.3%, respectively), substance P (SP) (21.6±2.4% and 37.7±7.5%, respectively), vasoactive intestinal polypeptide (VIP) (18.9±2.3% and 35.4±4.4%, respectively), neuronal nitric oxide synthase (nNOS) (15.3±2% and 32.9±7.7%, respectively), vesicular acetylcholine transporter (VAChT) (15±2% and 34.7±4.5%, respectively), leuenkephalin (LENK) (14.3±7.1% and 25.9±8.9%, respectively), and somatostatin (SOM) (12.4±3% and 31.8±7.3%, respectively). UBT-projecting neurons were also surrounded by VAChT-, CGRP-, LENK-, and nNOSIR fibers. The possible role of these neurons and fibers in the neural pathways of the UBT is discussed.

Downregulation of neuronal vasoactive intestinal polypeptide in Parkinson's disease and chronic constipation

Chronic constipation (CC) is a common and severe gastrointestinal complaint in Parkinsons disease (PD), but its pathogenesis remains poorly understood. This study evaluated functionally distinct submucosal neurons in relation to colonic motility and anorectal function in PD patients with constipation (PD/CC) vs both CC and controls.