Annamaria Grandis

CB(1) signaling in forebrain and sympathetic neurons is a key determinant of endocannabinoid actions on energy balance.

The endocannabinoid system (ECS) plays a critical role in obesity development. The pharmacological blockade of cannabinoid receptor type 1 (CB(1)) has been shown to reduce body weight and to alleviate obesity-related metabolic disorders. An unsolved question is at which anatomical level CB(1) modulates energy balance and the mechanisms involved in its action. Here, we demonstrate that CB(1) receptors expressed in forebrain and sympathetic neurons play a key role in the pathophysiological development of diet-induced obesity. Conditional mutant mice lacking CB(1) expression in neurons known to control energy balance, but not in nonneuronal peripheral organs, displayed a lean phenotype and resistance to diet-induced obesity. This phenotype results from an increase in lipid oxidation and thermogenesis as a consequence of an enhanced sympathetic tone and a decrease in energy absorption. In conclusion, CB(1) signaling in the forebrain and sympathetic neurons is a key determinant of the ECS control of energy balance.

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.

Sympathetic innervation of the ileocecal junction in horses.

The distribution and chemical phenotypes of sympathetic and dorsal root ganglion (DRG) neurons innervating the equine ileocecal junction (ICJ) were studied by combining retrograde tracing and immunohistochemistry. Immunoreactivity (IR) for tyrosine hydroxylase (TH), dopamine beta‐hydroxylase (DBH), neuronal nitric oxide synthase (nNOS), calcitonin gene‐related peptide (CGRP), substance P (SP), and neuropeptide Y (NPY) was investigated. Sympathetic neurons projecting to the ICJ were distributed within the celiac (CG), cranial mesenteric (CranMG), and caudal mesenteric (CaudMG) ganglia, as well as in the last ganglia of the thoracic sympathetic chain and in the splanchnic ganglia. In the CG and CranMG 91 ± 8% and 93 ± 12% of the neurons innervating the ICJ expressed TH‐ and DBH‐IR, respectively. In the CaudMG 90 ± 15% and 94 ± 5% of ICJ innervating neurons were TH‐ and DBH‐IR, respectively. Sympathetic (TH‐IR) fibers innervated the myenteric and submucosal ganglia, ileal blood vessels, and the muscle layers. They were more concentrated at the ICJ level and were also seen encircling myenteric plexus (MP) and submucosal plexus (SMP) descending neurons that were retrogradely labeled from the ICJ. Among the few retrogradely labeled DRG neurons, nNOS‐, CGRP‐, and SP‐IR nerve cells were observed. Dense networks of CGRP‐, nNOS‐, and SP‐IR varicosities were seen around retrogradely labeled prevertebral ganglia neurons. The CGRP‐IR fibers are probably the endings of neurons projecting from the intestine to the prevertebral ganglia. These findings indicate that this crucial region of the intestinal tract is strongly influenced by the sympathetic system and that sensory information of visceral origin influences the sympathetic control of the ICJ. J. Comp. Neurol. 518:4046–4066, 2010.

Prospective Evaluation of Techniques for Differentiating Shoulder Pathology As a Source of Forelimb Lameness in Medium and Large Breed Dogs

OBJECTIVE To assess lameness evaluation, shoulder abduction angles, radiography, and ultrasonography for determining presence, location, and severity of forelimb pathology. STUDY DESIGN Prospective cohort study. ANIMALS Dogs >or=20 kg (n=30). METHODS Each dog was assigned lameness scores. Shoulder abduction angles were determined. Radiographs of shoulders and elbows were subjectively graded for pathology. One investigator unaware of dog history (lameness, radiographic findings) performed ultrasonographic assessment of shoulders with subjective grading of pathology. Another investigator unaware of dog history (lameness, radiographic, ultrasonographic findings) performed arthroscopic assessment of shoulders with subjective grading of pathology. Elbows were disarticulated and evaluated for gross pathology. Histologic pathology scoring of shoulder tissues was performed. Data were compared for differences among groups, sensitivities, specificities, positive and negative predictive values, and positive and negative likelihood ratios were calculated. RESULTS Twenty-seven forelimbs were considered clinically normal, 26 had shoulder pathology, 5 had elbow pathology, and 2 had pathology of both the shoulder and elbow. Dogs with shoulder pathology were twice as likely to be lame compared with dogs with elbow pathology. Limbs with medial shoulder instability had significantly higher abduction angles than normal limbs and those with elbow pathology. Radiographs were clinically useful for diagnosing elbow, but not shoulder, pathology. Ultrasonography was clinically useful for diagnosing shoulder pathology other than instability. Abduction angles, ultrasonographic evaluation, and arthroscopic assessments had strong, significant correlations with reference standards. CONCLUSIONS Clinically relevant diagnostic techniques yielded characteristic, repeatable differences in objective and subjective assessments for distinguishing presence, location, and severity of forelimb lameness in dogs. CLINICAL RELEVANCE The diagnostic approach to forelimb lameness in dogs should include shoulder pathology as a differential with multiple assessments used to determine the clinical cause of lameness.

Intrinsic innervation of the horse ileum

This paper describes the morphology and distribution of the enteric nervous system (ENS) cells and fibres immunoreactive for choline acetyltransferase (ChAT), neuronal nitric oxide synthase (nNOS), substance P (SP), calcitonin gene-related peptide (CGRP), NF200kDa (NF200), and S100 protein. The percentages of subclasses of enteric neurons in the total neuronal population were investigated by the use of anti-PGP 9.5 or anti-NSE antibodies. ChAT-IR myenteric plexus (MP) and submucosal plexus (SMP) neurons were 66+/-7% and 74+/-15%, respectively, whereas those cells expressing nNOS-IR were 38+/-7% and 5+/-1%, respectively. MP and SMP neurons expressing both phenotypes were also present. SP-IR was expressed by 14+/-13% of MP and 66+/-8% of SMP neurons whereas CGRP-IR was observed only in the SMP (43+/-6%). NF200-IR was expressed by 61+/-15% and 91+/-10% of the MP and SMP neurons, respectively. The majority of the CGRP-IR SMP neurons expressed also SP-IR. Almost all SP-IR neurons in both the plexuses were cholinergic. The present study quantifies the main neuronal subpopulations of the ENS of the horse ileum; these data might be utilized to understand the neuronal modifications which occur in several gastrointestinal tract disorders.

Collaterals of recurrent laryngeal nerve fibres innervate the thymus: a fluorescent tracer and HRP investigation of efferent vagal neurons in the rat brainstem

The origin and course of efferent vagal fibers, which innervate the rat thymus, were investigated by a fluorescent retrograde double labeling method, using Fast blue (FB) and Diamidino yellow dihydrochloride (DY) as tracers. In the same animal, one tracer was injected into the cranial portion of the right lobe of the thymus and the other dye was deposited around the cut end of the right recurrent laryngeal nerve. The neuronal population giving origin to the recurrent nerve was mapped by using retrograde labeling with HRP applied to the central stump of the nerve. The HRP retrograde axonal transport showed that most efferent vagal fibers of the recurrent nerve have their perikarya in the nucleus retroambigualis (NRA), nucleus ambiguus (NA), and to a lesser extent in the nucleus retrofacialis (NRF). In fluorescent retrograde double labeling of thymus and recurrent laryngeal nerve both single and double labeled cells were found. The cells labeled by the injections into the thymus were colocalized with the neurons labeled by the tracer deposited in the recurrent laryngeal nerve to the NRA, NA, and NRF. Moreover along the rostrocaudal extent of the NRF and NA double labeled cells were present, showing that some of the thymic efferents are collaterals of the recurrent nerve fibers. Our experiments shown that some thymic vagal fibres originate from neurons of nucleus dorsalis nervi vagi (NDV) as demonstrated both by HRP and FB injected thymuses. The possible role of these efferents in thymic function is briefly discussed.

Viscerotopic representation of the subdiaphragmatic tracts of the digestive apparatus within the vagus complex in the sheep.

The distribution in the brainstem and cervical spinal cord of neurons supplying the reticulum and the reticular groove, the rumen, the omasum, the abomasum, and the small and large intestine was investigated in the sheep using the fluorescent retrograde tracer technique. Only the reticulum and reticular groove were represented in the dorsal motor nucleus of the vagus nerve (DMNX), in the nucleus ambiguus (NA), and in the nucleus retroambigualis (NRA). The other forestomach, the abomasum and the small intestine were supplied by the DMNX only, with the exception of the rumen which was also innervated by the NRA. Some reticular formation neurons were found labeled after the injection of the tracer into the reticulum, the reticular groove, and the rumen. We present evidence that the reticular groove is the part of the forestomach having the widest representation, and also the richest innervation.

Immunohistochemical Localization of Substance P And Cholecystokinin in the Dorsal Root Ganglia and Spinal Cord of the Bottlenose Dolphin (Tursiops truncatus)

The presence of substance P (SP) and cholecystokinin (CCK) immunoreactive neurons was examined in the bottlenose dolphin dorsal root ganglia (DRGs) and spinal cord by immunohistochemical techniques. SP‐positive and CCK‐immunoreactive neurons were respectively ∼50% and 1% of the total number of ganglion cells examined and especially belonged to small and medium‐sized cell populations. Using double labeling techniques we observed that SP‐ and CCK‐immunoreactivity coexisted in a very low number of primary afferent neurons (2.7%). Few SP‐immunoreactive (IR) neurons (2.7%) were also CCK‐positive. On the contrary, 65% of CCK‐immunoreactive neurons contained SP. Interestingly, we observed CCK‐immunoreactive satellite glial cells located around large cell class somata. Virtually no SP‐IR and CCK‐positive neurons were surrounded by peripheral CCK‐immunoreactive satellite glial cells. The SP‐IR and CCK‐positive nerve fibers were particularly conspicuous in the superficial layers of the spinal cord. The present study indicates that SP and CCK only partially overlap in the thoracic, lumbar, and caudal DRGs of the bottlenose dolphin, suggesting that the majority of SP‐IR ganglion neurons are lacking in CCK‐immunoreactivity. The role of SP‐containing DRG neurons is discussed also in relation to the huge vascular spinal retia mirabilia typical of cetaceans. Anat Rec, 293:477–484, 2010.

Endoscopic examination of the nasolacrimal duct in ten horses.

REASONS FOR PERFORMING STUDY No endoscopic examination of the nasolacrimal duct has been described before. In contrast with other imaging techniques, endoscopy provides a direct inspection of the intralumen and ductal mucosa in standing sedation. OBJECTIVES To provide a reference against which the endoscopic and clinical features of obstructive nasolacrimal disease in the horse may be compared. METHODS Endoscopic examination of the nasolacrimal duct was performed in 10 French Thoroughbred bay mares with a 3 mm shaft diameter flexible fibrescope. The duct was divided into 3 zones (1, 2 and 3) from the nostril to the lacrimal sac. RESULTS Endoscopic examination of the nasolacrimal duct appeared to be quite easy to perform in Zones 1 and 2, while the ease or difficulty of examining Zone 3 depended on the weight and size of the horse for the position of the endoscope in the lacrimal canal. CONCLUSIONS Endoscopic inspection has provided the possibility of a novel diagnostic approach to conditions of the nasolacrimal duct in horses. POTENTIAL RELEVANCE Endoscopic examination of the nasolacrimal duct could help to diagnose and treat nasolacrimal diseases by performing sample withdrawal and duct irrigation.

Intrinsic innervation of the ileocaecal junction in the horse: Preliminary study

REASON FOR PERFORMING STUDY In horses, morpho-functional studies related to the enteric nervous system (ENS) controlling the sphincters are lacking. OBJECTIVES To investigate immunohistochemically the morphology, distribution, density, phenotypes and projections of neurons controlling the ileocaecal junction (ICJ). METHODS Two young horses were anaesthetised and underwent midline laparotomy. The neuronal retrograde fluorescent tracer Fast Blue (FB) was injected into the wall of the ICJ. A post surgical survival time of 30 days was used. Following euthanasia, the ileum and a small portion of caecum were removed. Cryosections were used to investigate the immunoreactivity (IR) of the neurons innervating the ICJ for choline acetyltransferase (ChAT), neuronal nitric oxide synthase (nNOS), substance P (SP), calcitonin gene-related peptide (CGRP) and neurofilament NF200kDa (NF). RESULTS Ileal FB-labelled neurons innervating the ICJ were located in the myenteric plexus (MP) and submucosal plexus (SMP) up to 48 cm and 28 cm, respectively, from the point of the FB injections. Descending MP and SMP neurons were nitrergic (54 +/- 11% and 68 +/- 4%, respectively), cholinergic (60 +/- 19% and 82 +/- 11%, respectively), NF-IR (54 +/- 9% and 78 +/- 21%, respectively), and SP-IR (about 20% in both the plexuses). CGRP-IR was expressed only by SMP descending neurons (45 +/- 21%). In both the plexuses descending neurons coexpressing nNOS- and ChAT-IR were also observed (25 +/- 11% and 61 +/- 27%, respectively). CONCLUSIONS The presence of ileal long projecting neurons innervating the ICJ suggests that they are critical for its modulation. Consequently, in bowel diseases in which the resection of the terminal jejunum and proximal ileum are required, it is preferable, whenever possible, to conserve the major portion of the ileum. POTENTIAL RELEVANCE The knowledge of the phenotype of ENS neurons of the ileum might be helpful for developing pharmaceutical treatment of the ICJ motility disorders.