Signal amplification by combining two advanced immunohistochemical techniques.

The immunohistochemical techniques known as EnVision trade mark + System (EVS) and Mirror Image Complementary Antibody (MICA) were recently introduced into laboratory practice because of their high sensitivity. In this paper these techniques were compared and their sequences combined to obtain a new method possibly more sensitive than the original ones. The immunohistochemical staining employing the avidin-biotin complex (ABC), largely used as routine, was adopted as a term of comparison. Samples from the small and large intestine of pigs and sheep were fixed in Bouin and embedded in Paraplast. The primary antibodies utilized were directed against the neuronal nitric oxide synthase (nNOS), vasoactive intestinal polypeptide (VIP) and chromogranin A (Cr A). Targets of these antibodies were nerve structures of the intestinal wall, as well as endocrine cells scattered in the mucosa of the bowel, defined neuroendocrine cells or paraneurons. The EVS method appeared as slightly superior to the MICA method regarding sensitivity of detection. The EVS/MICA (combined) method resulted four/eight times more effective than the original techniques regarding sensitivity of detection and staining intensity, both at low and high dilutions of the primary antibodies. Of these latter, immunopositive structures were still clearly identifiable, at a dilution of 1:256,000. Such efficiency could be explained by the high number of revealing molecules of peroxidase contained in the new sequence. The application of the combined method is recommended when a small quantity of tissue antigens needs to be detected immunohistochemically.

Nitric oxide-containing neurons in the bovine gut, with special reference to their relationship with VIP and galanin.

The presence and distribution of nicotinamide dinucleotide phosphate diaphorase (NADPH-d)-containing neurons have been studied by means of NADPH-d histochemistry in different regions of the adult cow gut, from the esophagus to the rectum. NADPH-d and nitric oxide synthase (NOS) were constantly recognized to be colocalized in the same neuron. The colocalization of vasoactive intestinal polypeptide (VIP) and galanin in such nitrergic neurons was also studied by means of combined histochemical and immunofluorescence techniques. NADPH-d-positive neurons were present along the myenteric plexus of the entire gut, and in the submucous plexus from the abomasum to the rectum. Notably, they formed two types of nerve networks in the submucous connective tissue of the jejunum-ileum. NADPH-d-positive innervation of the muscle layers occurred throughout the tract, and sometimes a clear correspondence was noted between the number of reactive fibres and the thickness of the muscle. Nitrergic fibres also occurred in the mucosa and often were in relation to glands and blood vessels. The nitrergic neurons varied in size, shape, and intensity of staining, and often their terminals were seen to surround unstained perikarya. Various types of neurons were recognized on the basis of their chemical content; one of them contained galanin, VIP and NOS simultaneously. The present results suggest that the nitrergic neurons of the bovine gastrointestinal tract play roles presumably for controlling the motility of the gut and the conduction of interneuronal impulses.

The relationships of nicotinamide adenine dinucleotide phosphate-d to nitric oxide synthase, vasoactive intestinal polypeptide, galanin and pituitary adenylate activating polypeptide in pigeon gut neurons.

The distribution of nicotinamide adenine dinucleotide phosphate (NADPH)-d neurons and their relationship with nitric oxide synthase (NOS), vasoactive intestinal polypeptide (VIP), pituitary adenylate activating polypeptide (PACAP) and galanin (Gal) were examined in the gastrointestinal (GI) tract of the pigeon Columbia livia. NADPH-d-histochemistry, indirect immunofluorescence and confocal analysis were applied to cryosections. Western blot analysis was also applied on pigeon gut. NADPH-d neurons were found throughout the pigeon GI tract and they were evident in the myenteric, circular muscle and submucous plexuses. Positive varicose nerve fibres were also distributed within the longitudinal muscle layers and in the lamina propria of the mucosa. The stomach was the segment richest in positivities. The copresence VIP/Gal/NOS as well as PACAP/VIP were revealed in some NADPH-d-neurons. We suppose that the nitrergic nerve population of the pigeon GI tract belong to the muscle motility regulation as an inhibitory descending nerve pathway. Moreover the presence of VIP, Gal and PACAP in some NADPH-d-containing neurons enhances the inhibitory actions of these neurotransmitters whereas PACAP and Gal role is actually unknown.

Galanin in the lizard oviduct: its distribution and relationships with estrogen, VIP and oviposition.

The distribution of neurons containing galanin immunoreactivity (Gal/IR) has been detected in the oviduct of the lizard Podarcis s. sicula during the main phases of its sexual cycle and after 17beta-estradiol treatment. Indirect immunofluorescence technique was applied both to cryostatic sections and whole mount preparations, and Western blot analysis, with an antibody directed against mammalian galanin (Gal), was performed with lizard oviduct extracts. Colocalization of Gal with vasoactive intestinal polypeptide (VIP) was also studied as well as Gal effects on egg deposition. In the quiescent oviduct of non-reproductive females, scanty Gal/IR fibres were found in the uterine-vaginal segment. During the reproductive period a gradual increase of positive nerve fibres and cell bodies were found distally in the lizard oviduct and the vagina revealed a reactive nerve population denser than elsewhere. Gal-IR nerve structures were present either in the musculature or mucosa and in the intermuscular layer they were organized in a nerve network. In the oviduct of non-reproductive females, 17beta-estradiol administration induced a significant increase of neurons containing Gal/IR. This hormone could be involved in the egg laying by means of galanin action and this hypothesis is supported by the induction of premature oviposition in pre-ovulatory females after Gal administration. Western blot analysis validates this peptide as true Gal, recognising one protein band with a molecular weight (3.2 kDa), similar to that of porcine Gal. Double labelling studies showed the co-presence of Gal and VIP in some neurons.

NADPH-diaphorase and NOS enzymatic activities in some neurons of reptilian gut and their relationships with two neuropeptides.

The distribution of neurons containing the enzymes NADPH-diaphorase (NADPH-d) and nitric oxide synthase (NOS) has been studied in the gastrointestinal tract of lizard (Podarcis s. sicula) and snake (Thamnophis sirtalis). The techniques employed were the NADPH-d/nitroblue tetrazolium histochemical method, and the indirect immunofluorescence applied to cryostat sections and to whole-mount preparations. The colocalization of NADPH-d with NOS, with vasoactive intestinal polypeptide (VIP) and with galanin (Gal) was also studied, and a Western blot analysis using an antibody directed against mammalian Gal was performed on lizard stomach extracts. NADPH-d positive nerve cell bodies and fibres were found in the myenteric and submucous plexuses throughout the gastrointestinal tract of both reptiles. These nerve structures were also present in the other intramural nerve plexuses, although in smaller quantities. Both in lizard and snake, the stomach revealed a positive nerve population that was more dense than elsewhere in the gut. The population of the NADPH-d-positive neurons observed in the lizard was larger than that observed in the snake. The distribution of both populations was similar to those that have been described in the gut of several mammalian and non-mammalian vertebrates. Both in lizard and snake, a one-to-one correspondence was noted between NOS- and NADPH-d-containing nerve cell bodies, and the nitrergic neurons containing Gal appeared to be more numerous than those containing VIP. Western blot analysis recognised a single band with a molecular weight (3.4 kDa) very similar to that of porcine Gal. It is hypothesised that at least some of the nitrergic neurons of the lizard and snake gut are inhibitory motor neurons innervating the circular smooth musculature. In addition, the colocalization of NOS and VIP in neurons enhances their inhibitory action. The role of the neurons containing both NOS and Gal remains unknown.

NADPH-diaphorase-, nitric oxide synthase- and VIP-containing nerve structures in the hen oviduct: a histochemical and immunohistochemical study.

The enzyme NADPH-diaphorase (NADPH-d; a marker of NO producing or nitrergic neurons) and the neuropeptide VIP have been detected in the nerve structures of the hen oviduct by histochemical and immunohistochemical techniques performed on cryostatic sections and whole mount preparations. In the upper four segments of the oviduct, i.e. the infundibulum, magnum, isthmus and shell gland, nitrergic and VIP-positive nerve structures were particularly numerous in the intermuscular and mucosal layers, and were represented by fibres and cell bodies showing mainly a perivascular distribution. Functionally, such perivascular structures were related to the blood flow regulation. Different types of nitrergic pyrenophora were recognized in the walls of the shell gland on the basis of their peculiar morphology. In the distal zone of the oviduct, the vagina, nitrergic and VIP-positive nerve fibres were widely diffused in the circular muscle, which was particularly thick in this segment. The source of at least part of such fibres was probably represented by large nerve cell bodies scattered in the layer and containing NO and VIP colocalized. Functionally these cells retained inhibitory motor neurons causing relaxation of the vaginal smooth muscle.

Neuroendocrine cells in the female urogenital tract of the pig, and their immunohistochemical characterization.

A systematic search for neuroendocrine (NE) cells in the urogenital organs of the pig was carried out by means of Linder's argyrophil method and immunohistochemical techniques. The occurrence, distribution and immunohistochemical character of NE cells (paraneurons) were studied in the vaginal vestibulum, vagina, uterus, oviduct, ovary, urethra, urinary bladder and ureter. In the vestibular glands paraneurons were found to be the most numerous, while a moderate number of these cells occurred in the uterine horn and in the urethra. A distinctly smaller number of paraneurons was present in the oviduct and only occasional NE cells were observed in the urinary bladder. Immunohistochemistry was performed by using the peroxidase-antiperoxidase procedure. Different subpopulations of paraneurons were distinguishable. Chromogranin A-positive paraneurons were found in the vestibular glands, uterine horns, oviducts, urethra and urinary bladder. Somatostatin positivity was observed in NE cells of the vestibular gland, uterine horn, oviduct and urethra. The subpopulation of serotonin-positive paraneurons was present in the vestibular gland and urethra. Bombesin, vasoactive intestinal polypeptide, cholecystokinin, substance P, nitric oxide synthase, beta-endorphin, insulin, adrenocorticotropic hormone, oxytocin and thyroid-stimulating hormone antibodies gave negative reactions in the studied NE cells.

CCK-containing paraneurons in human adenomatous prostate.

The presence of CCK-containing neuroendocrine cells in human adenomatous prostates, and the colocalization of CCK together with serotonin in the same cell, have been demonstrated by means of an immunohistochemical technique and by a double labeling immunofluorescent staining. CCK-containing neuroendocrine cells had a focal distribution in the prostates and sometimes showed dendrite-like cytoplasmic processes. The major part of CCK (96.55%) colocalized with serotonin. CCK probably stimulates muscle contraction and endocrine/exocrine secretions in the urogenital tract.

VIP-immunoreactive nerve structures of the gastrointestinal tract in the developing and adult domestic duck.

The first appearance and distribution of VIP-containing nerve structures were studied in the gut of embryonal, newly hatched and adult domestic ducks. Immunoreactive structures appeared in the mesenchyme of the gizzard-duodenum junction (antrum) at 5 days of incubation, and soon after diffused both cranially and caudally. Early in development, VIP-positive cell bodies were more numerous than positive fibres, appearing 3 days before the latter. Later on, the positive cell bodies decreased in number while the fibres increased. In the foregut, VIP-positive structures firstly appeared in the myenteric plexus while in the mid- and hindgut they appeared simultaneously in the myenteric and submucous-plexuses. The gastrointestinal VIP-innervation in newly hatched ducks differed from that of the adult because of the incomplete development of its mucosal component. The findings obtained indicated following: The time interval from 13 to 17 days of incubation seems critical for the growth of the gastrointestinal VIP-containing neurons. The molecular factors regulating the appearance of neuronal phenotypes are probably synthesized early in the avian gizzard development. VIP is widely diffused in the rectum where it may play important roles in regulating the onset and synchronism of peristaltic and anti-peristaltic movements.

Immunocytochemistry of paraneurons in the female urethra of the horse, cattle, sheep, and pig.

The aim of this study is to describe the presence of neuroendocrine (NE) cells (paraneurons), producing biogenic amines and/or peptidergic hormones, in the female urethra of cattle, sheep, pigs, and horses, by means of histochemical and double labeling immunofluorescent techniques. 5-Hydroxy-tryptamine-, chromogranin A-, cholecystokinin- and somatostatin-containing NE cells are present in the urethral epithelium of all the species studied, with the unique exception of the lack of somatostatin cells in the horse. Paraneurons containing 5-hydroxytryptamine colocalized with chromogranin A or cholecystokinin were also found in all subjects. Such active substances are hypothesized to play a role in the contraction of the urethral musculature, emission of urogenital fluids, and inhibition of endocrine and exocrine secretions.

Serotonin-, somatostatin- and chromogranin A-containing cells of the urethro-prostatic complex in the sheep. An immunocytochemical and immunofluorescent study.

The urethral and prostatic epithelial of the sheep contain a large number of amine- and/or peptide-producing neuroendocrine cells (NE), also called paraneurons. Four different cell types have been immunohistochemically recognised among them. The first contains the amine serotonin, the second the protein chromogranin A, the third the amine and the protein together and the fourth the hormone somatostatin. Serotonin-producing cells are elongated in shape and often show cytoplasmic dendrite-like processes directed towards the basal membrane and/or the lumen. Chromogranin A-containing cells are polymorphic and constitute the more numerous NE subpopulation. Cells containing both the bioactive substances seem to be less numerous than the chromogranin A cells and slightly more frequent than the serotonin cells. All these cell types are diffused along the whole urethro-prostatic complex and show their highest density in the collicular zone. Somatostatin-containing cells often show a unique cytoplasmic extension directed towards the basal membrane and are rare. It is supposed that the presence of serotonin in the urogenital tract is functionally correlated with the emission of urine and/or semen, while somatostatin is associated with the inhibition of local exocrine and/or endocrine secretions.

Histopathological heterogeneity and cytopathological similarity of findings in different muscles of two brothers affected by rigid spine syndrome.

The pathological changes in muscles biopsied from 2 brothers with rigid spine syndrome are reported. The findings ranged from marked fascicular atrophy and fibrosis to hypotrophy of small groups of fibres and vacuolation in most fibres. The presence of vacuoles and deposits of accumulated material seemed to be common to all the biopsies. These findings, compared with those reported in the literature, confirmed the histopathological heterogeneity of this syndrome but proposed also the hypothesis that similar elementary lesions of muscle fibres can account for the initiation of the pathological process, developing asynchronously in different muscles because of their different activity.

Chromogranin- and somatostatin-containing neuroendocrine cells in the porcine uterus. An immunocytochemical study.

Endocrine cells scattered in organic mucosae were defined "Neuroendocrine" (NE) cells because they constitute a section of the Diffuse Neuroendocrine System (DNES). Such cells have never been evidentiated in the normal endometrium. By means of histochemical and immunohistochemical techniques, NE cells, some of which contain the hormone somatostatin, are described in the glandular epithelium of the uterine horn in non-pregnant, non-castrated, young and adult sows. As is known, the uterine horn is the organ of pregnancy in the pig. The localization, distribution and morphology of the uterine NE and somatostatin-containing cells are reported and the importance of their function, in the pregnant and non-pregnant porcine uterus, is discussed.

Urogenital paraneurons in several mammals.

Amines and/or peptide-producing cells, deserving to be called paraneurons, were demonstrated in the urethro-prostatic complex of the man, rabbit, buffalo and sheep and in the uterine horns of the pig, horse and mouse, by means of histochemical, immunohistochemical, immunofluorescent and double labeling immunofluorescent techniques. In particular, the urethro-prostatic complex of the sheep contains cells producing serotonin, chromogranin A and somatostatin. Often the amine and the "marker"-protein were colocalized in the same cells. Chromogranin A- and somatostatin-containing cells were found in the uterine horns of the pig. Serotonin probably plays a role in the contraction/relaxation of the musculature of the male urogenital tract, thus regulating the urinary and spermatic flow. The function of somatostatin cells in the urogenital tract is unknown. The presence and direction of their cytoplasmic processes lead us to hypothesize a double endocrine/paracrine modality of action.

Staining of neuroendocrine cells by Linder's argyrophil method.

Linder's argyrophil method, recently developed to stain nervous structures, is useful in the histochemical study of amine- and/or peptide-producing neuroendocrine (APUD) cells. On sections from various organs of four animal species Linder's method worked well and rapidly stained the neuroendocrine cells yellow, red or black; it stained black nervous structures against a pale yellow background. Double staining of single sections from Bouin-fixed gastric mucosa of rabbits demonstrated the correspondence of both Linder- and Grimelius-positive cells. Rapidity of application, intensity of impregnation and reproducibility in results are the best features of Linder's method when applied to the study of the neuroendocrine system.

[Ataxia-telangiectasia: an optic and electron morphocytologic study of a conjunctival biopsy]. / Ataxie-télangiectasie: étude morphocytologique optique et électronique d'une biopsie conjonctivale.

The authors present a case of ataxia-telangiectasia also known as Louis-Bar Syndrome. The rarity of the disease and the dearth of extensive literature about the subject submitting this particular case to optic and electronic study and to report the results. The description, that comes out from our histologic and submicroscope study of the conjunctiva, attests the change of the conduct, of the diameter and conjunctival flow of the microvessels. This, in turn touches upon the cytological question behind the cause of changes in the relationships of vessels and surrounding tissues.

The effect of diet on the ophthalmological, clinical and biochemical aspects of Richner-Hanhart syndrome: a morphological ultrastructural study of the cornea and the conjunctiva.

Type II tyrosinemia (Richner-Hanhart syndrome) is a familial aminoacid disorder, clinically characterized by ocular changes (keratitis), palmo-plantar hyperkeratosis, no constant mental changes with mental deterioration, abnormal urinary excretion and high serum tyrosine level in consequence of the absence of tyrosine-aminotransferase. Almost 20 families have been described in the literature of which 50% are of Italian origin, suggesting that this disorder is particularly frequent in our country. We report a family with 2 affected members with typical clinical and biochemical findings (keratitis, palmo-plantar hyperkeratosis, abnormal urinary and serum tyrosine concentrations), not suffering from mental retardation. Clinical symptoms completely disappeared after the decrease of urinary and serum tyrosine levels following a tyrosine- and phenylalanine-free diet. These cases are compared with those reported in literature, and the usefulness of diet for the improvement of clinical and metabolic symptoms is discussed.

Innervation pattern of intrinsic laryngeal musculature of the rabbit.

The intrinsic laryngeal musculature of rabbit was studied using various neurohistological techniques. The vegetative and sensory nerve terminals are present in abundance, while the proprioceptors are seemingly absent. Furthermore, simple motor endplates, sometimes furnished with ultraexpansional fibers, complex endplates, or 'en grappe' are described. Their abundance within a musculature lacking in muscle-spindles is indicative of a possible vicarious function.

Hepatolenticular degeneration: histological study of conjunctiva and observation of chalazii during D-penicillamine treatment.

Three patients with hepatolenticular degeneration (Wilson's disease) are described. Bilateral blepharoconjunctivitis and Kayser-Fleischer ring was observed in all 3 patients. In 2 patients, long-lasting, bilateral chalazii appeared during chronic treatment with D-penicillamine. Histochemical and ultrastructural studies of pre-treatment conjunctival biopsies showed no copper storage in the tissue thus supporting the hypothesis that copper storage in Descemet's membrane derives from the anterior chamber.