NADPH-d activity in rat thymus after the application of retinoid acid.

The aim of this work was to determine the localization of nicotinamide-adenine dinucleotide phosphate-diaphorase (NADPH-d) activity as the marker for synthesis of nitric oxide synthase (NOS) in the rat thymus after the application of retinoid acid (RA) on 1st, 7th, 14th and 21st days of gestation. The given results can build the basis for understanding of the role of NOS in rat thymus. NADPH-d positive cells were represented with dark-blue color and were localized on corticomedullar junction of the thymus. These cells were of different intensity of coloring and were shaped in oval, circle or irregular forms. NADPH-d positive nerve fibers were observed in perivascular topography. They were marked more strongly in the case of control group. The result of application of RA to gravid rats was that the birth weights of newborn rats and their thymuses were smaller, but without statistically significance.

Effect of retinoic acid on the nitrergic innervation of meibomian glands in rats.

The purpose of this study was to investigate the effect of prenatal administration of retinoic acid (RA) on the development of nicotinamide adenine dinucleotide diaphorase (NADPH-d) positive structures in the rat Meibomian glands. One mg/kg of RA was applied to pregnant Wistar rats intraperitonaelly during the gestational period in each of the 12th-14th embryonic days (totally 3 mg/kg). Sections of the central upper eyelids were investigated in rat pups on the 14th postnatal day. They were processed histochemically for NADPH-d, to study the presence and distribution of nitric oxide synthase (NOS) positive nerve structures. NADPH-d staining of Meibomian glands was compared in two groups of rat pups. In the control group, eyelids of 14 day-old rats were studied with no experimental intervention. The second group consisted of rat pups which were prenatally administered the excess of RA. Histochemical analysis of control eyelids revealed numerous NADPH-d well-stained acini of Meibomian glands arranged tightly into groups. Intensively stained vessels and NADPH-d/NOS-positive nerve fibers bordered acini of Meibomian glands. These structures were present in the submucosal layer as well. The analysis of RA group showed less numerous, shrunken acini of Meibomian glands that were seen not only smaller in size, but also in density of their staining and the amount of nitrergic nerve fibers around acini were considerably lowered. In the submucosa differences were noticed compared to the control group, there were numerous NADPH-d stained vessels accompanied by NADPH-d/NOS-positive nerve fibers. The excess of RA during the prenatal period may influence on the development and morphology of NADPH-d positive structures of rat's Meibomian glands.

Influence of surgical and chemical orchidectomy on weight and distribution of AChE-nerve fibres in thymuses of adult rats.

The thymus is a crossroad between the immune and neuroendocrine systems. As such, it is innervated by acetylcholinesterase (AChE)-positive fibres of the vagus, the recurrent laryngeal and the phrenic nerves. It is well know, that the innervations density of the thymus increases with age. In our study, adult rats were orchidectomized (surgically and chemically by the application of a luteinizing hormone-releasing hormone). The density of AChE-positive nerve fibres in thymuses, as well as the weight of thymuses was examined. The authors found that both surgical and chemical orchidectomy result in macroscopic and microscopic regeneration of the atrophied thymuses. In regenerated rat's thymuses after orchidectomy the density of AChE-positive nerve fibres was markedly higher in comparison with the control animals. The distribution, as well as the density of AChE-positive nerve fibres in regenerated thymuses after orchidectomy evokes the images of its innervations like in young animals before age-related involution. The authors also found a markedly higher weight of thymuses of orchidectomized rats in comparison with the control groups. In recent study the authors proved that after 8 weeks surgical orchidectomy leads to the regeneration of thymic AChE-positive innervation and chemical orchidectomy by administration of luteinizing hormone-releasing hormone after 4 weeks of adult rats.

NADPH-diaphorase expression in the rat jejunum after intestinal ischemia/reperfusion.

The purpose of this study was to analyze the nicotinamide adenine dinucleotide phosphate - diaphorase (NADPH-d) activity in the rat jejunum after a mesenteric ischemia/reperfusion injury. Nitric oxide, synthetised from L-arginine by the enzyme nitric oxide synthase, is a nonadrenergic noncholinergic relaxant neurotransmitter of the intestinal smooth muscle. It plays an important role in the process of plasticity after the ischemia/reperfusion injury. Experimental animals were divided in two groups: the control group and the ischemic/reperfusion group, with different period of the reperfusion. The NADPH-d histochemical method has been used as a marker for the nitric oxide synthase. NADPH-d activity has been rapidly decreased in the neurons of both enteric nervous systems in plexuses of the jejunum after 1 h mesenteric ischemia and 1 h reperfusion. Differences were predominantly detected in the myenteric plexus; they were seen in change of the neuronal shape, in the arrangement of neurons and in intensity of their staining. The NADPH-d positivity was absent in the intestinal crypts. After 1 h ischemia and 24 h reperfusion, the NADPH-d activity was gradually increased, but it was lower in comparison with the control group. On the 30th day following the ischemia/reperfusion there were no changes in NADPH-d positivity compared with the control animals. These results indicated that the jejunal ischemia/reperfusion has affected the neurons of the enteric nervous system of adult rats and resulted in the early decrease of NADPH-d positivity 1 h of the reperfusion insult. The gradual increasing of NADPH-d activity in 24 h following the reperfusion could be considered as a result of the plasticity process. On the 30(th) day after the ischemia/reperfusion all histochemical changes were returned to the control levels.

Immunohistochemical evaluation of Pi class glutathione S-transferase expression in invasive breast carcinoma.

OBJECTIVES: The aim of our work was to determine the expression of Pi class glutathione S-transferase (GSTP1) in 43 samples of invasive breast carcinoma and compare results versus normal breast cells. BACKGROUND: Breast cancer is the commonest cancer in women. Despite advances in early detection and more efficacious adjuvant chemotherapy, a part of patients with early-stage have reccurent disease. In these cases the development of resistance to therapy is observed. The glutathione S-transferases (GSTs) are potentially involved in tumour chemoresistance. METHODS: Enzyme immunohistochemical method was chosen for the detection of GSTP1 and its expression was compared in breast cancer cells versus normal breast cells. RESULTS: We have found that majority (63%) of breast carcinomas shows GSTP1 positivity (nuclear and cytoplasmic immunoreactivity). It is expected that GSTP1 positive tumours would show a poorer prognosis than GSTP1 negative ones. CONCLUSION: Immunohistochemistry is a useful method for investigating the expression and cellular localization of GSTP1 within tumours. It may be applied to a routine clinical test and it can serve as a marker for resistance to chemotherapy (Tab. 1, Fig. 4, Ref. 20). Full Text in free PDF www.bmj.sk.

Distribution of NADPH-diaphorase and AChE activity in the anterior leaflet of rat mitral valve.

The mitral valve, as an active flap, forms the major part of the left ventricular inflow tract and therefore plays an important function in many aspects of left ventricular performance. The anterior leaflet of this valve is the largest and most ventrally placed of two leaflets that come together during ventricular systole to close the left atrioventricular orifice. Various neurotransmitters are responsible for different functions including controlling valve movement, inhibiting or causing the failure of impulse conduction in the valve and the sensation of pain. Nitric oxide acts as a gaseous free radical neurotransmitter, neuromediator and effective cardiovascular modulator. Acetyl-choline is known to function as a typical neurotransmitter. Histochemical methods for detection of nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d), as an indirect nitric oxide-synthase marker, and method for detection of acetylcholinesterase (AChE) were used. Both methods were performed on the same valve sample. A widespread distribution of nerve fibres was observed in the anterior leaflet of the mitral valve. The fine NADPH-d positive (nitrergic) nerve fibres were identified in all zones of valve leaflet. AChE positive (cholinergic) nerve fibres were identified forming dense network and fibres organized in stripes. Endocardial cells and vessels manifested heavy NADPH-d activity. Our observations suggest a different arrangement of nitrergic and cholinergic nerve fibres in the anterior leaflet of the mitral valve. The presence of nitrergic and cholinergic activity confirms the involvement of both neurotransmitters in nerve plexuses and other structures of mitral valve.

NADPH-diaphorase expression in the meibomian glands of rat palpebra in postnatal development.

In the current study, we aimed at investigating the presence of nitric oxide synthase (NOS) positive nerve fibers in rat meibomian glands (MGs) at various stages of development. There is good evidence to suggest that nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) is a surrogate for neuronal nitric oxide synthase (NOS). Sections of the central, upper eyelids of Wistar rats were processed histochemically for NADPH-d to investigate the presence and distribution of NOS-positive nerve fibers at the following time points: day 1 and weeks 1, 2 and 3 post partum, and in adult controls. At day 1, MG acini were lightly stained and located at a distance from the mucosal border. Vessels were accompanied by intensely stained NADPH-d positive nerve fibers. At the week 1 time point, both the vessels and the NADPH-d positive fibers were still present, but less numerous. MGs were now closer to the mucosa, so that the submucosa was thinner. The acini were mostly pale but occasionally darker. At week 3, there were fewer blood vessels in both the submucosa and within the septa. Darker acini were more common than lightly stained acini. NADPH-d positive dots were observed in the vicinity of the MGs. At the week 3 time point, MGs were adjacent to the mucosal border and stained more intensely than at earlier times; almost all acini were stained. The microscopic appearances were almost identical with those of adult palpebra. Submucosal and septal blood vessels and NADPH-d positive nerve fibers were less numerous. NADPH-d histochemical staining confirmed differences in the density of stained nerve fibers at different developmental stages. The greatest density of NADPH-d -positive nerve fibers occurred in 1-day-old rats whereas they were less numerous in adult rat eyelids. Nerves innervating MGs utilize nitric oxide (NO) as a neurotransmitter mostly in early developmental stages and this need thereafter decreases and stabilizes at 3 weeks postnatally.

Multidrug resistance proteins in renal cell carcinoma.

A large number of renal cancer patients show poor or partial response to chemotherapy and the precise mechanism has not been understood yet. MDR is the principal mechanism by which many cancers develop resistance to chemotherapeutic drugs and is associated with the elevated expression of MDR proteins. These are divided into two groups: ABC transporters and non-ABC transporters. The aim of our study was to determine the expression of MDR1/Pgp, MRP1 and LRP in 47 samples of renal cell carcinomas using immunohistochemical assay. Our results were analysed in relation to nuclear grade and other clinical and pathological parameters to see the possible correlation between the expression of MDR proteins and factors mentioned above. The majority of renal carcinoma specimens showed positivity for MDR proteins. In this regard, 21 % of samples revealed positive results for MDR1, 62 % for MRP1 and 76.6 % for LRP protein. Furthermore, our study displayed significant differences between MDR1, LRP and nuclear grade. On the other hand, no association was found between MRP1 and nuclear grade, as well as between the expression of three MDR proteins and other clinically relevant parameters.

Immunohistochemical detection of MDR proteins in Wilms' tumour.

OBJECTIVES: The aim of our work was to determine the expression of three MDR proteins (MDR1/Pgp, MRP1 and LRP/MVP) in 15 tissue samples of nephroblastoma (Wilms' tumour). BACKGROUND: The majority of Wilms' tumours respond well to chemotherapy and are successfully cured, but a small subset displays resistance to therapy. The molecular mechanisms of drug resistance in this tumour type of childhood are still poorly analyzed. In our opinion, the elucidation of reasons for therapy failure in nephroblastomas is urgently needed before cure becomes a reality for children with this cancer. METHODS: To demonstrate these proteins the enzyme indirect immunohistochemical method was used. The brown colour of the diaminobenzidine reaction product allowed us to define the distribution of stain clearly. CONCLUSION: Our immunohistochemical analysis did not demonstrate any expression of MDR1 in all cases of nephroblastoma (14 cases were after pre-operative chemotherapy, 1 case wasn't). The analysis of MRP1 and LRP expression in our set revealed 60% positivity for MRP1 and 26.7% positivity for LRP. The ability to recognize the multidrug resistance phenotype might assist in choosing specific chemotherapeutic regimens to improve prognosis and therapy (Tab. 2, Fig. 2, Ref. 20). Full Text (Free, PDF) www.bmj.sk.

Neuronal nitric oxide synthase in the rabbit spinal cord visualised by histochemical NADPH-diaphorase and immunohistochemical NOS methods.

The NADPH-diaphorase (NADPH-d) staining method is widely used in the investigation of both the central and peripheral nervous systems. Neuronal nitric oxide synthase (nNOS) has previously been shown to be responsible for the NADPH-d activity in neurons. However, NADPH-d activity does not always fully represent the enzyme nNOS. We investigated the distribution of NADPH-d activity and nNOS protein in the rabbit spinal cord for all groups of neurons and Rexed's laminae. In most laminae the distribution of NADPH-d activity was identical to nNOS immunoreactivity. Both were present in the dorsal horn and in pericentral areas of the spinal cord, but some differences existed. The superficial part of the dorsal horn (laminae I-III) stained more intensely for NADPH-d than for nNOS. However, the most prominent difference was seen in the lateral part of the dorsal horn--the lateral collateral pathway (LCP). The LCP stained strongly for NADPH-d activity, while nNOS staining was absent. Although there is an excellent correlation between NADPH-d staining and nNOS immunohistochemical staining in the spinal cord in general, the presence of staining differences necessitates the use of immunohistochemistry for some specialized applications.

Comparative study of the topographic localization of NADPH diaphorase positive cells in the rabbit and pheasant thymuses.

Nicotinamide adenine dinucleotide hydrogen phosphatediaphorase (NADPHd) histochemistry was used as a marker for nitric oxide synthase (NOS). In the rabbit thymus, NADPHd staining was observed between the capsule and corticomedullary junction in radially oriented blood vessels in the cortex. The outer surface of the thymic lobule and interlobular septa showed adipocytes clumped together. There was a high density of NADPHd positive cells in the medulla, without a sharp boundary in corticomedullary space. In addition to radially oriented blood vessels in the cortex, they were also found as solitary profiles with well stained walls in the medulla. Neuronal plexuses were localized in perivascular topography. In the pheasant thymus, NADPHd positive cells were present as clusters which were distributed in the medulla and the corticomedullar area. NADPHd positive nerve fibres were localized in perivascular topography.

Alterations of the vessel wall innervation during diabetes mellitus.

Coronary and valvular heart disease during diabetes mellitus (DM) are major contributors of morbidity and mortality in the diabetic population. Relatively little atention has been given to the study of heart valve nerve structures in different pathological processes. In this study we have demonstrated the presence of possible morphological alterations in vessels of the anterior cusp of the rat mitral valve during 8-12 weeks DM. A histochemical method was used for the detection of NADPH-diaphorase (NADPH-d), which is the indirect NO-synthase marker. Arterioles and fine capillaries were localized in the attachment zone of the anterior cusp. Perivascular nerve fibres were identified running in the tunica adventitia. A marked dilatation of the vessels was seen in diabetes in comparison with control samples. No NADPH-d positive nerve fibres were observed in the tunica adventitia. It can be presumed that metabolic changes in the vessel walls during DM reflect modified neurotransmission of NO by means of their excessive overproduction of NOS (endothelial--eNOS) in endothelial cells. (Fig. 6, Ref. 32.).

Immunohistochemical detection of LRP protein in the normal human lung.

In this study, we have determined the LRP (lung resistance-related protein) by immunohistochemical method. LRP belongs to proteins which cause the multidrug resistance (MDR). It has been found in various normal human tissues, where it plays a protective role against toxic compounds. Multidrug-resistant cells distribute the cytotoxic drug into the perinuclear region and then redistribute it back into the cytoplasm. It is just a hypothesis today that LRP can mediate drug resistance by regulating both the cytoplasmic redistribution and the nucleocytoplasmic transport of drugs. In order to detect LRP we have used the paraffin-embedded sections of the normal human lung tissue. LRP was predominantly located in two regions: 1) in bronchial epithelial cells and 2) in alveolar macrophages. Positive cells were coloured brown and showed strong reactivity. Negative control included the omitting of primary antibody and replacing it by buffer solution. Bronchial epithelial cells and alveolar macrophages stayed uncoloured, i.e. unreactive. (Fig. 5, Ref. 17.).

Comparative analysis of NADPH-diaphorase positive neurons in the rat, rabbit and pheasant thoracic spinal cord. A histochemical study.

The distribution of NADPH-diaphorase (NADPH-d) activity was investigated and compared in the rat, rabbit and pheasant thoracic spinal cord. The investigation of all spinal cord regions (laminae) in three experimental species revealed marked differences in the distribution of NADPH-d activity. Cross sectional analysis of the spinal cord of the rat, rabbit and pheasant confirmed differences in the shape of the gray matter in all examined species. More detailed investigation of Rexed's laminas showed similar distribution of NADPH-d activity in the spinal cord of the rat and rabbit, which were different when compared with the spinal cord of the pheasant. Ventral horn of the rat and rabbit showed no labelling whereas in pheasant this area possessed a number of scattered, intensively stained neurons. In the location of autonomic preganglionic neurons, differences were found as well. In the rat there was seen a number of densely packed, clearly dark blue coloured neurons. Similarly, these neurons were present in the rabbit spinal cord but they were less numerous. No staining was found in this region of pheasant. Pericentral area (lamina X) and intermediate zone (laminaVII) revealed the presence of NADPH-d positive neurons in all examined species although they differed in number and shape of their bodies. The dorsal horn showed the presence of NADPH-d staining in all three animals but its distribution was different in medio-lateral direction. It can be suggested that observed differencies in the presence and distribution of NADPH-d activity across the examined species may reflect different fylogenetic development.

[Detection of peptidergic and nitrergic structures in the spinal ganglia of rabbits]. / Dôkaz prítomnosti peptidergických a nitrergických struktúr v spinálnych gangliách králika.

In this study we have demonstrated the presence of neuropeptide substance P and non-peptide neurotransmitter NO (nitric oxide) in the dorsal root ganglia of rabbit. NADPH-diaphorase histochemical staining was used for the detection of NO and immunohistochemical method for the detection of substance P.A particular number of dorsal root ganglion (DRG) cells were stained by SP and NADPH-d reaction. The presence of SP and NADPH-diaphorase positive cells varied depending upon spinal level of DRGs. Positively stained neurons were only small or medium-sized. Cells of large diameter profiles showed no staining. Substance P immunoreactive cells were stained brown and dark brown, the intensity of NADPH-d staining varied from light to very dark blue. In some DRGs cells, there was a very significant neuronal co-localization of immunoreactivity for SP and reactivity for NADPH-d. In summary, DRG cells appear to express diaphorase and substance P activity, and some of them contain both neurotransmitters. Recent studies analysing the participation of NO in the regulation of SP release in the spinal cord suggest, that the DRGs neurons may display a close interaction between NO and SP. (Fig. 14, Ref. 39.)

[Coexistence of cholinergic and nitrergic neurotransmitters in the spinal cord of rabbits]. / Koexistencia cholínergického a nitrergického neurotransmitera v mieche králika.

Nitric oxide (NO) plays a major role as a neuronal messenger molecule. NO has been assumed to act as a retrograde signalling molecule that modulates transmitter release. Acetylcholine (ACh) is known to function as a typical neurotransmitter. In the present work the presence of both transmitters (NO and ACh) and their possible relations in the rabbit spinal cord were examined. In our experiments histochemical methods for the visualisation of acetylcholinesterase (AChE) and NADPH diaphorase (NADPH-d) were used. Both histochemical methods were performed separately and together on the same sections of the thoracic spinal cord. NADPH-d positive dark blue stained neurons were mainly detected in superficial and deep layers of dorsal horn, preganglionic autonomic neurons and pericentral area (1). The presence of AChE positive amber yellow neurons was confirmed mostly in motoneurons located in ventral horns and then in neurons of the intermediate zone. Except for the above mentioned also double-labeled neurons containing both yellow and dark blue histochemical product were noticed. Their presence was confirmed in the intermediate zone and in the pericentral area. Thus, the coexistence of NADPH-d and AChE was confirmed in the area of interneurons. These observations suggest that NO may play a role in the control of cholinergic neuronal activity and that NO can be involved in the modulation of synaptic transmission. (Fig. 9, Ref. 21.)

[New pedagogic methods in anatomy: experience at Cambridge University]. / Nové pedagogické prístupy v anatómii: skúsenosti z Univerzity v Cambridge.

The expansion of knowledge in basic medical sciences is not linked to the time assigned for the teaching of anatomy to medical undergraduates. The question of "basic knowledge" in teaching anatomy during medical training arises as a need for education of future clinical doctors. Nowadays, two extreme views in teaching anatomy can be recognized: one adopted some pure anatomists who feel their existence threatened even by the idea of any reduction in their field, and one by some morphologists exclusively interested in cellular biology, who consider that classical anatomy is of no interest, since it has been exhausted as a field for research. An intermediate position is taken by some clinicians, who maintain that anatomy is indispensable but seek a severe reduction in the content to what they consider to be necessary. The above mentioned need for clinicians was reflected in recommendations of Education Committee of the General Medical Council (GMC) which in short, could be characterized by: the substantial reduction of factual information, the increase of student learning and the emphasis of clinically applied anatomy with its integration to the general medical education. GMC delegated the Department of Anatomy at the University of Cambridge by the developing of the new anatomy course. This new course was for the first time introduced in school year 1998-1999. In this study are presented ways and methods of undergraduate anatomy teaching at the University of Cambridge. These educational principles could serve as a model for teaching anatomy during its transformation in other medical faculties.

Partial colocalization of NADPH-diaphorase and acetylcholinesterase positivity in spinal cord neurons.

The freely diffusible radical, nitric oxide (NO), has been assumed to act as a retrograde signaling molecule that modulates transmitter release. Acetylcholine (ACh) is known to function as a typical neurotransmitter. In the present work we have examined the presence of both transmitters (NO and ACh) and their possible relations in the rabbit spinal cord. In our experiments we have used histochemical methods for the visualization of acetylcholinesterase (AChE) and NADPH diaphorase (NADPH-d) which label neurons that express nitric oxide synthase (NOS). Both histochemical methods were performed separately or together on the same sections of the thoracic spinal cord. NADPH-d positive dark blue stained neurons were seen mostly in superficial and deep layers of the dorsal horn, preganglionic autonomic neurons and pericentral area. The presence of AChE positive amber yellow neurons was confirmed mostly in motoneurons located in the ventral horns and in neurons of the pericentral and intermediate zone. Besides the above mentioned neurons, also double-labeled neurons were found which contained both the yellow and dark blue histochemical product. Their presence was confirmed in the intermediate zone and in the pericentral area. Thus, the co-existence of NADPH-d and AChE occurred in the location of interneurons. Our observations suggest that NO may play a role in the control of cholinergic neuronal activity and that NO can be involved in the modulation of synaptic transmission.

[Laminar distribution of NADPH-diaphorase in the thoracic spinal cord in pheasants]. / Laminárna distribúcia NADPH-diaforázy v hrudníkovej mieche bazanta.

BACKGROUND: The distribution of NADPH-diaphorase (NADPH-d) activity was investigated in the spinal cord of pheasants. MATERIAL AND METHODS: Histochemical method for visualization of NADPH-d was used in this study. This method is considered to be a good marker for NO synthase. RESULTS: The investigation of NADPH-d activity in laminae of the thoracic spinal cord of pheasants revealed the presence of scattered intensively stained neurons in laminae VIII and IX of the ventral horn. In the location of autonomic preganglionic neurons, no presence of NADPH-d positivity was noticed. The pericentral area (lamina X) and intermediate zone (lamina VII) showed NADPH-d positive neurons located more dorsally with larger distance from the central canal. In superficial layers of the dorsal horn (lamina I and II) marked differences were seen in the distribution of NADPH-d activity through the medio-lateral direction. CONCLUSION: In summary, it can be suggested that the observed presence of NADPH-d activity may reflect the utilization of NO in the thoracic part of the spinal cord in pheasants. (Fig. 5, Ref. 20.)