Mekbeb Afework

Colocalization of nitric oxide synthase and NADPH-diaphorase in rat adrenal gland

The distribution and colocalization of nitric oxide synthase (NOS) and reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase was studied in the neuronal elements of the adrenal gland of the rat. Ganglion cells and many nerve fibres in the gland showed both NOS-immunoreactivity and NADPH-diaphorase staining. The adrenal cortical cells showed NADPH-diaphorase staining but were not immunoreactive for NOS. Positive labelling for both NADPH-diaphorase and NOS was found in bundles and in single fibres with varicosities, preferentially located around the noradrenaline (NA)-storing cells. Adrenaline (A)-storing cells and ganglion cells in the medulla, along with the cortical cells and blood vessels in the zona glomerulosa, received relatively fewer positive fibres.

Distribution and colocalization of nitric oxide synthase and NADPH-diaphorase in adrenal gland of developing, adult and aging Sprague-Dawley rats

The distribution and colocalization of nitric oxide synthase and nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-diaphorase) was investigated in the adrenal gland of developing, adult and aging rats with the use of immunohistochemical and histochemical techniques. Nitric oxide synthase-immunoreactive neurons within the adrenal gland were found from the 20th day of gestation onwards. During early development the neurons were found as small clusters of smaller-size cells compared to those observed in the adult gland. Their number reached that of adult level by the 4th day after birth, and in the glands from aging rats a 28.6% increase was observed. Whilst no immunofluorescence was seen in chromaffin cells during early development, some cells from glands of aging rats showed nitric oxide synthase-immunoreactivity with varying intensity. The immunoreactive neurons from postnatal rat adrenals were also positive for NADPH-diaphorase, whilst those in prenatal rats were negative or lightly stained. Nitric oxide synthase-immunoreactive nerve fibres were present in all adrenal glands examined from the 16th day of gestation onwards. A considerable degree of variation in the distribution of immunoreactive fibres both in medulla and outer region of cortex at the different age groups was observed and described. Most, but not all, nitric oxide synthase-immunoreactive nerve fibres also showed NADPH-diaphorase staining.

Distribution of P2X receptors in the rat adrenal gland

Abstract. The distribution of each of the seven subtypes of ATP-gated P2X receptors was investigated in the adrenal gland of rat utilizing immunohistochemical techniques with specific polyclonal antibodies to unique peptide sequences of P2X1–7 receptors. A small number of chromaffin cells showed positive immunoreaction for P2X5 and P2X7, with the relative occurrence of P2X7 -immunoreactive chromaffin cells exceeding that of P2X5 . The preganglionic nerve fibres that form terminal plexuses around some chromaffin cells showed P2X1 immunoreactivity. Intrinsic adrenal neurones were observed to be positively stained for P2X2 and P2X3 receptors. P2X2 immunoreactivity occurred in several neurones found singly or in groups in the medulla, while only a small number of neurones were immunoreactive for P2X3 . Adrenal cortical cells were positively immunostained for P2X4–7 . Immunoreactivity for P2X4 was confined to the cells of the zona reticularis, while P2X5–7 immunoreactivities occurred in cells of the zona fasciculata. The relative occurrence of immunoreactive cortical cells of the zona fasciculata was highest for P2X6 , followed by P2X7 and then P2X5 . The smooth muscle of some capsular and subcapsular blood vessels showed P2X2 immunoreactivity. The specific and widespread distribution of P2X receptor subtypes in the adrenal gland suggests a significant role for purine signalling in the physiology of the rat adrenal gland.

Calretinin immunoreactivity in adrenal glands of developing, adult and ageing Sprague-Dawley rats

Localization of calretinin immunoreactivity in the rat adrenal gland was studied using immunohistochemical methods. Calretinin‐immunoreactive adrenal chromaffin cells and nerve fibres were found at all of the ages examined from embryonic day 16 up to 2‐yr‐old. Immunoreactive chromaffin cells showed a decrease in number with increasing age, and in the adult and ageing rats they were found among the adrenaline‐storing chromaffin cells. Calretinin immunofluorescence was also observed in a few ganglion cells of the adult and ageing rats, and in most of the cortical cells at embryonic day 16. The density of immunoreactive nerve fibres in the gland showed a progressive increase with age to a peak by day 4 after birth and then a gradual decrease afterwards; they became associated mainly with some of the ganglion cells in the adult rat. As revealed by double labelling of calretinin immunoreactivity in adrenal sections from the adult rat with NADPH‐diaphorase histochemistry, no co‐localization existed between calretinin and NADPH‐diaphorase; however, a small number of neuronal cell bodies which contained nitric oxide synthase were found heavily surrounded with calretinin‐immunoreactive nerve fibres. In ageing rats, an increased density of calretinin‐immunoreactive nerve fibres was seen associated largely with the chromaffin cells.

The intra-adrenal distribution of intrinsic and extrinsic nitrergic nerve fibres in the rat

The intra-adrenal distribution of nitric oxide synthase (NOS)-immunoreactive nerve fibres was studied in rats subjected to various denervations. Splanchnic nerve section eliminated the NOS-immunoreactive nerve fibres which innervate adrenal chromaffin and neuronal cells. It did not affect those innervating blood vessels and zona glomerulosa, which instead were affected by adrenal demedullation. Guanethidine, 6-hydroxydopamine (6-OHDA) and capsaicin treatments, however, did not produce any change. These results suggest that nitrergic nerves which innervate adrenal medullary cells are extrinsic (largely preganglionic sympathetic), whilst those innervating the zona glomerulosa and the majority of adrenal vessels are intrinsic, and that they do not belong to nerves sensitive to the sympathetic nerve neurotoxins, guanethidine and 6-OHDA, or the sensory neurotoxin, capsaicin.

Colocalization of neuropeptides and NADPH-diaphorase in the intra-adrenal neuronal cell bodies and fibres of the rat

Colocalization of vasoactive intestinal peptide, neuropeptide Y, calcitonin gene-related peptide, substance P, and tyrosine hydroxylase, respectively, with NADPH-diaphorase staining in rat adrenal gland was investigated using the double labelling technique. All vasoactive intestinal peptide- and some neuropeptide Y-immunoreactive intrinsic neuronal cell bodies seen in the gland were double stained with NADPH-diaphorase. Double labelling also occurred in some nerve fibres immunoreactive to vasoactive intestinal peptide and neuropeptide Y in the medulla and cortex. No colocalization of calcitonin gene-related peptide, substance P or tyrosine hydroxylase immunoreactivity with NADPH-diaphorase staining was observed. However, nerve fibres with varicosities immunoreactive for all the neuropeptides examined were closely associated with some of the NADPH-diaphorase-stained neuronal cell bodies. Thus, in rat adrenal gland, nitric oxide is synthesized in all ganglion cells containing vasoactive intestinal peptide and in some containing neuropeptide Y, but not in those containing calcitonin gene-related peptide, substance P or tyrosine hydroxylase.

Age-related changes in the localization of P2X (nucleotide) receptors in the rat adrenal gland

Observation of the changes in the occurrence and distribution of nucleotide (P2X) receptors in the adrenal gland during development and ageing, and correlation with the changes in adrenal status at similar stages may give morphological insights into the functions of purine nucleotides in the gland. Age‐related changes in the localization of all seven subtypes of the P2X receptor in the adrenal gland of rat were therefore investigated immunohistochemically. In the adrenal glands of prenatal rats, immunoreactivity to P2X receptor subtypes was not observed. In glands of the postnatal rat at the developmental stages studied, only immunoreactivity for the P2X5 receptor subtype was observed. A small number of faintly P2X5‐immunoreactive chromaffin cells were found in the adrenal glands of 1‐day‐old rats; the frequency of localization and intensity of staining of immunoreactive cells had increased by day 4 and was further increased at day 7. P2X5 immunoreactivity was not observed in the adrenal glands from 14‐ and 21‐day‐old rats. At 8 weeks of age, immunoreactivity with a specific distribution for each of the seven receptor subtypes was observed. Except for the P2X4 receptor, adrenal glands at 24 months showed a similar pattern of immunoreactivity for the receptor subtypes as that observed at 8 weeks. Immunoreactivity for P2X4 was first observed in the adrenal cortical cells of the zona reticularis at 8 weeks, but was absent in 24‐month‐old rats. However, several P2X4‐immunoreactive chromaffin cells appeared at 24 months. Such immunoreactive cells were not seen in rats of any of the other ages studied. It was concluded that the greater expression of P2X5 receptor at an early developmental stage and of P2X4 in ageing might reflect functional roles for purines in cellular proliferation and/or differentiation, and in cellular degeneration, respectively, in adrenal glands of rat.

Changes in P2Y2 receptor localization on adrenaline- and noradrenaline-containing chromaffin cells in the rat adrenal gland during development and aging

Using immunohistochemistry, the occurrence and age‐related changes of the P2Y2 receptor was investigated in the adrenal gland of rat at different ages, ranging from embryonic day E16 to 22 months. Immunoreactivity for the P2Y2 receptor was present in chromaffin cells and nerve fibres at all ages examined. Double labeling with the antibody against phenyl ethanolamine‐N‐methyltransferase, which marks adrenaline‐producing chromaffin cells, revealed that only a few of the P2Y2‐immunoreactive cells were adrenaline producing at embryonic day E16, the vast majority being noradrenaline‐containing cells. However, immunoreactivity for adrenaline‐containing cells in the P2Y2 receptor‐labeled chromaffin cells increased with increasing age and at 1 week post‐natal almost all chromaffin cells were positive for both P2Y2 and phenyl ethanolamine‐N‐methyltransferase, while noradrenaline‐containing cells were minimal. At 2 weeks, there was a dramatic drop in P2Y2‐immunoreactive chromaffin cells and this was maintained in adult rats, noradrenaline‐containing cells dominating. In the aging rat adrenals, P2Y2 receptor‐immunoreactivity was localized in subpopulations of both adrenaline and noradrenaline‐producing cells. Intrinsic neurones were also visible that were positively labeled with the P2Y2 receptor antibody in the adrenals of both adult and aging rats. P2Y2‐immunoreactive nerve fibres formed a plexus around the adrenal cortical cells of zona glomerulosa in the post‐natal, but not in adult or aging rats.

Localization of P2X Receptors in the Guinea Pig Adrenal Gland

The distribution of each of the seven subtypes of adenosine 5′-triphosphate (ATP)-gated P2X receptors was investigated in the adrenal gland of guinea pig utilizing immunohistochemical techniques. The occurrence of positive immunoreactivity with specific distribution was observed for antibodies against P2X1, P2X2, P2X5 and P2X6 receptors, whereas no immunoreactivity was found for antibodies against P2X3, P2X4 and P2X7 receptors. Immunoreactivity for P2X1 occurred in cells of the inner region of the zona reticularis of the cortex, Several P2X2-immunoreactive connective tissue-like elements were located between groups of cortical cells of the zona reticularis. Bundles and terminals of nerve fibres as well as intrinsic neurones gave positive immunoreactivity for P2X5. The immunoreactive nerve fibres appeared to belong to both extrinsic preganglionic sympathetic and intrinsic immunoreactive neurones. Chromaffin cells were immunoreactive for the P2X6 receptor antibody. The widespread and specific distribution of P2X receptor subtypes in the adrenal gland suggests significant roles for purinergic signalling in the physiology of the guinea pig adrenal gland.

Toxicological evaluation of methanol leaves extract of Vernonia bipontini Vatke in blood, liver and kidney tissues of mice.

BACKGROUND Various medicinal plants have been studied using modern scientific approaches. These plants have a variety of properties and various biological components that can be used to treat various diseases. However, harmful effects of plants are common clinical occurrence. OBJECTIVE This study was designed to investigate toxicological assessment of acute and chronic methanol leaf extract of Vernonia bipontini Vatke (V.bipontini V) on blood, liver and kidney tissues of mice. METHODS Lethal dose (LD) at which 50% of experimental mice died and long term toxicity of methanolic leaf extract of V. bipontini V were determined. Some hematological and biochemical parameters were evaluated. Then, liver and kidney tissues of each animal were taken and processed for light microscopy. RESULTS Almost all mice treated with 800mg/kg methanol leaf extract of V. bipontini V showed swellings on the left part of abdominal region related to location of spleen, mild diarrhea and enlargement of spleen. The LD50 of the methanol leaf extract of V. bipontini V was 2130.6±1.5mg/kg. Treatment with 800mg/kg body weight of methanol leaf extract significantly decreased body, liver and kidney weights, red blood cells (RBC), haemoglobin (Hgb), mean cell haemoglobin (Mch), Mchc, platelet and significantly increased serum aspartate transferance (AST), vatanine tranferance(ALT) and alkaline phosphate (ALP) levels while 400mg/kg dose had no effect on these parameters. The reduced organ weights did not correlate with loss of body weight at 800mg/kg of methanol leaf extract of the plant. Light microscope observations of liver tissue of mice treated with 800mg/kg of the methanol leaf extract revealed dilated sinusoids, nuclear enlargement, lots of bi-nucleation of hepatocytes, peripheral cramped chromatin, shrinkages (single cell death) of hepatocytes, fragmentation of hepatocytes while no histopathological changes were observed in liver and kidney of mice treated at 400mg/kg. Kidney tissue sections of mice did not show significant histopathological changes at 400mg/kg. However, at 800mg/kg kidney sections showed increased cellularity of glomerulus, urinary space obliteration and enlarged macula densa. CONCLUSION This study suggests that the methanol leaf extract may have been phytotoxic to liver that resulted in a rise in serum AST, ALT and ALP levels.