Neuronal Nitric Oxide Synthase Antibody Research Articles

The Effect of Pneumonic Pasteurellosis on Apoptosis and Nitric Oxide Synthase in the Lungs in Calves

Pneumonic Pasteurellosis (PP) is an infectious disease caused by Pasteurella multocida and Mannheimia haemolytica, mostly observed in cattle, sheep and calves. PP is characterized by fibrinous bronchopneumonia and pleuritis in the lungs. In this study, it was aimed to determine Caspase-3, Caspase-9, inducible nitric oxide synthase and neuronal nitric oxide synthase expressions by immunohistochemical methods in the lungs suffered from PP. For this purpose, twenty lung tissues were collected from calves with PP. For the Control Group, ten lungs of calves were collected from Aksaray Slaughterhouse. After necropsies of calves were confirmed to be PP by bacteriological examinations. Then the routine histological process was performed to tissues, and stained by Hematoxylin & Eosin for histopathology, and Caspase-3, Caspase-9, inducible nitric oxide synthase and neuronal nitric oxide synthase antibody staining for immunohistochemistry. The immunohistochemical findings indicated that Caspase-3, Caspase-9, inducible nitric oxide synthase and neuronal nitric oxide synthase positive reactions were seen in alveolar, bronchial and bronchiolar epithelia, and desquamated inflammatory cells in the lumens. In addition, the peripheral neural extensions were immunopositive for neuronal nitric oxide synthase and vascular endothelial cell were positive for inducible nitric oxide synthase. The findings can contribute to a better understanding of expressions of molecules such as Caspase and nitric oxide synthase. These results show that apoptosis and nitric oxide synthase expressions have triggered by airway epithelia and inflammatory cells in the lungs with Pneumonic Pasteurellosis in calves.

Robust, 3-Dimensional Visualization of Human Colon Enteric Nervous System Without Tissue Sectioning

Small, 2-dimensional sections routinely used for human pathology analysis provide limited information about bowel innervation. We developed a technique to image human enteric nervous system (ENS) and other intramural cells in 3 dimensions. Using mouse and human colon tissues, we developed a method that combines tissue clearing, immunohistochemistry, confocal microscopy, and quantitative analysis of full-thickness bowel without sectioning to quantify ENS and other intramural cells in 3 dimensions. We provided 280 adult human colon confocal Z-stacks from persons without known bowel motility disorders. Most of our images were of myenteric ganglia, captured using a 20× objective lens. Full-thickness colon images, viewed with a 10× objective lens, were as large as 4× 5 mm2. Colon from 2 pediatric patients with Hirschsprung disease was used to show distal colon without enteric ganglia, as well as a transition zone and proximal pull-through resection margin where ENS was present. After testing a panel of antibodies with our method, we identified 16 antibodies that bind to molecules in neurons, glia, interstitial cells of Cajal, and muscularis macrophages. Quantitative analyses demonstrated myenteric plexus in 24.5% ± 2.4% of flattened colon Z-stack area. Myenteric ganglia occupied 34% ± 4% of myenteric plexus. Single myenteric ganglion volume averaged 3,527,678 ± 573,832 mm3 with 38,706 ± 5763 neuron/mm3 and 129,321 ± 25,356 glia/mm3. Images of large areas provided insight into why published values of ENS density vary up to 150-fold-ENS density varies greatly, across millimeters, so analyses of small numbers of thin sections from the same bowel region can produce varying results. Neuron subtype analysis revealed that approximately 56% of myenteric neurons stained with neuronal nitric oxide synthase antibody and approximately 33% of neurons produce and store acetylcholine. Transition zone regions from colon tissues of patients with Hirschsprung disease had ganglia in multiple layers and thick nerve fiber bundles without neurons. Submucosal neuron distribution varied among imaged colon regions. We developed a 3-dimensional imaging method for colon that provides more information about ENS structure than tissue sectioning. This approach could improve diagnosis for human bowel motility disorders and may be useful for other bowel diseases as well.

Feasibility of intraperitoneal placental-derived mesenchymal stem cell injection in stomachs of diabetic mice.

Diabetic gastropathy is associated with loss of interstitial cells of Cajal and autonomic neuropathy. Effective management for diabetic gastropathy is still unavailable. This study was aimed to confirm the pathogenetic changes in diabetic gastropathy and to examine the effect of treatment with placental-derived mesenchymal stem cells (PDMSCs) in stomachs of animal models. Fourteen non-obese diabetic/ShiLtJ mice of 8weeks were bled until week 30. Diabetes mellitus developed in 10 out of 14 mice, which all survived with insulin. The mice were grouped into three groups: nondiabetic group (n=4), diabetic sham group (n=5), and diabetic PDMSC group (n=5) all of which were treated with intraperitoneal PDMSCs injection at week 30. All mice were killed at week 34, and the stomachs were examined by immunohistochemical stain with c-kit and neuronal nitric oxide synthase antibodies. The number of c-kit positive cells in stomach decreased significantly in the diabetic sham group compared with that in the nondiabetic group (21.2±6.7 vs 88.0±29.3, P=0.006) but increased with PDMSC treatment (21.2±6.7 vs 64.0±15.1, P=0.02). The positive rate of neuronal nitric oxide synthase in neural plexus was also significantly lower in the diabetic sham group than in the nondiabetic group (22.3%±18.5% vs 48.0%±22.7%, P=0.003) but increased with PDMSC treatment (22.3%±18.5% vs 43.3%±20.5%, P=0.03). Interstitial cells of Cajal and neural plexus decreased in stomachs of mice with diabetes mellitus but were significantly repaired with intraperitoneal injection of PDMSC.

Baseline Erectile Function Alters the Cavernous Nerve Quantity and Distribution Around the Prostate

We clarified the distribution of neuronal nitric oxide synthase positive nerve fibers around the prostate and factors contributing to fiber quantity. We investigated 46 hemispheres of 23 nonnerve sparing radical prostatectomy specimens from patients with available preoperative International Index of Erectile Function and maximum penile circumferential change data. We performed immunohistochemical staining with neuronal nitric oxide synthase antibody, and divided the prostatic hemisphere into 6 zones to assess the distribution of neuronal nitric oxide synthase positive nerve fibers at the apex, midportion and base of the prostate. Multivariate analysis of the quantity of overall neuronal nitric oxide synthase positive nerve fibers used the variables of specimen weight, patient age, body mass index, International Index of Erectile Function and maximum penile circumferential change. A maximum penile circumferential change of 20 mm or less was considered objective erectile dysfunction. Median patient age was 68 years (range 57 to 74). Median International Index of Erectile Function-erectile function domain score was 12 (range 0 to 24) and median maximum penile circumferential change was 25.0 mm (range 2.70 to 38.3). Of the neuronal nitric oxide synthase positive nerve fibers 65% were distributed in a 3 to 5 o'clock sextant (p <0.001). Logistic regression analysis revealed that maximum penile circumferential change was the single significant variable (p = 0.019). The fiber count was greater in the objective erectile dysfunction group (median 1,500, range 382 to 2,760) than in the nonerectile dysfunction group (median 649, range 156 to 2,916) (p = 0.009). The fiber count was significantly different between the 2 groups in the 3 to 6 o'clock area, especially at the apex. Baseline erectile function greatly impacts cavernous nerve quantity and distribution. Cavernous nerve preservation in the neurovascular bundle, especially at the apex, is still essential for patients with erectile dysfunction.

A combination of tumor necrosis factor‐α and neuronal nitric oxide synthase antibodies applied topically over the traumatized spinal cord enhances neuroprotection and functional recovery in the rat

The possibility that neutralization of nitric oxide synthase and tumor necrosis factor alpha (TNF-alpha) in the cord using their antiserum will induce neuroprotection and improve functional outcome following spinal cord injury (SCI) was examined in a rat model. The SCI was induced in rats by a unilateral incision of the right dorsal horn at the T10-11 segments under equithesin anesthesia. TNF-alpha and/or neuronal nitric oxide synthase (nNOS) antibodies were applied over the traumatized spinal cord at 10-90 minutes after injury and functional recovery and cord pathophysiology were examined at five hours. Topical application of TNF-alpha antiserum at 10 min followed by NOS antiserum at 20 min after SCI significantly improved functional recovery and attenuated blood-spinal cord barrier (BSCB) disturbances, edema formation, and cord pathology. These neuroprotective effects were also seen when the NOS antiserum was applied 10 min after injury followed by TNF-alpha antiserum at 30 min after trauma. However, when TNF-alpha antiserum was applied 1 h after injury and NOS antiserum was given either before or after TNF-alpha antiserum, no neuroprotective effects were observed. Interestingly, neuronal injury was tightly correlated with nNOS expression in the cord in antibody treated groups. These novel observations suggest that early blockade of TNF-alpha and nNOS expression within 20-30 min after SCI is beneficial in nature. This indicates that TNF-alpha and nitric oxide play synergistic roles in the pathophysiology of SCI and combined antibodies therapy has added neuroprotective values in spinal trauma.

Immunohistochemical distribution of c-Kit-positive cells and nitric oxide synthase-positive nerves in the guinea-pig small intestine

Anatomical relationships between c-Kit-positive cells and nitric oxide synthase-positive nerves in the small intestine were examined by double-labeling immunohistochemistry. Cryosections and whole mount preparations of the guinea-pig small intestine were double-immunolabeled using anti-c-Kit and neuronal nitric oxide synthase antibodies, and were observed using confocal laser scanning microscopy. The c-Kit-like immunoreactivity constituted dense reticular networks in the deep muscular plexus and myenteric plexus of the intestinal wall. The nitric oxide synthase-like immunoreactivity occurred in the circular muscle layer, most densely at the deep muscular plexus, as well as within the ganglion strands or connecting strands of the myenteric plexus. Close association between c-Kit-like immunoreactivity and nitric oxide synthase-like immunoreactivity was evident in the deep muscular plexus. Specimens immunolabeled with the anti-nitric oxide synthase antibody were further examined under transmission electron microscopy. Axon profiles with nitric oxide synthase-like immunoreactivity lay closely adjacent to the interstitial cells in the deep muscular plexus as well as to smooth muscle cells of the circular muscle layer, whereas there was a considerable distance (>500 nm) between interstitial cells and axon profiles with nitric oxide synthase-like immunoreactivity in the myenteric plexus. These results suggest that the interstitial cells in the deep muscular plexus serve as mediators of the nitrergic neurotransmission to the musculature in the small intestine, playing a role in the regulation of intestinal movement.