Autophagy promotes survival of retinal ganglion cells after optic nerve axotomy in mice.

Autophagy is an essential recycling pathway implicated in neurodegeneration either as a pro-survival or a pro-death mechanism. Its role after axonal injury is still uncertain. Axotomy of the optic nerve is a classical model of neurodegeneration. It induces retinal ganglion cell death, a process also occurring in glaucoma and other optic neuropathies. We analyzed autophagy induction and cell survival following optic nerve transection (ONT) in mice. Our results demonstrate activation of autophagy shortly after axotomy with autophagosome formation, upregulation of the autophagy regulator Atg5 and apoptotic death of 50% of the retinal ganglion cells (RGCs) after 5 days. Genetic downregulation of autophagy using knockout mice for Atg4B (another regulator of autophagy) or with specific deletion of Atg5 in retinal ganglion cells, using the Atg5(flox/flox) mice reduces cell survival after ONT, whereas pharmacological induction of autophagy in vivo increases the number of surviving cells. In conclusion, our data support that autophagy has a cytoprotective role in RGCs after traumatic injury and may provide a new therapeutic strategy to ameliorate retinal diseases.

Hirschsprung's disease: the "Swiss roll" technique revisited.

PURPOSE: During pull-through for Hirschsprung's disease (HSCR), the assessment of innervation is mainly based on the presence of ganglion cells when conventional Hematoxylin and Eosin (HE) staining is used. In hypoganglionosis, the evaluation is difficult. We adapted a standardized methodology for the examination of resected bowel after HSCR surgery, using the technique described by Moolenbeek on rodent intestine and later by Meier-Ruge in children. We have analysed the entire innervation of surgically resected bowels and compared the results with the follow up of patients. METHODS: Three longitudinal strips of colon were harvested from the mesenteric, anti-mesenteric and intermediate part in the whole length of resected colon of six patients with HSCR. Each strip was divided into two parts. One of the contiguous strips was assessed with HE and Hematoxylin-Phloxin-Safran, and the other one with acetylcholinesterase (AChE) histochemistry. We analyzed the distribution of ganglion cells and nerve arrangement along the strips with both techniques and compared the results obtained in the three different regions of the bowel. RESULTS: There was no significant difference in the pattern of innervation circumferentially. There was a correlation between a progressive increase of AChE activity and nerve hypertrophy and a decrease of ganglion cells from the proximal to the distal part of the resected colon in the submucosa and the myenteric plexus. Nerve hypertrophy and AChE-positive reaction in the mucosa were found at the resection border in patients who presented postoperative complications. CONCLUSIONS: Simultaneous assessment of nerve cells, nerve fibers and AChE activity is important in the evaluation of the innervation of the bowel segment proximal to the aganglionic zone. The method described is feasible and can be adapted to older children and adults with larger bowels. These results point out the importance of assessing nerve fibers in intraoperative biopsies during pull-through procedures to prevent uncomplete surgical bowel resection.

Calbindin-immunoreactive sensory neurons in dissociated dorsal root ganglion cell cultures of chick embryo: role of culture conditions.

Immunoreactivity to calbindin D-28k, a vitamin D-dependent calcium-binding protein, is expressed by neuronal subpopulations of dorsal root ganglia (DRG) in the chick embryo. To determine whether the expression of this phenotypic characteristic is maintained in vitro and controlled by environmental factors, dissociated DRG cell cultures were performed under various conditions. Subpopulations of DRG cells cultured at embryonic day 10 displayed calbindin-immunoreactive cell bodies and neurites in both neuron-enriched or mixed DRG cell cultures. The number of calbindin-immunoreactive ganglion cells increased up to 7-10 days of culture independently of the changes occurring in the whole neuronal population. The presence of non-neuronal cells, which promotes the maturation of the sensory neurons, tended to reduce the percentage of calbindin-immunoreactive cell bodies. Addition of horse serum enhanced both the number of calbindin-positive neurons and the intensity of the immunostaining, but does not prevent the decline of the subpopulation of calbindin-immunoreactive neurons during the second week of culture; on the contrary, the addition of muscular extract to cultures at 10 days maintained the number of calbindin-expressing neurons. While calbindin-immunoreactive cell bodies grown in culture were small- or medium-sized, no correlation was found between cell size and immunostaining density. At the ultrastructural level, the calbindin immunoreaction was distributed throughout the neuroplasm. These results indicate that the expression of calbindin by sensory neurons grown in vitro may be modulated by horse serum-contained factors or interaction with non-neuronal cells. As distinct from horse serum, muscular extract is able to maintain the expression of calbindin by a subpopulation of DRG cells.

Angiotensin II is an endogenous neurotransmitter for rat and human mesenteric resistance blood vessels

Angiotensin II (Ang II) is one of the most potent vasoconstrictors. We document here the innervation of rat and human mesenteric resistance arteries (MRA) by angiotensinergic neurons of the rat and human sympathetic coeliac ganglia. Angiotensinogen (Ang-N)-mRNA and angiotensin converting enzyme-mRNA but no renin-mRNA were detected by using quantitative real time polymerase chain reaction in total RNA extracts of rat coeliac ganglia. In the same extracts, cathepsin D-mRNA was detected: This protease also cleaves Ang I from Ang-N and could therefore account for the generation of neuronal Ang peptides in the absence of renin. In situ hybridization confirmed the presence of Ang-N-mRNA in the cytoplasm of rat coeliac ganglia. By using solid-phase extraction, high performance liquid chromatography and subsequent radioimmunoassay, Ang II and its metabolites were detected in rat and also in human coeliac ganglia. Immunoreactivity for Ang II was demonstrated in rat and human coeliac ganglia neurons and their projections innervating MRA. In addition, segmental angiotensinergic innervation of MRA was also observed. By means of confocal laser scanning microscopy we were able to demonstrate the presence of angiotensinergic synapses en passant along side of vascular smooth muscle cells. Our findings could indicate that Ang II is synthesized inside the neurons of sympathetic coeliac ganglia and may act as an endogenous neurotransmitter locally in MRA.

Acute retroperitoneal bleeding due to inferior mesenteric artery aneurysm: case report

Background: Visceral artery aneurysms (VAA), although uncommon, are increasingly being detected. We describe a case of spontaneous retroperitoneal hemorrhage from a ruptured IMA aneurysm associated with stenosis of the superior mesenteric artery (SMA) and celiac trunk, successfully treated with surgery. Methods: A 65-year-old man presented with abdominal pain and hypovolemic shock. Abdominal CT scan showed an aneurysm of the inferior mesenteric artery with retroperitoneal hematoma. In addition, an obstructive disease of the superior mesenteric artery and celiac axis was observed. Results: Upon emergency laparotomy a ruptured inferior mesenteric artery aneurysm was detected. The aneurysm was excised and the artery reconstructed by end-to-end anastomosis. Conclusions This report discusses the etiology, presentation, diagnosis and case management of inferior mesenteric artery aneurysms

Impact of Prophylactic Cranial Irradiation Timing on Brain Relapse Rates in Patients With Stage IIIB Non-Small-Cell Lung Carcinoma Treated With Two Different Chemoradiotherapy Regimens.

PURPOSE: To retrospectively assess the influence of prophylactic cranial irradiation (PCI) timing on brain relapse rates in patients treated with two different chemoradiotherapy (CRT) regimens for Stage IIIB non-small-cell lung cancer (NSCLC). METHODS AND MATERIALS: A cohort of 134 patients, with Stage IIIB NSCLC in recursive partitioning analysis Group 1, was treated with PCI (30 Gy at 2 Gy/fr) following one of two CRT regimens. Regimen 1 (n = 58) consisted of three cycles of induction chemotherapy (ICT) followed by concurrent CRT (C-CRT). Regimen 2 (n = 76) consisted of immediate C-CRT during thoracic radiotherapy. RESULTS: At a median follow-up of 27.6 months (range, 7.2-40.4), 65 patients were alive. Median, progression-free, and brain metastasis-free survival (BMFS) times for the whole study cohort were 23.4, 15.4, and 23.0 months, respectively. Median survival time and the 3-year survival rate for regimens 1 and 2 were 19.3 vs. 26.1 months (p = 0.001) and 14.4% vs. 34.4% (p < .001), respectively. Median time from the initiation of primary treatment to PCI was 123.2 (range, 97-161) and 63.4 (range, 55-74) days for regimens 1 and 2, respectively (p < 0.001). Overall, 11 (8.2%) patients developed brain metastasis (BM) during the follow-up period: 8 (13.8%) in regimen 1 and 3 (3.9%) in regimen 2 (p = 0.03). Only 3 (2.2%) patients developed BM at the site of first failure, and for 2 of them, it was also the sole site of recurrence. Median BMFS for regimens 1 and 2 were 17.4 (13.5-21.3) vs. 26.0 (22.9-29.1 months), respectively (p < 0.001). CONCLUSION: These results suggest that in Stage IIIB NSCLC patients treated with PCI, lower BM incidence and longer survival rates result from immediate C-CRT rather than ITC-first regimens. This indicates the benefit of earlier PCI use without delay because of induction protocols.

Intrinsically photosensitive retinal ganglion cell function in relation to age: a pupillometric study in humans with special reference to the age-related optic properties of the lens.

BACKGROUND: The activity of melanopsin containing intrinsically photosensitive ganglion retinal cells (ipRGC) can be assessed by a means of pupil responses to bright blue (appr.480 nm) light. Due to age related factors in the eye, particularly, structural changes of the lens, less light reaches retina. The aim of this study was to examine how age and in vivo measured lens transmission of blue light might affect pupil light responses, in particular, mediated by the ipRGC. METHODS: Consensual pupil responses were explored in 44 healthy subjects aged between 26 and 68 years. A pupil response was recorded to a continuous 20 s light stimulus of 660 nm (red) or 470 nm (blue) both at 300 cd/m2 intensity (14.9 and 14.8 log photons/cm2/s, respectively). Additional recordings were performed using four 470 nm stimulus intensities of 3, 30, 100 and 300 cd/m2. The baseline pupil size was measured in darkness and results were adjusted for the baseline pupil and gender. The main outcome parameters were maximal and sustained pupil contraction amplitudes and the postillumination response assessed as area under the curve (AUC) over two time-windows: early (0-10 s after light termination) and late (10-30 s after light termination). Lens transmission was measured with an ocular fluorometer. RESULTS: The sustained pupil contraction and the early poststimulus AUC correlated positively with age (p=0.02, p=0.0014, respectively) for the blue light stimulus condition only.The maximal pupil contraction amplitude did not correlate to age either for bright blue or red light stimulus conditions.Lens transmission decreased linearly with age (p<0.0001). The pupil response was stable or increased with decreasing transmission, though only significantly for the early poststimulus AUC to 300 cd/m2 light (p=0.02). CONCLUSIONS: Age did not reduce, but rather enhance pupil responses mediated by ipRGC. The age related decrease of blue light transmission led to similar results, however, the effect of age was greater on these pupil responses than that of the lens transmission. Thus there must be other age related factors such as lens scatter and/or adaptive processes influencing the ipRGC mediated pupil response enhancement observed with advancing age.

Intrinsically photosensitive retinal ganglion cells.

The recent discovery of melanopsin-expressing retinal ganglion cells that mediate the pupil light reflex has provided new insights into how the pupil responds to different properties of light. These ganglion cells are unique in their ability to transduce light into electrical energy. There are parallels between the electrophysiologic behavior of these cells in primates and the clinical pupil response to chromatic stimuli. Under photopic conditions, a red light stimulus produces a pupil constriction mediated predominantly by cone input via trans-synaptic activation of melanopsin-expressing retinal ganglion cells, whereas a blue light stimulus at high intensity produces a steady-state pupil constriction mediated primarily by direct intrinsic photoactivation of the melanopsin-expressing ganglion cells. Preliminary data in humans suggest that under photopic conditions, cones primarily drive the transient phase of the pupil light reflex, whereas intrinsic activation of the melanopsin-expressing ganglion cells contributes heavily to sustained pupil constriction. The use of chromatic light stimuli to elicit transient and sustained pupil light reflexes may become a clinical pupil test that allows differentiation between disorders affecting photoreceptors and those affecting retinal ganglion cells.

Differentiation of postmitotic neuroblasts into substance P-immunoreactive sensory neurons in dissociated cultures of chick dorsal root ganglion.

Counts performed on dissociated cell cultures of E10 chick embryo dorsal root ganglia (DRG) showed after 4-6 days of culture a pronounced decline of the neuronal population in neuron-enriched cultures and a net gain in the number of ganglion cells in mixed DRG cell cultures (containing both neurons and nonneuronal cells). In the latter case, the increase in the number of neurons was found to depend on NGF and to average 119% in defined medium or 129% in horse serum-supplemented medium after 6 days of culture. The lack of [3H]thymidine incorporation into the neuronal population indicated that the newly formed ganglion cells were not generated by proliferation. On the contrary, the differentiation of postmitotic neuroblasts present in the nonneuronal cell compartment was supported by sequential microphotographs of selected fields taken every hour for 48-55 hr after 3 days of culture. Apparently nonneuronal flat dark cells exhibited morphological changes and gradually evolved into neuronal ovoid and refringent cell bodies with expanding neurites. The ultrastructural organization of these evolving cells corresponded to that of primitive or intermediate neuroblasts. The neuronal nature of these rounding up cell bodies was indeed confirmed by the progressive expression of various neuronal cell markers (150 and 200-kDa neurofilament triplets, neuron specific enolase, and D2/N-CAM). Besides a constant lack of immunoreactivity for tyrosine hydroxylase, somatostatin, parvalbumin, and calbindin-D 28K and a lack of cytoenzymatic activity for carbonic anhydrase, all the newly produced neurons expressed three main phenotypic characteristics: a small cell body, a strong immunoreactivity to MAG, and substance P. Hence, ganglion cells newly differentiated in culture would meet characteristics ascribed to small B sensory neurons and more specifically to a subpopulation of ganglion cells containing substance P-immunoreactive material.

Multidetector CT Features of Mesenteric Vein Thrombosis.

Mesenteric vein thrombosis (MVT) accounts for 5%-15% of all mesenteric ischemic events and is classified as either primary or secondary. Primary MVT is idiopathic, whereas secondary MVT can result from a variety of underlying diseases and risk factors, including primary hypercoagulable states or prothrombotic disorders, myeloproliferative neoplasms, cancer (most frequently of the pancreas or liver), diverse inflammatory conditions, recent surgery, portal hypertension, and miscellaneous causes such as oral contraceptives or pregnancy. Clinical symptoms of MVT are rather nonspecific and are mainly characterized by abdominal pain. The mortality rate for MVT remains high, since even now the diagnosis is often delayed. Multidetector computed tomography (CT) is the modality of choice in this context. Although venous bowel ischemia occurs only infrequently with MVT, radiologists should be familiar with its multidetector CT features. Familiarity with the possible causes of MVT, the underlying pathogenic mechanisms associated with MVT, and the correlation between multidetector CT features and these pathogenic mechanisms is necessary to optimize medical management and improve patient care. © RSNA, 2012.

Brain spectrin isoforms during development of chick dorsal root ganglion cells in vivo and in vitro

Brain spectrin is one of the major cytoskeletal proteins associated with the plasma membrane. In many tissues this protein occurs in a variety of isoforms, for which at least three have been described in the brain: i) brain spectrin 240/235 is localized in neurons most prominently in axons and is present early during brain development. ii) Brain spectrin 240/235E is immunologicaly related to erythrocyte spectrin and restricted to somato-dendritic regions in neurons and to glia. It appears late in brain development. iii) A third form, brain spectrin 240/ 235A, is found exclusively in astrocytes. In this study we have investigated the appearance and distribution of brain spectrins 240/235 and 240/235E during embryonic chick dorsal root ganglia development in vivo and in vitro. This system provides a unique model due to the lack of dendrites on developing sensory neurons. Both isoforms first appeared at embryonic day 6. Brain spectrin 240/235 increased transiently around embryonic day 10 and 14, and was first expressed in ventrolateral neurons. It was localized abundantly in perikarya and their axons. This somato-axonal distribution pattern found in situ was also observed in vitro. In contrast, brain spectrin 240/235E only slightly increased between E6 and E15 and remained unchanged thereafter. It was localized mainly in small neurons of the mediodorsal area, where it was found as punctate staining in the cytoplasm, forming first a nuclear cap and in subsequent stages becoming distributed evenly throughout cytoplasm. This brain spectrin isoform was absent from axons, both in situ and in vitro. In conclusion, this study suggests i) that brain spectrin 240/235 may contribute towards the outgrowth, elongation and possibly maintenance of axonal processes, ii) that brain spcctrin 240/235E could be involved in the stablization of the cytoarchilecture of cell bodies in a sclected population of ganglion cells, and iii) that isoform expression of brain spectrin 240/235E in DRG cells may depend on environmental factors.

NADPH-diaphorase-positive ganglion cells of the rat adrenal gland: age- and sex-related changes in their number, size, and distribution.

The rat adrenal gland contains ganglion cells able to synthesize nitric oxide (NO). This messenger molecule controls and modulates adrenal secretory activity and blood flow. The present study analyzed the number, size, and distribution of NO-producing adrenal neurons in adulthood and during postnatal development by means of beta-nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry. This method reliably visualizes the enzyme responsible for NO generation. The reactive neurons per adrenal gland were 350-400 in both male and female adult rats. The positive nerve cell bodies were mostly located in the medulla, few being detected within the cortex and the subcapsular region. Dual labeling with anti-microtubule-associated protein 2 antibody, specific for neuronal elements, confirmed this distribution. Anti-microtubule-associated protein 1b antibody identified a subset of NADPH-d-positive neurons, displaying different degrees of maturation according to their position within the adrenal gland. At birth, there were about 220 NADPH-d-labeled neurons per adrenal gland in both sexes. As confirmed by dual immunocytochemical labeling, their great majority was evenly distributed between the cortex and the subcapsular region, the medulla being practically devoid of stained neurons. After birth, the number of adrenal NADPH-d-positive ganglion cells displayed a strong postnatal increase and reached the adult-like distribution after 1-2 months. During the period of increase, there was a transient difference in the numbers of these cells in the two sexes. Thus we present here evidence of plasticity in the number, size, and distribution of NADPH-d-positive adrenal neurons between birth and adulthood; in addition, we describe transient sex-related differences in their number and distribution during the 2nd postnatal week, which are possibly related to the epigenetic action of gonadal hormones during this period.

Block of the superior cervical ganglion, description of a novel ultrasound-guided technique in human cadavers.

OBJECTIVE.: Injection of opioids to the superior cervical ganglion (SCG) has been reported to provide pain relief in patients suffering from different kinds of neuropathic facial pain conditions, such as trigeminal neuralgia, postherpetic neuralgia, and atypical facial pain. The classic approach to the SCG is a transoral technique using a so-called "stopper" to prevent accidental carotid artery puncture. The main disadvantage of this technique is that the needle tip is positioned distant from the actual target, possibly impeding successful block of the SCG. A further limitation is that injection of local anesthetics due to potential carotid artery puncture is contraindicated. We hypothesized that the SCG can be identified and blocked using ultrasound imaging, potentially increasing precision of this technique. INTERVENTIONS.: In this pilot study, 20 US-guided simulated blocks of the SCG were performed in 10 human cadavers in order to determine the accuracy of this novel block technique. After injection of 0.1 mL of dye, the cadavers were dissected to evaluate the needle position and coloring of the SCG. RESULTS.: Nineteen of the 20 needle tips were located in or next to the SCG. This corresponded to a simulated block success rate of 95% (95% confidence interval 85-100%). In 17 cases, the SCG was completely colored, and in two cases, the caudal half of the SCG was colored with dye. CONCLUSIONS.: The anatomical dissections confirmed that our ultrasound-guided approach to the SCG is accurate. Ultrasound could become an attractive alternative to the "blind" transoral technique of SCG blocks.