Cardiovascular responses to noradrenaline microinjection in the ventrolateral periaqueductal gray of unanesthetized rats

The periaqueductal gray area (PAG) is a mesencephalic area involved in cardiovascular modulation. Noradrenaline (NA), a neurotransmitter involved in central blood pressure control, is present in the rat PAG. We report here on the cardiovascular effects caused by NA microinjection into the ventrolateral PAG (vlPAG) of unanesthetized rats and the peripheral mechanism involved in their mediation. NA microinjection in the vlPAG of unanesthetized rats evoked dose-related pressor and bradycardiac responses. No significant cardiovascular responses were observed in urethane-anesthetized rats. The pressor response was potentiated by pretreatment with the ganglion blocker pentolinium (5 or 10 mg/kg, intravenously). Pretreatment with the vasopressin antagonist dTyr(CH(2))(5) (Me)AVP (50 mu g/kg, intravenously) blocked the pressor response evoked by the NA microinjection into the vlPAG. Additionally, circulating vasopressin content was found to be significantly increased after NA microinjection in the vlPAG. The results suggest that activation of noradrenergic synapses within the vlPAG modulates vasopressin release in unanesthetized rats. (c) 2007 Wiley-Liss, Inc.

15d-prostaglandin J(2) inhibits inflammatory hypernociception: Involvement of peripheral opioid receptor

The 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) is an endogenous ligand of peroxisome proliferator-activated receptors gamma (PPAR-gamma) and is now recognized as a potent anti-inflammatory mediator. However, information regarding the influence of 15d-PGJ(2) on inflammatory pain is still unknown. In this study, we evaluated the effect of 15d-PGJ(2) upon inflammatory hypernociception and the mechanisms involved in this effect. We observed that intraplantar administration of 15d-PGJ(2) (30-300 ng/paw) inhibits the mechanical hypernociception induced by both carrageenan (100 mu g/paw) and the directly acting hypernociceptive mediator, prostaglandin E-2 (PGE(2)). Moreover, 15d-PGJ(2) [100 ng/temporomandibular joint (TMJ)] inhibits formalininduced TMJ hypernociception. On the other hand, the direct administration of 15d-PGJ(2) into the dorsal root ganglion was ineffective in blocking PGE(2)- induced hypernociception. In addition, the 15d-PGJ(2) antinociceptive effect was enhanced by the increase of macrophage population in paw tissue due to local injection of thioglycollate, suggesting the involvement of these cells on the 15d-PGJ(2)-antinociceptive effect. Moreover, the antinociceptive effect of 15d-PGJ(2) was also blocked by naloxone and by the PPAR-gamma antagonist 2-chloro-5-nitro-N-phenylbenzamide (GW9662), suggesting the involvement of peripheral opioids and PPAR-gamma receptor in the process. Similar to opioids, the 15d-PGJ(2) antinociceptive action depends on the nitric oxide/cGMP/protein kinase G (PKG)/K-ATP(+) channel pathway because it was prevented by the pretreatment with the inhibitors of nitric-oxide synthase (N-G-monomethyl-L-arginine acetate), guanylate cyclase] 1H-(1,2,4)-oxadiazolo(4,2-alpha) quinoxalin-1- one[, PKG [indolo[2,3-a]pyrrolo[3,4-c]carbazole aglycone (KT5823)], or with the ATP-sensitive potassium channel blocker glibenclamide. Taken together, these results demonstrate for the first time that 15d-PGJ(2) inhibits inflammatory hypernociception via PPAR-gamma activation. This effect seems to be dependent on endogenous opioids and local macrophages.

Differential regulation of TRP channels in a rat model of neuropathic pain

Neuropathic pain is a chronic disease resulting from dysfunction of the nervous system often due to peripheral nerve injury. Hypersensitivity to sensory Stimuli (mechanical, thermal or chemical) is a common source of pain in patients and ion channels involved in detecting these Stimuli are possible candidates for inducing and/or maintaining the pain. Transient receptor potential (TRP) channels expressed on nociceptors respond to different sensory stimuli and a few of them have been studied previously in the models of neuropathic pain. Using real-time PCR for quantification of all known TRP channels we identified several TRP channels, which have not been associated with nociception OF neuropathic pain before, to be expressed in the DRG and to be differentially regulated after spared nerve injury (SNI). Of all TRP channel members, TRPML3 showed the most dramatic change in animals exhibiting neuropathic pain behaviour compared to control animals. fit situ hybridisation showed a widespread increase of expression ill neurons of small, medium and large cell sizes, indicating expression ill multiple subtypes. Co-localisation of TRPML3 with CGRP, NF200 and IB4 staining confirmed a broad Subtype distribution. Expression studies during development showed that TRPML3 is all embryonic channel that is induced upon nerve injury in three different nerve injury models investigated. Thus. the current results link for the first time a re-expression of TRPML3 with the development of neuropathic pain conditions. In addition, decreased mRNA levels after SNI were seen for TRPM6, TRPM8, TRPV1, TRPA1, TRPC3, TRPC4 and TRPC5. (C) 2009 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.

Dorsal root ganglionectomy for the diagnosis of sensory neuropathies. Surgical technique and results

Background: Inflammatory diseases stand out among sensory neuronopathies because, in their active phase, they can be treated with immunosuppressive agents. Immunosuppressive therapy may present severe adverse effects and requires previous inflammatory activity confirmation. Sensory neuronopathies are diagnosed based on clinical and EMG findings. Diagnostic confirmation and identification of inflammatory activity are based on sensory ganglion histopathological examination. We describe the surgical technique used for dorsal root ganglionectomy in patients with clinical/EMG diagnosis of sensory neuronopathies. Methods: The sensory ganglion was obtained from 15 patients through a small T7-T8 hemilaminectomy and foraminotomy to expose the C7 root from its origin to the spinal nerve bifurcation. In 6 patients, the dural cuff supposed to contain the ganglion was resected en bloc; and in 9 patients, the ganglion was obtained through a longitudinal incision of the dural cuff and microsurgical dissection from the ventral and dorsal roots and radicular arteries. All ganglia were histopathologically examined. Results: No ganglion was found in the dural cuff in 2 patients submitted to en bloc removal, and the ganglion was removed in all patients who underwent microsurgical dissection. All but 2 patients that had ganglion examination presented a neuronopathy of nerve cell loss, 3 with mononuclear inflammatory infiltrate. These patients underwent immunosuppressive therapy, and 2 of them presented clinical improvement. No surgical complications were observed. Conclusions: Microsurgical dorsal root ganglionectomy for diagnosing inflammatory sensory ganglionopathies was effective and safe. Although safe, en bloc resection of the proximal dural cuff was not effective for this purpose. (c) 2008 Published by Elsevier Inc.

Neuroprotective effects of oral lamotrigine administration on rabbit retinas after pars plana vitrectomy and silicone oil injection

Purpose: To investigate potential retinal neuroprotective effects of oral lamotrigine in rabbits after pars plana vitrectomy (PPV) and intravitreal silicone oil injection (SOI). Methods: Twelve New Zealand rabbits (weight, 2.0-2.5 kg) underwent PPV with SOI on the right eye. For 30 days postoperatively, 6 rabbits received a daily oral dose of lamotrigine (25 mg/kg), and 6 rabbits received a daily oral dose of water. The animals were killed 30 days after surgery. All retinas were processed histologically, immunostained using glial fibrillary acidic protein (GFAP), and analyzed by fluorescence microscopy. Retina sections from all groups were analyzed by TUNEL for the presence of apoptosis and stained with hematoxylin-eosin for morphologic analysis and retina cell density measurements in each layer using a Zeiss Axiophot microscope and KS 400 software. Results: Retinas from water-operated eyes showed a significant decrease in cell density associated with cell death compared with retinas from water-control eyes; cell density was reduced by 56% in the outer nuclear layer (ONL), 49% in the inner nuclear layer (INL), and 64% in the ganglion cell layer (GCL). Lamotrigine-operated retinas showed a reduction in cell death when compared with water-operated retinas; cell death was reduced by 52% in the ONL, 25% in the INL, and 56% in the GCL. Water-operated retinas showed TUNEL-positive cells and GFAP immunofluorescence throughout Muller cell processes; lamotrigine-operated retinas showed no TUNEL-positive cells and decreased GFAP staining when compared with water-operated retinas. Conclusions: PPV with SOI was associated with apoptosis of retinal cells and activation of glial cells in rabbit eyes. Oral lamotrigine administration provided protection against these effects.

Immunolocalization of nitric oxide synthase isoforms in human archival and rat tissues, and cultured cells

Nitric oxide (NO) exerts important physiological and pathological roles in humans. The study of NO requires the immunolocalization of its synthesizing enzymes, neuronal, endothelial and inducible NO synthases (NOS). NOS are labile to formalin-fixation and paraffin-embedding, which are used to prepare human archival tissues. This lability has made NOS immunohistochemical studies difficult, and a detailed protocol is not yet available. We describe here a protocol for the immunolocalization of NOS isoforms in human archival cerebellum and non-nervous tissues, and in rat tissues and cultured cells. Neuronal NOS antigenicity in human archival and rat nervous tissue sections was microwave-retrieved in 50 mM Tris-HCl buffer, pH 9.5, for 20 min at 900W. Neuronal NOS was expressed in stellate, basket, Purkinje and granule cells in human and rat cerebellum. Archival and frozen human cerebellar sections showed the same neuronal NOS staining pattern. Archival cerebellar sections not subjected to antigen retrieval stained weakly. Antigenicity of inducible NOS in human lung was best retrieved in 10 mM sodium citrate buffer, pH 6.0, for 15 min at 900W. Inflammatory cells in a human lung tuberculoma were strongly stained by anti-inducible NOS antibody. Anti-endothelial NOS strongly stained kidney glomeruli. Cultured PC12 cells were strongly stained by anti-neuronal NOS without antigen retrieving. The present immunohistochemistry protocol is easy to perform, timeless, and suitable for the localization of NOS isoforms in nervous and non-nervous tissues, in human archival and rat tissues. It has been extensively used in our laboratory, and is also appropriate for other antigens. (C) 2011 Elsevier B.V. All rights reserved.

Drosophila melanogaster lipins are tissue-regulated and developmentally regulated and present specific subcellular distributions

Lipins constitute a novel family of Mg2+-dependent phosphatidate phosphatases that catalyze the dephosphorylation of phosphatidic acid to yield diacylglycerol, an important intermediate in lipid metabolism and cell signaling. Whereas a single lipin is detected in less complex organisms, in mammals there are distinct lipin isoforms and paralogs that are differentially expressed among tissues. Compatible with organism tissue complexity, we show that the single Drosophila Lpin1 ortholog (CG8709, here named DmLpin) expresses at least three isoforms (DmLpinA, DmLpinK and DmLpinJ) in a temporal and spatially regulated manner. The highest levels of lipin in the fat body, where DmLpinA and DmLpinK are expressed, correlate with the highest levels of triacylglycerol (TAG) measured in this tissue. DmLpinK is the most abundant isoform in the central nervous system, where TAG levels are significantly lower than in the fat body. In the testis, where TAG levels are even lower, DmLpinJ is the predominant isoform. Together, these data suggest that DmLpinA might be the isoform that is mainly involved in TAG production, and that DmLpinK and DmLpinJ could perform other cellular functions. In addition, we demonstrate by immunofluorescence that lipins are most strongly labeled in the perinuclear region of the fat body and ventral ganglion cells. In visceral muscles of the larval midgut and adult testis, lipins present a sarcomeric distribution. In the ovary chamber, the lipin signal is concentrated in the internal rim of the ring canal. These specific subcellular localizations of the Drosophila lipins provide the basis for future investigations on putative novel cellular functions of this protein family.

Stereological and allometric studies on neurons and axo-dendritic synapses in the superior cervical ganglia of rats, capybaras and horses

The superior cervical ganglion (SCG) in mammals varies in structure according to developmental age, body size, gender, lateral asymmetry, the size and nuclear content of neurons and the complexity and synaptic coverage of their dendritic trees. In small and medium-sized mammals, neuron number and size increase from birth to adulthood and, in phylogenetic studies, vary with body size. However, recent studies on larger animals suggest that body weight does not, in general, accurately predict neuron number. We have applied design-based stereological tools at the light-microscopic level to assess the volumetric composition of ganglia and to estimate the numbers and sizes of neurons in SCGs from rats, capybaras and horses. Using transmission electron microscopy, we have obtained design-based estimates of the surface coverage of dendrites by postsynaptic apposition zones and model-based estimates of the numbers and sizes of synaptophysin-labelled axo-dendritic synaptic disks. Linear regression analysis of log-transformed data has been undertaken in order to establish the nature of the relationships between numbers and SCG volume (V(scg)). For SCGs (five per species), the allometric relationship for neuron number (N) is N=35,067xV (scg) (0.781) and that for synapses is N=20,095,000xV (scg) (1.328) , the former being a good predictor and the latter a poor predictor of synapse number. Our findings thus reveal the nature of SCG growth in terms of its main ingredients (neurons, neuropil, blood vessels) and show that larger mammals have SCG neurons exhibiting more complex arborizations and greater numbers of axo-dendritic synapses.

Hypertrophy and neuron loss: structural changes in sheep SCG induced by unilateral sympathectomy

Recently, superior cervical ganglionectomy has been performed to investigate a variety of scientific topics from regulation of intraocular pressure to suppression of lingual tumour growth. Despite these recent advances in our understanding of the functional mechanisms underlying superior cervical ganglion (SCG) growth and development after surgical ablation, there still exists a need for information concerning the quantitative nature of the relationships between the removed SCG and its remaining contralateral ganglion and between the remaining SCG and its modified innervation territory. To this end, using design-based stereological methods, we have investigated the structural changes induced by unilateral ganglionectomy in sheep at three distinct timepoints (2, 7 and 12 weeks) after surgery. The effects of time, and lateral (left-right) differences, were examined by two-way analyses of variance and paired t-tests. Following removal of the left SCG, the main findings were: (i) the remaining right SCG was bigger at shorter survival times, i.e. 74% at 2 weeks, 55% at 7 weeks and no increase by 12 weeks, (ii) by 7 weeks after surgery, the right SCG contained fewer neurons (no decrease at 2 weeks, 6% fewer by 7 weeks and 17% fewer by 12 weeks) and (iii) by 7 weeks, right SCG neurons were also larger and the magnitude of this increase grew substantially with time (no rise at 2 weeks, 77% by 7 weeks and 215% by 12 weeks). Interaction effects between time and ganglionectomy-induced changes were significant for SCG volume and mean perikaryal volume. These findings show that unilateral superior cervical ganglionectomy has profound effects on the contralateral ganglion. For future investigations, it would be interesting to examine the interaction between SCGs and their innervation targets after ganglionectomy. Is the ganglionectomy-induced imbalance between the sizes of innervation territories the milieu in which morphoquantitative changes, particularly changes in perikaryal volume and neuron number, occur? Mechanistically, how would those changes arise? Are there any grounds for believing in a ganglionectomy-triggered SCG cross-innervation and neuroplasticity? (C) 2011 ISDN. Published by Elsevier Ltd. All rights reserved.

Histologic and immunohistochemical evaluation of intestinal innervation in dogs with and without intussusception

Objective-To assess viability of innervation in bowel segments appearing macroscopically viable from dogs with intussusception. Animals-7 dogs without gastrointestinal dysfunction that had been euthanized for reasons unrelated to the study (control dogs) and 13 dogs with intussusception that underwent enterectomy and intestinal anastomosis (affected dogs). Procedures-A total of 31 samples of intestinal tissue were obtained from the control dogs; 28 samples were obtained from affected dogs during surgery. Samples were histologically and immunohistochemically prepared and subjectively scored for degree of vacuolization and staining, respectively. Other data collected included mean muscle cell density of circular and longitudinal muscular layers, ratio between areas of muscular layers, mean number of myenteric plexuses, mean ganglion cell density of myenteric plexuses, and degree of degeneration in neuronal plexuses as estimated through synaptophysin and neuron-specific enolase (NSE) immunoreactivity. Results-Mean muscle cell density of longitudinal muscular layers, ratio between areas of muscular layers, and synaptophysin immunoreactivity did not differ significantly between affected and control dogs; values of all other variables did. Correlations were evident between mean ganglion cell density in myenteric plexuses and mean muscle cell density in circular muscular layers, degree of neuronal degeneration in myenteric plexuses and NSE immunoreactivity, and degree of neuronal degeneration in myenteric plexuses and mean ganglion cell density of myenteric plexuses. Conclusions and Clinical Relevance-Innervation may be impaired in bowel segments that appear macroscopically viable. Therefore, careful evaluation of preserved surgical margins during enterectomy and enteroanastomosis and monitoring of digestive function after surgery are important. (Am J Vet Res 2010;71:636-642)

PRECLINICAL INVESTIGATION OF THE RETINAL BIOCOMPATIBILITY OF SIX NOVEL VITAL DYES FOR CHROMOVITRECTOMY

Purpose: To investigate the retinal biocompatibility of six novel vital dyes for chromovitrectomy. Methods: An amount of 0.05 mL of 0.5% and 0.05% light green (LG), fast green (FG), Evans blue (EB), brilliant blue (BriB), bromophenol blue (BroB), or indigo carmine (IC) was injected intravitreally in the right eye, whereas in the left eye balanced salt solution was applied for control in rabbits` eyes. Clinical examination, fluorescein angiography, histology with light microscopy, and transmission electron microscopy were performed after 1 and 7 days. Retinal cell layers were evaluated for morphologic alterations and number of cells. The electroretinographic changes were assessed at baseline, 24 hours and 7 days. Results: Fluorescein angiography disclosed hypofluorescent spots only in the 0.5% EB group. Light microscopy and transmission electron microscopy disclosed slight focal morphologic changes in eyes exposed to 0.05% IC, FG, BriB, similar to the control at 1 and 7 days. In the lower dose groups, EB, LG, and BroB caused substantial retinal alterations by light microscopy. At the higher dose, BroB and EB produced diffuse cellular edema and vacuolization within the ganglion cells, bipolar cells, and photoreceptors. FG and IC at 0.5% caused slight retinal alterations similar to balanced salt solution injection. LG at 0.5% caused diffuse vacuolization of bipolar cells after 1 and 7 days. Injection of 0.5% EB caused a significant decrease in neuroretinal cell counts in comparison to control eyes in the 7-day examination (P < 0.05). Electroretinography revealed intermittent prolonged latency and decreased amplitude in eyes injected with 0.5% EB, LG, BriB, and BroB, while at the lower dose, only LG and EB induced few functional changes. Conclusion: The progressive order of retinal biocompatibility, from safest to most toxic, was IC, FG, BriB, BroB, LG, EB.

Rat forming incisor requires a rigorous ECM remodeling modulated by MMP/RECK balance

Reversion-inducing-cysteine-rich protein with Kazal motifs (RECK) is a single membrane-anchored MMP-regulator and regulates matrix metalloproteinases (MMP) 2, 9 and 14. In turn, MMPs are endopeptidases that play a pivotal role in remodeling ECM. In this work, we decided to evaluate expression pattern of RECK in growing rat incisor during, specifically focusing out amelogenesis process. Based on different kinds of ameloblasts, our results showed that RECK expression was conducted by secretory and post-secretory ameloblasts. At the secretory phase, RECK was localized in the infra-nuclear region of the ameloblast, outer epithelium, near blood vessels, and in the stellate reticulum. From the transition to the maturation phases, RECK was strongly expressed by non-epithelial immuno-competent cells (macrophages and/or dendritic-like cells) in the papillary layer. From the transition to the maturation stage, RECK expression was increased. RECK mRNA was amplified by RT-PCR from whole enamel organ. Here, we verified the presence of RECK mRNA during all stages of amelogenesis. These events were governed by ameloblasts and by non-epithelial cells residents in the enamel organ. Concluding, we found differential expression of MMPs-2, -9 and RECK in the different phases of amelogenesis, suggesting that the tissue remodeling is rigorously controlled during dental mineralization.

Localization of neurotransmitters and calcium binding proteins to neurons of salamander and mudpuppy retinas

We wished to identify the different types of retinal neurons on the basis of their content of neuroactive substances in both larval tiger salamander and mudpuppy retinas, favored species for electrophysiological investigation. Sections and wholemounts of retinas were labeled by immunocytochemical methods to demonstrate three calcium binding protein species and the common neurotransmitters, glycine, GABA and acetylcholine. Double immunostained sections and single labeled wholemount retinas were examined by confocal microscopy. Immunostaining patterns appeared to be the same in salamander and mudpuppy. Double and single cones, horizontal cells, some amacrine cells and ganglion cells were strongly calbindin-immunoreactive (IR). Calbindin-IR horizontal cells colocalized GABA. Many bipolar cells, horizontal cells, some amacrine cells and ganglion cells were strongly calretinin-IR. One type of horizontal cell and an infrequently occurring amacrine cell were parvalbumin-IR. Acetylcholine as visualized by ChAT-immunoreactivity was seen in a mirror-symmetric pair of amacrine cells that colocalized GABA and glycine. Glycine and GABA colocalized with calretinin, calbindin and occasionally with parvalbumin in amacrine cells. (C) 2001 Elsevier Science Ltd. All rights reserved.

Functional development of the inferior colliculus (IQ and its relationship with the auditory brainstem response (ABR) in the tammar wallaby (Macropus eugenii)

To discover the developmental relationship between the auditory brainstem response (ABR) and the focal inferior colliculus (IC) response, 32 young tammar wallabies were used, by the application of simultaneous ABR and focal brainstem recordings, in response to acoustic clicks and tone bursts of seven frequencies. The ic or the tammar wallaby undergoes a rapid functional development from postnatal day (PND) 114 to 160. The earliest (PND 114) auditory evoked response was recorded from the rostral IC. With development, more caudal parts of the IC became functional until age about PND 127, when all parts of the IC were responsive to sound. Along a dorsoventral direction, the duration of the IC response decreased, the peak latency shortened, while the amplitude increased, reaching a maximum value at the central IC, then decreased. After PND 160, the best frequency (BF) of the ventral IC was the highest, with values between 12.5 and 16 kHz, the BF of the dorsal IC was the lowest, varying between 3.2 and 6.4 kHz, while the BF of the central IC was between 6.4 and 12.5 kHz. Between PND 114 and 125, the IC response did not have temporal correlation with the ABR. Between PND 140 and 160, only the early components of the responses from the ventral and central IC correlated with the P4 waves of the ABR. After PND 160, responses recorded from different depths of the IC had a temporal correlation with the ABR. (C) 2001 Published by Elsevier Science B.V.

Differential perturbation of neuronal and glial glutamate transport systems in retinal ischaemia

Glutamate is the major excitatory neurotransmitter in the retina and is removed from the extracellular space by an energy-dependent process involving neuronal and glial cell transporters. The radial glial Muller cells express the glutamate transporter, GLAST, and preferentially accumulate glutamate. However, during an ischaemic episode, extracellular glutamate concentrations may rise to excitotoxic levels. Is this catastrophic rise in extracellular glutamate due to a failure of GLAST? Using immunocytochemistry, we monitored the transport of the glutamate transporter substrate, D-aspartate, in the retina under normal and ischaemic conditions. Two models of compromised retinal perfusion were compared: (1) Anaesthetised rats had their carotid arteries occluded for 7 days to produce a chronic reduction in retinal blood flow. Retinal function was assessed by electroretinography. D-aspartate was injected into the eye for 45 min, Following euthanasia, the retina was processed for D-aspartate. GLAST and glutamate immunocytochemistry. Although reduced retinal perfusion suppresses the electroretinogram b-wave, neither retinal histology, GLAST expression, nor the ability of Muller cells to uptake D-aspartate is affected. As this insult does not appear to cause excitotoxic neuronal damage, these data suggest that GLAST function and glutamate clearance are maintained during periods of reduced retinal perfusion. (2) Occlusion of the central retinal artery for 60 min abolishes retinal perfusion, inducing histological damage and electroretinogram suppression. Although GLAST expression appears to be normal. its ability to transport D-aspartate into Muller cells is greatly reduced. Interestingly, D-aspartate is transported into neuronal cells, i.e. photoreceptors, bipolar and ganglion cells. This suggests that while GLAST is vitally important for the clearance of excess extracellular glutamate, its capability to sustain inward transport is particularly susceptible to an acute ischaemic attack. Manipulation of GLAST function could alleviate the degeneration and blindness that result from ischaemic retinal disease. (C) 2001 Elsevier Science Ltd, All rights reserved.