Oxidative stress damage circumscribed to the central temporal retinal pigment epithelium in early experimental non-exudative age-related macular degeneration.

Non-exudative age-related macular degeneration (NE-AMD) represents the leading cause of blindness in the elderly. The macular retinal pigment epithelium (RPE) lies in a high oxidative environment because its high metabolic demand, mitochondria concentration, reactive oxygen species levels, and macular blood flow. It has been suggested that oxidative stress-induced damage to the RPE plays a key role in NE-AMD pathogenesis. The fact that the disease limits to the macular region raises the question as to why this area is particularly susceptible. We have developed a NE-AMD model induced by superior cervical ganglionectomy (SCGx) in C57BL/6J mice, which reproduces the disease hallmarks exclusively circumscribed to the temporal region of the RPE/outer retina. The aim of this work was analyzing RPE regional differences that could explain AMD localized susceptibility. Lower melanin content, thicker basal infoldings, higher mitochondrial mass, and higher levels of antioxidant enzymes, were found in the temporal RPE compared with the nasal region. Moreover, SCGx induced a decrease in the antioxidant system, and in mitochondria mass, as well as an increase in mitochondria superoxide, lipid peroxidation products, nuclear Nrf2 and heme oxygenase-1 levels, and in the occurrence of damaged mitochondria exclusively at the temporal RPE. These findings suggest that despite the well-known differences between the human and mouse retina, it might not be NE-AMD pathophysiology which conditions the localization of the disease, but the macular RPE histologic and metabolic specific attributes that make it more susceptible to choroid alterations leading initially to a localized RPE dysfunction/damage, and secondarily to macular degeneration.

Superior cervical gangliectomy induces non-exudative age-related macular degeneration in mice.

Non-exudative age-related macular degeneration, a prevalent cause of blindness, is a progressive and degenerative disease characterized by alterations in Bruch's membrane, retinal pigment epithelium, and photoreceptors exclusively localized in the macula. Although experimental murine models exist, the vast majority take a long time to develop retinal alterations and, in general, these alterations are ubiquitous, with many resulting from non-eye-specific genetic manipulations; additionally, most do not always reproduce the hallmarks of human age-related macular degeneration. Choroid vessels receive sympathetic innervation from the superior cervical ganglion, which, together with the parasympathetic system, regulates blood flow into the choroid. Choroid blood flow changes have been involved in age-related macular degeneration development and progression. At present, no experimental models take this factor into account. The aim of this work was to analyze the effect of superior cervical gangliectomy (also known as ganglionectomy) on the choroid, Bruch's membrane, retinal pigment epithelium and retina. Adult male C57BL/6J mice underwent unilateral superior cervical gangliectomy and a contralateral sham procedure. Although superior cervical gangliectomy induced ubiquitous choroid and choriocapillaris changes, it induced Bruch's membrane thickening, loss of retinal pigment epithelium melanin content and retinoid isomerohydrolase, the appearance of drusen-like deposits, and retinal pigment epithelium and photoreceptor atrophy, exclusively localized in the temporal side. Moreover, superior cervical gangliectomy provoked a localized increase in retinal pigment epithelium and photoreceptor apoptosis, and a decline in photoreceptor electroretinographic function. Therefore, superior cervical gangliectomy recapitulated the main features of human non-exudative age-related macular degeneration, and could become a new experimental model of dry age-related macular degeneration, and a useful platform for developing new therapies.

Origins of the sympathetic innervation to the nasal-associated lymphoid tissue (NALT): an anatomical substrate for a neuroimmune connection.

The participation of sympathetic nerve fibers in the innervation of the nasal-associated lymphoid tissues (NALT) was investigated in hamsters. Vesicular monoamine transporter 2 (VMAT2), an established sympathetic marker, is expressed in all neurons of superior cervical ganglia (SCG). In addition, VMAT2 -immunoreactive nerve fibers were localized in the NALT as well as in adjacent anatomical structures of the upper respiratory tract. Unilateral surgical ablation of the SCG abolished VMAT2 innervation patterns ipsilaterally while the contra lateral side is unaffected. These results provide the anatomical substrate for a neuroimmune connection in the NALT.

A standardized surgical technique for rat superior cervical ganglionectomy.

Superior cervical ganglionectomy (SCGx) is a valuable microsurgical model to study the role of the sympathetic nervous system in a vast array of physiological and pathological processes, including homeostatic regulation, circadian biology and the dynamics of neuronal dysfunction and recovery after injury. Despite having several experimental applications in the rat, a thorough description of a standardized procedure has never been published. Here, we provide a brief review of the principal features and experimental uses of the SCGx, the surgical anatomy of the neck and sympathetic cervical chain, and a step-by-step description of how to consistently remove the superior cervical ganglia through the omohyoid muscle or the carotid triangle. Furthermore, we suggest procedures and precautions to be taken during and after surgery to optimize results and describe tools to validate surgical success. We expect that the following standardized and optimized protocol will allow researchers to organize knowledge into a cohesive framework in those areas where the SCGx is applied.

Effect of unilateral superior cervical ganglionectomy on mandibular bone in rats.

OBJECTIVE: To assess the effect of local sympathectomy on mandibular bone during tooth eruption in rats. METHODS: The effect of a unilateral superior cervical ganglionectomy (Gx) on morphometry of ipsi- and contralateral mandible and volumetric bone density (as assessed by peripheral computed tomography) was examined 30 days after surgery. RESULTS: Only a few mandibular morphometric parameters decreased significantly after denervation in rats subjected to a unilateral Gx and a contralateral sham-operation. Mandibular volumetric bone density decreased significantly after sympathetic denervation. In a second experiment, carried out under conditions of unilateral unimpeded eruption of incisors performed ipsilaterally or contralaterally to a unilateral Gx, a significant interaction "denervation x type of eruption" was found for most morphometric parameters. Further analysis indicated higher morphometric indexes in denervated mandibles than in the innervated ones under impeded incisor eruption conditions, and lower morphometric indexes in denervated mandibles than in the innervated ones under unimpeded incisor eruption conditions. Unimpeded eruption augmented total volumetric bone density providing the innervation was intact and caused opposite effects on cortical volumetric bone density in the presence of innervation (increase) or absence of innervation (decrease). Trabecular volumetric bone density decreased significantly after sympathetic denervation. CONCLUSION: The results support a role of the sympathetic nervous system in the regulation of bone remodeling.

Effect of unilateral superior cervical ganglionectomy on mandibular incisor eruption rate in rats.

To assess the effect of sympathectomy on rat tooth eruption, the effect of a unilateral superior cervical ganglionectomy (SCGx) on eruption rate of ipsi- and contralateral lower incisors was examined. Two experiments were performed. In a first experiment, the eruption rate of ipsilaterally denervated incisors was similar to that of contralaterally innervated incisors, when assessed for up to 28 days after surgery. In a second experiment, under conditions of unilateral unimpeded eruption of incisors performed ipsilaterally or contralaterally to a unilateral SCGx, a significantly lower eruption rate of denervated incisors at the impeded eruption side, and a significantly higher eruption rate of denervated incisors at the unimpeded side were observed, when computed every 2 days. Significant differences in individual Student's t tests at every time interval occurred mainly during the first and the last week of examination. When average daily eruption rate was computed in weekly intervals, a significant interaction between SCGx and the side of impeded or unimpeded eruption was found in a factorial ANOVA, that is, for each of the 4 weeks of examination, sympathetically denervated incisors showed lower eruption rates at the impeded eruption side, and higher eruption rates at the unimpeded side. These results indicate that incisor eruption is not modified by a local sympathetic denervation unless the contralateral lower rat incisor is cut out of occlusion.

Effect of unilateral superior cervical ganglionectomy on bone mineral content and density of rat's mandible.

To assess the effect of a local sympathectomy on bone metabolism, the effect of a unilateral superior cervical ganglionectomy (Gx) on growth and bone mineral content and density of the ipsi- and contralateral mandibles was examined in female rats. A significant increase in the hemi-mandibular bone ipsilateral to Gx was found as compared to the contralateral, sham-operated side 30 days, but not 15 days, after surgery. Bone mineral content of the hemi-mandibular bones was significantly lower in the side ipsilateral to Gx in the group of rats killed on the 30th day after surgery. Since no difference in areas between innervated and denervated hemi-mandibles was found, bone mineral density was also significantly lower in the hemi-mandible ipsilateral to Gx. The results further support that a regional sympathectomy causes qualitative alterations in bone modeling and remodeling, leading to bone resorption.

Pineal metabolic reaction to retinal photostimulation in ganglionectomized rats.

The aim of the present work was to test the pineal gland metabolic reactivity to nocturnal retinal short term photic stimulation in superior cervical ganglionectomized rats. The experimental support for this work is the appearance of a transitory post synaptic hyperactivity in the pineal gland, during the anterograde degenerating process of the conarii sympathetic nerve fibers after surgical removal of the cell body. In this situation the pineal gland is deafferented from the peripheral sympathetic nervous system keeping intact, however, the direct central connections to the deep pineal/lamina intercalaris region (DP). The results show a blockade of the pineal noradrenergic stimulatory process due to the retinal photostimulation. The inactivation of N-acetyltransferase led to a true metabolic shift to the oxidative pathway resulting in a decrease of the amount of N-acetylserotonin and an increase of the amount of serotonin, 5-hydroxyindoleacetic acid and 5-hydroxytryptophan. This inhibitory process brought into action by retinal illumination is dependent on the direct central neural connections to the pineal gland, since rats that were lesioned in the DP, previously to ganglionectomy, did not show any alteration on the indolic content of the pineal gland when subjected to nocturnal retinal photostimulation.

Aspectos morfológicos do músculo masseter do coelho após simpatectomia cervical / Morphological aspects of masseter muscle of rabbits after cervical sympatectomy

Este trabalho teve como objetivos analisar o músculo masseter do coelho (O. cuniculus), após a retirada do gânglio simpático cervical superior direito, quanto aos aspectos histológicos, histoquímicos, imuno-histoquímicos e ultraestruturais. Foram utilizados 2 grupos de animais: 30 com simpatectomia e 10 sem simpatectomia - grupo controle. Os coelhos controles foram sacrificados 6, 12, 22 e 32 meses após o início do experimento, e os com simpatectomia, 3, 6, 9, 12, 15 e 18 meses após a retirada do gânglio simpático. Após o sacrifício, foram retirados os músculos masseteres direito e esquerdo dos animais com simpatectomia e controles; foi estudada a porçäo superficial do músculo, subdividida em 3 regiöes: próxima à inserçäo proximal - "anterior", regiäo mediana e próxima à inserçäo distal - "posterior". Os fragmentos foram congelados em n-hexana, e cortes histológicos foram submetidos às seguintes coloraçöes e reaçöes: HE, Tricrômico de Gomori, PAS, SDH, NADH-TR, ATPase miofibrilar (mATPase), Esterase Inespecífica e Imunohistoquímica, utilizando-se anticorpos específicos para a miosina "fast", (tipo IIa) e "slow", (tipo I). Outros fragmentos musculares foram coletados para a análise ultra-estrutural. O músculo masseter dos grupos controles, apresentou aspectos morfológicos similares, em ambos os lados: as fibras musculares mostraram morfologia geral preservada, à microscopia óptica e eletrônica; distribuiçäo em mosaico; frequência maior de fibras tipo IIa, seguida por fibras do tipo I, e menor frequência de fibras IIb. Nos grupos com simpatectomia, foram observadas lesöes nas fibras musculares, nos animais dos grupos de 6, 12 e em maior grau, no grupo de 18 meses. Em geral, houve predominíio de fibras tipo I, nos grupos com 3, 6, 9, 12 e 15 meses; no grupo de 18 meses, houve predomínio de fibras tipo IIa, sendo observada frequência maior de fibras IIb, em relaçäo aos demais grupos, com ou sem simpatectomia.

Diurnal rhythms in ornithine decarboxylase activity and norepinephrine and acetylcholine synthesis and acetylcholine synthesis of rat submaxillary lymph nodes: effect of pinealectomy, superior cervical ganglionectomy and melatonin replacement.

The effect of pinealectomy, superior cervical ganglionectomy and melatonin replacement on diurnal variations in submaxillary lymph node ornithine decarboxylase activity, tyrosine hydroxylase activity and [3H]choline conversion to [3H]acetylcholine were examined in rats subjected to pinealectomy, bilateral superior cervical ganglionectomy or their respective sham-operations, and treated with Freund's complete adjuvant or its vehicle. In both immunized and nonimmunized sham-operated rats, significant diurnal variations in ornithine decarboxylase activity were detectable, with a maximum at 13.00 h (vehicle) or at 17.00 h (Freund's adjuvant). In rats subjected to pinealectomy, ornithine decarboxylase activity decreased by about half, still exhibiting significant diurnal variations with a maximum at 13.00 h. Abolition of circadian rhythmicity and depression of ornithine decarboxylase activity to about one third of controls were found in submaxillary lymph nodes of bilaterally superior cervical ganglionectomized rats. Administration of melatonin (30 micrograms/animal) in the late evening during 11 days counteracted the depressed levels and suppressed the amplitude of diurnal rhythmicity of ornithine decarboxylase in pinealectomized or bilaterally superior cervical ganglionectomized rats, as well as augmented enzyme activity in sham-operated controls. The amplitude and mean levels of 24-hour rhythms in submaxillary lymph node tyrosine hydroxylase activity and [3H]choline conversion to acetylcholine (that attained their maxima at 21.00-1.00 and 13.00-17.00 h, respectively) decreased significantly after pinealectomy, these effects being significantly counteracted by melatonin injection. Melatonin augmented tyrosine hydroxylase activity and acetylcholine synthesis in sham-pinealectomized rats. The results are compatible with the view that the pineal gland plays a role in circadian changes of immune responsiveness in lymphoid tissue via an immunopotentiating effect of melatonin on lymph node cell proliferation.