Superior Cervical Ganglion Research Articles

The Positive Effects of Poria cocos Extract on Quality of Sleep in Insomnia Rat Models.

Sleep disorders may have various causes and can incur mental and/or physical symptoms, and affect an individual’s quality of life. In this study, we confirm that the Poria cocos extract (PCET) can improve sleep quality and structure by promoting inhibitory neurotransmission via the γ-aminobutyric acid (GABA) type A (GABAA) receptors based on the mechanisms revealed in the experiment with superior cervical ganglion neurons. Pentobarbital-induced sleep tests were conducted in order to determine whether the PCET extract improves the sleep quality and structure in normal ICR mice. Sleep latency and duration were checked with the righting reflex. To simulate the state of awakening as well as a normal sleep state, caffeine was administered orally before the PCET diet. After oral gavage of PCET, sleep latency was decreased, and total sleep duration was increased in normal and caffeine-induced sleep disturbance state. In the ACTH-induced sleep disturbed models, administration of PCET significantly reduced the sleep latency and increased the non-REM sleep duration, which was analyzed in real-time EEG by implanting wireless electrodes in SD rats. PCET was found to improve the sleep quality under a normal sleep state through the GABAA receptor; it also promoted and improved the sleep quality and sleep structure in both the arousal activation state and stress-based sleep disturbance.

Implication of P2Y12 receptor in uc.48+-mediated abnormal sympathoexcitatory reflex via superior cervical ganglia in myocardial ischemic rats

Purinergic 2Y12 (P2Y12) receptor antagonists are used as platelet aggregation inhibitors. Long non-coding RNAs (lncRNAs) play an important role in neuropathological events. Satellite glial cells (SGCs) in the superior cervical ganglia (SCGs) encircle the somata of neurons. This study explored if the upregulated P2Y12 receptor in SCGs was relevant to lncRNA uc.48+ during myocardial ischemia (MI). The results showed that upregulation of P2Y12 receptor was accompanied by increased expression of uc.48+ in the SCGs of MI rats which displayed abnormal changes in cervical sympathetic nerve activity, blood pressure, heart rate, electrocardiograms and cardiac tissue structure. The P2Y12 antagonist clopidogrel improved abnormal alterations in cardiac function and tissue structure in MI rats. Short hairpin RNA (shRNA) against uc.48+ significantly inhibited P2Y12 receptor upregulation and its co-expression with glial fibrillary acidic protein (GFAP) in SCGs, and ameliorated the cardiac dysfunction in MI rats. By contrast, overexpression of uc.48+ increased the expression of P2Y12 in SCGs and enhanced cervical sympathetic nerve activity in control rats. Direct interaction between uc.48+ and the P2Y12 receptor was predicted using the bioinformatic tool CatRAPID and confirmed by RNA immunoprecipitation. Moreover, overexpression of the P2Y12 receptor reversed the protective effect of uc.48+ shRNA on cardiac dysfunction in MI rats. Uc.48 shRNA treatment also inhibited the enhanced rise of intracellular free Ca2+ level ([Ca2+]i) evoked by the P2Y12 agonist 2-methylthio-adenosine-5′-diphosphate (2-MeSADP) in SGCs of SCGs after oxygen-glucose deprivation (OGD) treatment. These data demonstrated that uc.48+ shRNA could counteract the P2Y12 upregulation and improve P2Y12-implicated cardiac dysfunction due to MI.

Protective Felix Culpa Effect of Superior Sympathetic Cervical Ganglion Degenerations on Prevention of Basilar Artery Spasm After Subarachnoid Hemorrhage: A Preliminary Experimental Study

Posterior cerebral blood flow is regulated by the basilar arteries (BAs). Vasospasm of BAs can occur after subarachnoid hemorrhage (SAH). Superior cervical sympathetic ganglia (SCG) fibers have a vasoconstrictor effect on the BA. We aimed to investigate the relationship between the degenerated neuron density of the SCG and the severity of BA vasospasm after experimental SAH. Twenty-four rabbits were used. Five were used as the control group, and 5 were used as the sham group. Experimental SAHs were performed in the remaining 14 animals (study group) by injecting homologous blood into the cisterna magna. After 3 weeks of injection, neuron densities of SCG and the severity of BA vasospasm index values (VSI) were examined histopathologically and compared statistically. The mean VSI was 0.669 ± 0.1129 in the control group, 0.981 ± 0.159 in the sham group, and 1.512 ± 0.298 in the study group. The mean degenerated neuronal density of SCG was 436 ± 79/mm3 in severe vasospasm (n= 3), 841 ± 101/mm3 in moderate vasospasm (n= 4), and 1.921 ± 849/mm3 in the less vasospasm detected animals (n= 6). This study shows an inverse relationship between the degenerated neuronal density in the SCG and VSI values. This finding indicates a diminished sympathetic input from the SCG, resulting in a beneficial effect (the felix culpa) by dilating the lumen diameter of the BA, so SCG degeneration after SAH protects the BA spasm.

Nearest Neighbor Density Analysis of the Stellate Ganglia

IntroductionThe so‐called heart‐brain axis is the neurological and physiological connection between the autonomic systems of both organs. The stellate ganglia and superior cervical ganglia are the location of the sympathetic neuronal cells that innervate the vasculature of the heart and brain, respectively. The paravertebral chain connects the ganglia on either side of the neck. Myocardial infarction is thought to cause remodeling of the sympathetic neurons in the stellate ganglia. In a recent qualitative study, the stellate ganglia were shown to remodel with regard to cell body number, size, density, and clustering in the presence of various cardiac diseases. We developed a tool to automatically quantify the nearest neighbor density of neurons weighted by size in pathology specimens of the stellate ganglia. This is the first step to quantifying sympathetic nervous system changes in the heart‐brain axis of cardiovascular pathology.MethodsBiopsies of the stellate ganglia were collected bilaterally from an opportunity sample of 17 willed body donors (34 total specimens) with and without histories of self‐reported myocardial infarction (MI) and/or cerebrovascular accident (CVA). The ganglia were stained with luxol fast blue and cresyl violet which allowed for histological analysis of local neurons. Images of the ganglia at its middle coronal slice after staining were processed using multiple libraries in python, primarily PIL and OpenCV. The images first underwent multiple filters: a high contrast filter and a smoothing filter. After this filter, the image passed through a color threshold that obtained the location of each neuron; the locations of each neuron were used to make a binary image. The binary image was passed through various parameters such as neuron area, radius of the minimum enclosing circle, and a ratio of the area of the neuron and area of the minimum enclosing circle. A heatmap was generated from this last image through a convolution calculation that takes into account the location of the neuron, size, and proximity of surrounding neurons‐ all variables that are needed to calculate nearest neighbor density. Quantitative and qualitative measurements are generated from this: a graphical map of high and low density areas and number of neurons per given threshold of density.ResultsA pipeline was created that generates heatmaps of the nearest neighbor density based on the location and size of neurons on the stellate ganglia. Quantitative data is also measured based on how many neurons are located in varying levels of density structures.ConclusionsHeatmaps of the nearest neighbor density and size of the neurons potentially show signs of cardiac diseases‐ regions of high density or patterns of where highly dense regions are located are indicators. Developments within computation pathology is an easily accessible and pioneering way for new technological innovation.

Single-Cell Quantification of Triple-AAV Vector Genomes Coexpressed in Neurons.

Adeno-associated viruses (AAVs) are one of the most widely used types of viral vectors for research and gene therapy. AAV vectors are safe, have a low immunogenic profile, and provide efficient and long-term transgene expression in a variety of tissues and organs targeted by a specific serotype. Despite these unique features, therapeutic applications, as well as basic research studies, of AAVs have been limited by their packaging capacity of less than 5 kb. Multiple strategies have been explored to deliver large genes. One strategy is to split large transgenes into two or three fragments and package them into separate AAV capsids, generating dual or triple AAV vectors. Combining the fragments potentially allows reconstitution of an mRNA transcript containing the complete sequence of transgene in the same cell. The success of AAVs as vectors for the delivery of large or multiple genes depends directly on the efficiency of co-transduction. Here, we describe a method to measure the efficacy of codelivery, quantifying the number of AAV vectors per cell. We detail how to calculate the average number of incoming AAV genomes in neurons, given the distribution of cell fluorescence across in vitro and in vivo experimental models. To validate the method, we simulated a triple AAV strategy using three fluorescent-protein-encoding genes. We provide a general protocol for constructing plasmids and producing and purifying AAV vectors. We also include a protocol for triple AAV vector co-transduction in primary neuronal cultures and mouse brain. The method can be applied to multiple organs and tissues for the treatment of disorders caused by mutations in multiple or large genes. These protocols will be useful for researchers working to develop and improve new gene delivery technologies. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Construction of AAV plasmids and production of AAVs Basic Protocol 2: AAV transduction of primary superior cervical ganglia (SCG) neuronal cultures Basic Protocol 3: Mouse surgery, AAV injection, and tissue collection and processing Basic Protocol 4: Image analysis and AAV genome quantification.

The Origin of Sympathetic Postganglionic Fibers in the Cervical Region of the Vagus Nerve

IntroductionThe vagus nerve (VN) is a cranial nerve that is mainly known for its parasympathetic function. Although a communication between the VN and sympathetic ganglia was described as early as 1858 by Henry Gray, the existence of sympathetic fibers within the VN is still under debate. Studies utilizing immunohistochemical verification with anti‐Tyrosine Hydroxylase (TH) antibody have found variable distribution of catecholaminergic activity in the cervical and thoracic trunks of the VN, ranging from 0% to 21.63%. We aimed to elucidate the presence and distribution of the postganglionic sympathetic element within the cervical region of the VN using immunohistochemical analysis with anti‐TH antibody. We evaluated macroscopic neural communications between cervical sympathetic chain ganglia (SCG) and the cervical VN as a potential source of intravagal catecholaminergic fibers.MethodsDissection of the VN, cervical chain ganglia, and any communicating branches was performed bilaterally on 20 fully embalmed cadavers (male n=11, female n=9) using standard dissection tools. All communications were histologically validated to confirm neural tissue and cervical VN samples were collected bilaterally in two locations. Tissue sections were stained with anti‐TH antibody and Luxol Fast Blue. Histological slides were viewed and photographed with a Leica DM 500 compound microscope. All images underwent quantitative analysis with ImageJ software to measure effective surface area (ESA) and TH‐positive areas.ResultsTwenty pairs of VN and SCG with associated neural communications were identified. Interconnections were found in 11 donors (55%), resulting in 13 total neural anastomoses between the VN and either the superior, middle, or inferior cervical ganglia. All measurements are reported as mean ± SEM. When comparing the right and left superior cervical vagus nerve ESA, the right side had a larger ESA than the left (2.27±0.26mm2 and 1.82±0.15mm2; p=0.039). TH Immunoreactivity was detected in all donors bilaterally. We observed TH positive fibers in three distribution patterns (Figure 1). There was a significant interaction of location and gender on the percentage of TH‐positive fibers. Females expressed a higher percentage of TH‐positive fibers on the right side (3.0 ±1.22% in females and 1.2 ±0.37% in males; p=0.044). The average ESA of communications on the right side was 0.15±0.07 mm2 and 0.04±0.009 mm2 on the left (p>0.05). The number of fascicles within each communication varied from one to seven (2.77±0.4956). Most fibers stained positive for TH, occupying 91.86% ±4.1% of the total ESA. Presence or size of communications did not influence number of TH positive fibers within the VN. We identified ganglion cells within the superior and inferior samples of the cervical VN (Figure 2).ConclusionTH‐positivity within the cervical VN and its communications with the SCG suggests a ganglionic sympathetic source. However, identification of ganglion cells within the VN suggests a postganglionic source that warrants further study. Investigating sympathetic activity of the VN and its source, aids in understanding its physiologic role.

The Role of the Superior Cervical Sympathetic Ganglion in Ischemia Reperfusion-Induced Acute Kidney Injury in Rats.

Acute kidney injury (AKI) has been found to be a serious clinical problem with high morbidity and mortality, and is associated with acute inflammatory response and sympathetic activation that subsequently play an important role in the development of AKI. It is well known that the sympathetic nervous system (SNS) and immune system intensely interact and mutually control each other in order to maintain homeostasis in response to stress or injury. Evidence has shown that the superior cervical sympathetic ganglion (SCG) participates in the bidirectional network between the immune and the SNS, and that the superior cervical ganglionectomy has protective effect on myocardial infarction, however, the role of the SCG in the setting of renal ischemic reperfusion injury has not been studied. Here, we sought to determine whether or not the SCG modulates renal ischemic reperfusion (IR) injury in rats. Our results showed that bilateral superior cervical ganglionectomy (SCGx) 14 days before IR injury markedly reduced the norepinephrine (NE) in plasma, and down-regulated the increased expression of tyrosine hydroxylase (TH) in the kidney and hypothalamus. Sympathetic denervation by SCGx in the AKI group increased the level of blood urea nitrogen (BUN) and kidney injury molecule-1 (KIM-1), and exacerbated renal pathological damage. Sympathetic denervation by SCGx in the AKI group enhanced the expression of pro-inflammatory cytokines in plasma, kidney and hypothalamus, and increased levels of Bax in denervated rats with IR injury. In addition, the levels of purinergic receptors, P2X3R and P2X7R, in the spinal cord were up-regulated in the denervated rats of the IR group. In conclusion, these results demonstrate that the sympathetic denervation by SCGx aggravated IR-induced AKI in rats via enhancing the inflammatory response, thus, the activated purinergic signaling in the spinal cord might be the potential mechanism in the aggravated renal injury.

Naringin Relieves Diabetic Cardiac Autonomic Neuropathy Mediated by P2Y14 Receptor in Superior Cervical Ganglion.

Diabetes mellitus (DM), an emerging chronic epidemic, contributes to mortality and morbidity around the world. Diabetic cardiac autonomic neuropathy (DCAN) is one of the most common complications associated with DM. Previous studies have shown that satellite glial cells (SGCs) in the superior cervical ganglia (SCG) play an indispensable role in DCAN progression. In addition, it has been shown that purinergic neurotransmitters, as well as metabotropic GPCRs, are involved in the pathophysiological process of DCAN. Furthermore, one traditional Chinese medicine, naringin may potently alleviate the effects of DCAN. Ferroptosis may be involved in DCAN progression. However, the role of naringin in DCAN as well as its detailed mechanism requires further investigation. In this research, we attempted to identify the effect and relevant mechanism of naringin in DCAN mitigation. We observed that compared with those of normal subjects, there were significantly elevated expression levels of P2Y14 and IL-1β in diabetic rats, both of which were remarkably diminished by treatment with either P2Y14 shRNA or naringin. In addition, abnormalities in blood pressure (BP), heart rate (HR), heart rate variability (HRV), sympathetic nerve discharge (SND), and cardiac structure in the diabetic model can also be partially returned to normal through the use of those treatments. Furthermore, a reduced expression of NRF2 and GPX4, as well as an elevated level of ROS, were detected in diabetic cases, which can also be improved with those treatments. Our results showed that naringin can effectively relieve DCAN mediated by the P2Y14 receptor of SGCs in the SCG. Moreover, the NRF2/GPX4 pathway involved in ferroptosis may become one of the principal mechanisms participating in DCAN progression, which can be modulated by P2Y14-targeted naringin and thus relieve DCAN. Hopefully, our research can supply one novel therapeutic target and provide a brilliant perspective for the treatment of DCAN.

Low-input Nucleus Isolation and Multiplexing with Barcoded Antibodies of Mouse Sympathetic Ganglia for Single-nucleus RNA Sequencing.

The cardiac autonomic nervous system is crucial in controlling cardiac function, such as heart rate and cardiac contractility, and is divided into sympathetic and parasympathetic branches. Normally, there is a balance between these two branches to maintain homeostasis. However, cardiac disease states such as myocardial infarction, heart failure, and hypertension can induce the remodeling of cells involved in cardiac innervation, which is associated with an adverse clinical outcome. Although there are vast amounts of data for the histological structure and function of the cardiac autonomic nervous system, its molecular biological architecture in health and disease is still enigmatic in many aspects. Novel technologies such as single-cell RNA sequencing (scRNA-seq) hold promise for the genetic characterization of tissues at single-cell resolution. However, the relatively large size of neurons may impede the standardized use of these techniques. Here, this protocol exploits droplet-based single-nucleus RNA sequencing (snRNA-seq), a method to characterize the biological architecture of cardiac sympathetic neurons in health and disease. A stepwise approach is demonstrated to perform snRNA-seq of the bilateral superior cervical (SCG) and stellate ganglia (StG) dissected from adult mice. This method enables long-term sample preservation, maintaining an adequate RNA quality when samples from multiple individuals/experiments cannot be collected all at once within a short period of time. Barcoding the nuclei with hashtag oligos (HTOs) enables demultiplexing and the trace-back of distinct ganglionic samples post sequencing. Subsequent analyses revealed successful nuclei capture of neuronal, satellite glial, and endothelial cells of the sympathetic ganglia, as validated by snRNA-seq. In summary, this protocol provides a stepwise approach for snRNA-seq of sympathetic extrinsic cardiac ganglia, a method that has the potential for broader application in studies of the innervation of other organs and tissues.

Urocortin-positive nerve fibres and cells are present in the human choroid

BackgroundChoroidal vascular regulation is mediated by the autonomic nervous system in order to gain proper blood flow control. While the mechanisms behind this control are unknown, neuroregulatory peptides are involved...

Cannabidiol activates neuronal Kv7 channels.

Cannabidiol (CBD), a chemical found in the Cannabis sativa plant, is a clinically effective antiepileptic drug whose mechanism of action is unknown. Using a fluorescence-based thallium flux assay, we performed a large-scale screen and found enhancement of flux through heterologously expressed human Kv7.2/7.3 channels by CBD. Patch-clamp recordings showed that CBD acts at submicromolar concentrations to shift the voltage dependence of Kv7.2/7.3 channels in the hyperpolarizing direction, producing a dramatic enhancement of current at voltages near -50 mV. CBD enhanced native M-current in mouse superior cervical ganglion starting at concentrations of 30 nM and also enhanced M-current in rat hippocampal neurons. The potent enhancement of Kv2/7.3 channels by CBD may contribute to its effectiveness as an antiepileptic drug by reducing neuronal hyperexcitability.

The sympathetic nervous system exacerbates carotid body sensitivity in hypertension.

The carotid bodies (CBs) of spontaneously hypertensive (SH) rats exhibit hypertonicity and hyperreflexia contributing to heightened peripheral sympathetic outflow. We hypothesized that CB hyperexcitability is driven by its own sympathetic innervation. To test this, the chemoreflex was activated (NaCN 50-100 µL, 0.4 µg/µL) in SH and Wistar rats in situ before and after: (i) electrical stimulation (ES; 30 Hz, 2 ms, 10 V) of the superior cervical ganglion (SCG), which innervates the CB; (ii) unilateral resection of the SCG (SCGx); (iii) CB injections of an α1-adrenergic receptor agonist (phenylephrine, 50 µL, 1 mmol/L), and (iv) α1-adrenergic receptor antagonist prazosin (40 µL, 1 mmol/L) or tamsulosin (50 µL, 1 mmol/L). ES of the SCG enhanced CB-evoked sympathoexcitation by 40-50% (P < 0.05) with no difference between rat strains. Unilateral SCGx attenuated the CB-evoked sympathoexcitation in SH (62%; P < 0.01) but was without effect in Wistar rats; it also abolished the ongoing firing of chemoreceptive petrosal neurones of SH rats, which became hyperpolarized. In Wistar rats, CB injections of phenylephrine enhanced CB-evoked sympathoexcitation (33%; P < 0.05), which was prevented by prazosin (26%; P < 0.05) in SH rats. Tamsulosin alone reproduced the effects of prazosin in SH rats and prevented the sensitizing effect of the SCG following ES. Within the CB, α1A- and α1B-adrenoreceptors were co-localized on both glomus cells and blood vessels. In conscious SH rats instrumented for recording blood pressure (BP), the CB-evoked pressor response was attenuated after SCGx, and systolic BP fell by 16 ± 4.85 mmHg. The sympathetic innervation of the CB is tonically activated and sensitizes the CB of SH but not Wistar rats. Furthermore, sensitization of CB-evoked reflex sympathoexcitation appears to be mediated by α1-adrenoceptors located either on the vasculature and/or glomus cells. The SCG is novel target for controlling CB pathophysiology in hypertension.

Порушення фізіологічних функцій нейронів верхнього шийного ганглія при цукровому діабеті

A large number of extra- and intramural ganglia in humans and animals exist. All pathways of central regulation of vegetative functions and peripheral reflex pathways pass through them, providing coordinated automatic activity of many organs and tissues. It is well known that sympathetic and sensory neurons are affected in the early stages of diabetes. Patients with diabetes often have autonomic neuropathies. They suffer from disorders of the cardiovascular system and vessels functions, from disorders of the thermoregulatory and pupilomotor functions. These disorders may be the result of the superior cervical ganglion neurons functional defects. This ganglion involves in homeostasis, innervates pineal gland, thyroid, vascular plexus, vestibular system, pupillary, carotid bodies, salivary and lacrimal glands, innervates vessels of the skull and brain. The superior cervical ganglion’s postganglionic axons also innervate the heart. However, disorders of the synaptic transmission in superior cervical ganglion neurons in diabetes remain insufficiently studied to date. Therefore, this article is about the physiological properties of the superior cervical ganglion neurons and their pathological changes in diabetes mellitus. The works about synaptic neurotransmission disorders in superior cervical ganglion neurons of animals with experimental diabetes mellitus are analyzed.

Pineal Gland Culture.

Pineal gland secretes the hormone melatonin at night with a circadian rhythm. The synthesis and secretion of melatonin are stimulated at night by norepinephrine released by sympathetic postganglionic neurons projecting from the superior cervical ganglia. Norepinephrine simultaneously activates α- and β-adrenoceptors, triggering melatonin synthesis.To study the regulation of melatonin production and secretion, it is very convenient to use an ex vivo preparation. Thus, it is possible to keep intact pineal glands in culture and to study the actions of agonists, antagonists, modulators, toxic agents, etc., in melatonin synthesis. Artificial melatonin synthesis stimulation in vitro is usually achieved by using a β-adrenergic agonist alone or in association with an α-adrenergic agonist. In this chapter, the methodology of cultured pineal glands will be described. Several papers were published by our group using this methodology, approaching the role played in melatonin synthesis control by angiotensin II and IV, insulin, glutamate, voltage-gated calcium channels, anhydroecgonine methyl ester (AEME, crack-cocaine product), monosodium glutamate (MSG), signaling pathways like NFkB, pathophysiological conditions like diabetes, etc.

Methods and Applications of Campenot Trichamber Neuronal Cultures for the Study of Neuroinvasive Viruses.

The development of compartmentalized neuron culture systems has been invaluable in the study of neuroinvasive viruses, including the alpha herpesviruses Herpes Simplex Virus 1 (HSV-1) and Pseudorabies Virus (PRV). This chapter provides updated protocols for assembling and culturing rodent embryonic superior cervical ganglion (SCG) and dorsal root ganglion (DRG) neurons in Campenot trichamber cultures. In addition, we provide several illustrative examples of the types of experiments that are enabled by Campenot cultures: (1) Using fluorescence microscopy to investigate axonal outgrowth/extension through the chambers, and alpha herpesvirus infection, intracellular trafficking, and cell-cell spread via axons. (2) Using correlative fluorescence microscopy and cryo electron tomography to investigate the ultrastructure of virus particles trafficking in axons.

Surgical Techniques and Nuances for Superior Cervical Ganglionectomy and Decentralization in Rats.

The sympathetic nervous system has been implicated in various physiological and pathological processes, including regulation of homeostatic functions, maintenance of the circadian rhythms, and neuronal disruption and recovery after injury. Of special interest is focus on the role of the superior cervical ganglion (SCG) in regulating the daily changes in pineal function. Removal of the superior cervical ganglion (SCGx) and decentralization have served as valuable microsurgical models to investigate the effects of surgical denervation on this glandor organ. In this chapter, we offer information about methodologies for performing SCGx along with decentralization and denervation procedures, including details about recommended equipment as well as tips that can improve these techniques.

Sıçan Subaraknoid Kanama Modelinde Bilateral Superior Servikal Ganglionektomi ve Melatonin Seviyeleri: Basit Önlemler Melatonin Düzeylerini Koruyabilir

Aim: Subarachnoid hemorrhage (SAH) is a serious disease, and it is thought that melatonin may have positive effects after SAH. Bilateral resection or blockage of superior cervical ganglions has constant effects on melatonin levels. Animal models with bilateral superior cervical ganglionectomy (SCG) show the role of superior cervical ganglion on melatonin and give clues about simple precautions which may help to prevent unfavorable outcomes in SAH patients. The aim of this study is to examine how melatonin levels change in SAH and SCG models. Material and Methods: Forty-two Sprague Dawley male rats weighing 200-250 g were used in the study and randomly divided into six groups. Arterial blood samples were collected 24 hours after the procedure in all groups. Serum melatonin levels of the groups were studied. Results: A significant difference in blood melatonin levels was observed between SAH and SCG groups, and against the control group. There was no significant difference between the melatonin levels in SCG group and SAH+SCG group (p=0.983). The mean blood melatonin level of the SAH group was higher than the SCG (p&amp;lt;0.001), SAH+SCG (p&amp;lt;0.001) and control groups (p=0.001). The mean blood melatonin levels of SAH+SCG and SCG groups were lower than the mean blood melatonin levels of the other groups and also the SAH group (p&amp;lt;0.001). Conclusion: Bilateral SCG significantly inhibited the abrupt increase of serum melatonin levels after SAH model in rats. Future studies aiming to address melatonin’s complex outcomes should take into account that minor exogenous factors may affect serum melatonin levels.

The Anterolateral Cervical Atlanto-Occipital Plexus: A Novel Finding with Application to Skull Base and Spine Surgery and Pain Disorders of the Head and Neck

We have described a novel nerve plexus of the upper neck. By exploring how the individual components of this specific anterolateral nerve plexus communicate with each other, patient care could be improved regarding the preoperative diagnosis, intraoperative navigation, and minimally invasive treatment. Using a surgical microscope, 11 adult cadaveric heads (22 cadaveric hemi-sides) were dissected. The region of the junction between the atlanto-occipital and craniocervical junctions was explored, specifically, the innervation of this region via adjacent nerve connections. Branches from these regional nerve sources were analyzed for interconnections, and, when found, these branches were documented and measured. A delicate nerve plexus was found overlying the anterolateral C1-C2 junction in all specimens. The plexus was contributed by the sympathetic trunk, vagus nerve, hypoglossal nerve, and C1 and C2 ventral rami. We termed this plexus the anterolateral cervical atlanto-occipital (ALCAO) plexus. On all but 2 cadaveric hemi-sides (91%), the C2 ventral ramus provided the most input into the plexus, with 1-2 branches. On 2 cadaveric hemi-sides, the C1 ventral ramus was the primary contributor and, on average, this nerve contributed 1-2 branches to the plexus. For 8 cadaveric hemi-sides (36.4%), the C1 fibers that are known to travel with the hypoglossal nerve and be distributed to the geniohyoid and thyrohyoid muscles arose from C1 nerve fibers that first traversed the ALCAO plexus. The sympathetic trunk contributed 1-4 lateral branches, with most of these arising superiorly from the superior cervical ganglion. The vagus nerve contributed 1-2 lateral branches and the hypoglossal nerve contributed 1-2 anteromedial branches. This plexus was located more or less lateral to the sympathetic trunk and superior cervical ganglion and medial to the transverse process of C1 and C2. The plexus innervated the rectus capitis lateralis, rectus capitis anterior, and lateral atlanto-occipital joint and, on 4 cadaveric hemi-sides, the atlantoaxial joint. Additionally, small branches were seen traveling to the anterior atlantoaxial and anterior atlanto-occipital membranes on 55% and 77.2% of the cadaveric hemi-sides, respectively. On 6 hemi-sides, very small branches from the ALCAO plexus ended in the periosteum over the anterolateral aspect of the anterior arch and transverse process of the C1 vertebra. It is important to recognize that the course of these interneural connections varies and could result in unforeseen complications during surgical procedures. A comprehensive knowledge of these neural connections will be useful when considering surgery and evaluating pathology of the neck and skull base.

Differential Regulation of Neurite Outgrowth and Growth Cone Morphology by 3D Fibronectin and Fibronectin-Collagen Extracellular Matrices.

The extracellular matrix (ECM) plays a critical role in development, homeostasis, and regeneration of tissue structures and functions. Cell interactions with the ECM are dynamic and cells respond to ECM remodeling by changes in morphology and motility. During nerve regeneration, the ECM facilitates neurite outgrowth and guides axons with target specificity. Decellularized ECMs retain structural, biochemical, and biomechanical cues of native ECM and have the potential to replace damaged matrix to support cell activities during tissue repair. To determine the ECM components that contribute to nerve regeneration, we analyzed neuron-ECM interactions on two types of decellularized ECM. One matrix was composed primarily of fibronectin (FN) fibrils, and the other FN-rich ECM also contained significant numbers of type I collagen (COL I) fibrils. Using primary neurons dissociated from superior cervical ganglion (SCG) explants, we found that neurites were extended on both matrices without a significant difference in average neurite length after 24h. The most distinctive features of neurites on the FN matrix were numerous short actin-filled protrusions and longer branches extending from neurite shafts. Very few protrusions and branches were detected on FN-COL matrix. Growth cone morphologies also differed with mostly filopodial growth cones on FN matrix whereas on FN-COL matrix, equivalent numbers of filopodial and slender growth cones were formed. Our work provides new information about how changes in major components of the ECM during tissue repair modulate neuron and growth cone morphologies and helps to define the contributions of neuron-ECM interactions to nerve development and regeneration.

Extracellular matrix stiffness negatively affects axon elongation, growth cone area and F-actin levels in a collagen type I 3D culture.

Three dimensional (3D) in vitro neuronal cultures can better reproduce physiologically relevant phenotypes compared to 2D-cultures, because in vivo neurons reside in a 3D microenvironment. Interest in neuronal 3D cultures is emerging, with special attention to the mechanical forces that regulate axon elongation and sprouting in three dimensions. Type I collagen (Col-I) is a native substrate since it is present in the extracellular matrix and hence emulates an in vivo environment to study axon growth. The impact of its mechanical properties needs to be further investigated. Here, we generated Col-I 3D matrices of different mechanical stiffness and evaluated axon growth in three dimensions. Superior cervical ganglion (SCG) explants from neonatal rats were cultured in soft and stiff Col-I 3D matrices and neurite outgrowth was assessed by measuring: maximum neuritic extent; neuritic halo area and fasciculation. Axonal cytoskeletal proteins were examined. Axon elongation in stiff Col-I 3D matrices was reduced (31%) following 24h in culture compared to soft matrices. In stiff matrices, neurites fasciculated and formed less dense halos. Consistently, almost no F-actin rich growth cones were recognized, and F-actin staining was strongly reduced in the axonal compartment. This study shows that stiffness negatively affects 3D neurite outgrowth and adds insights on the cytoskeletal responses upon mechanic interactions of axons with a 3D environment. Our data will serve to facilitate the development of model systems that are mechanically well-behaved but still mimic key physiologic properties observed in vivo.