visualizing-morphological-characteristics-neuromuscular-junction-rat

Visualizing the Morphological Characteristics of Neuromuscular Junction in Rat Medial Gastrocnemius Muscle

The neuromuscular junction (NMJ) is a complex structure serving for the signal communication from the motor neuron to the skeletal muscle and consists of ...

3d-visualization-retinal-vascular-pericytes-mice

3D Visualization of Retinal Vascular Pericytes in Mice by Immunostaining 

Retinal pericytes are essential for vascular development and stability of the retina, playing a key role in maintaining the integrity of the retinal ...

China Academy of Chinese Medical Sciences

China Academy Of Chinese Medical Sciences

This study aimed to investigate the sensory and motor innervation of "Taichong" (LR3) and "Ququan" (LR8) in the rat and provide an insight into the neural relationship between the different acupoints in the same meridian.

Comparison of Sensory and Motor Innervation Between the Acupoints LR3 and LR8 in the Rat With Regional Anatomy and Neural Tract Tracing.

Cluster of differentiation 31 (CD31), phalloidin and alpha smooth muscle actin (α-SMA) have been widely applied to label the cerebral blood vessels in the past years. Although CD31 is mainly used as endothelial marker in determining the cerebral capillaries, it seems likely that its labeling efficiency is closely correlated with the antibodies from the polyclonal or monoclonal one, as well as the conditions of blood vessels. In order to test this phenomenon, we compared the labeling characteristics of goat polyclonal anti-CD31 (gP-CD31) and mouse monoclonal anti-CD31 (mM-CD31) with those of phalloidin and α-SMA on the rat brain in health and ischemia/reperfusion (I/R) with the middle cerebral artery occlusion. By multiple immunofluorescence staining, it was found that gP-CD31 labeling expressed extensively on the cerebral capillaries forming the vascular networks on the normal and ischemic regions, but mM-CD31 labeling mainly presented on the capillaries in the ischemic region. In contrast to the vascular labeling with gP-CD31, phalloidin and α-SMA were mainly expressed on the wall of cortical penetrating arteries, and less on that of capillaries. By three-dimensional reconstruction analysis, it was clearly shown that gP-CD31 labeling was mainly located on the lumen side of vascular wall and was surrounded by phalloidin labeling and α-SMA labeling. These results indicate that gP-CD31 is more sensitive than mM-CD31 for labeling the cerebral vasculature, and is highly compatible with phalloidin and α-SMA for evaluating the cerebral vascular networks under the physiological and pathological conditions.

The immunolocalization of cluster of differentiation 31, phalloidin and alpha smooth muscle actin on vascular network of normal and ischemic rat brain.

The mouse model of multiple cerebral infarctions, established by injecting fluorescent microspheres into the common carotid artery, is a recent development in animal models of cerebral ischemia. To investigate its effectiveness, mouse models of cerebral infarction were created by injecting fluorescent microspheres, 45-53 µm in diameter, into the common carotid artery. Six hours after modeling, fluorescent microspheres were observed directly through a fluorescence stereomicroscope, both on the brain surface and in brain sections. Changes in blood vessels, neurons and glial cells associated with microinfarcts were examined using fluorescence histochemistry and immunohistochemistry. The microspheres were distributed mainly in the cerebral cortex, striatum and hippocampus ipsilateral to the side of injection. Microinfarcts were found in the brain regions where the fluorescent microspheres were present. Here the lodged microspheres induced vascular and neuronal injury and the activation of astroglia and microglia. These histopathological changes indicate that this animal model of multiple cerebral infarctions effectively simulates the changes of various cell types observed in multifocal microinfarcts. This model is an effective, additional tool to study the pathogenesis of ischemic stroke and could be used to evaluate therapeutic interventions. This study was approved by the Animal Ethics Committee of the Institute of Acupuncture and Moxibustion, China Academy of Chinese Medical Sciences (approval No. D2021-03-16-1) on March 16, 2021.

Histochemistry of microinfarcts in the mouse brain after injection of fluorescent microspheres into the common carotid artery.

Neural tract tracing is used to study neural pathways and evaluate neuronal regeneration following nerve injuries. However, it is not always clear which tracer should be used to yield optimal results. In this study, we examined the use of Alexa Fluor 488-conjugated cholera toxin subunit B (AF488-CTB). This was injected into the gastrocnemius muscle of rats, and it was found that motor, sensory, and sympathetic neurons were labeled in the spinal ventral horn, dorsal root ganglia, and sympathetic chain, respectively. Similar results were obtained when we injected AF594-CTB into the tibialis anterior muscle. The morphology and number of neurons were evaluated at different time points following the AF488-CTB injection. It was found that labeled motor and sensory neurons could be observed 12 hours post-injection. The intensity was found to increase over time, and the morphology appeared clear and complete 3-7 days post-injection, with clearly distinguishable motor neuron axons and dendrites. However, 14 days after the injection, the quality of the images decreased and the neurons appeared blurred and incomplete. Nissl and immunohistochemical staining showed that the AF488-CTB-labeled neurons retained normal neurochemical and morphological features, and the surrounding microglia were also found to be unaltered. Overall, these results imply that the cholera toxin subunit B, whether unconjugated or conjugated with Alexa Fluor, is effective for retrograde tracing in muscular tissues and that it would also be suitable for evaluating the regeneration or degeneration of injured nerves.

Alexa Fluor 488-conjugated cholera toxin subunit B optimally labels neurons 3-7 days after injection into the rat gastrocnemius muscle.

To visualize and compare the sensory and autonomic innervation of the local tissues at the sites of different traditional acupuncture points in the rat forehead and face by histochemical examination.

Sensory and autonomic innervation of the local tissues at traditional acupuncture point locations GB14, ST2 and ST6.

Microglia, the resident immune cells in the central nervous system, can monitor the microenvironment and actively respond to ischemic stroke and other brain injuries. In this procedure, microglia and neurons can cross-talk  transmembrane chemokine, Fractalkine (CX3CL1), to impact one another. We used a rat model of multifocal microinfarcts induced by the injection of fluorescent microspheres into the right common carotid artery and examined the morphological alteration of blood vessels, microglia, astrocytes, and neurons at 6 h, 1, 7, and 14 days after modeling, along with neurobehavioral tests and the staining of CX3CL1 in this study. Our results demonstrated that in the infarcted regions, astrocytes and microglia activated in response to neuronal degeneration and upregulation of cleaved caspase-3, which occurred concurrently with vascular alteration and higher expression of CX3CL1. We provided sequential histological data to shed light on the morphological changes after modeling, which would help in the identification of new targets and the choice of the ideal time window for therapeutic intervention in ischemic stroke.

Temporal alteration of microglia to microinfarcts in rat brain induced by the vascular occlusion with fluorescent microspheres.

Author Correction: The immunolocalization of cluster of differentiation 31, phalloidin and alpha smooth muscle actin on vascular network of normal and ischemic rat brain.

In order to demonstrate the intricate interconnection of pulmonary lymphatic vessels, blood vessels, and nerve fibers, the rat lung was selected as the target and sliced at the thickness of 100 μm for multiply immunofluorescence staining with lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), alpha smooth muscle actin (α-SMA), phalloidin, cluster of differentiation 31 (CD31), and protein gene product 9.5 (PGP9.5) antibodies. Taking the advantages of the thicker tissue section and confocal microscopy, the labeled pulmonary lymphatic vessels, blood vessels, and nerve fibers were demonstrated in rather longer distance, which was more convenient to reconstruct a three-dimensional (3D) view for analyzing their spatial correlation in detail. It was clear that LYVE-1 lymphatic vessels were widely distributed in pulmonary lobules and closely to the lobar bronchus. Through 3D reconstruction, it was also demonstrated that LYVE-1 lymphatic vessels ran parallel to or around the α-SMA venules, phalloidin arterioles and CD31 capillaries, with PGP9.5 nerve fibers traversing alongside or wrapping around them, forming a lymphatic, vascular and neural network in the lung. By this study, we provide a detailed histological view to highlight the spatial correlation of pulmonary lymphatic, vascular and neural network, which may help us for insight into the functional role of this network under the physiological and pathological conditions.

Three-dimensional visualization of the lymphatic, vascular and neural network in rat lung by confocal microscopy.

Peripheral nerve injury can cause changes in microglial cells on the spinal dorsal and ventral horns. This region-specific response implies that central microglial cells could be activated through both sensory and motor pathways. In order to further determine how peripheral nerve injury activates central microglial cells through neural pathways, the sciatic nerve was selected as the target for neural tract tracing and demyelination. Firstly, we used cholera toxin subunit B (CTB) to map the central sensory and motor territories of the sciatic nerve. Secondly, we applied lysophosphatidylcholine to establish the model of sciatic nerve demyelination and examined the distribution of activated microglial cells via immunofluorescence with ionized calcium-binding adapter molecule 1. It was shown that CTB labeling included the transganglionically labeled sensory afferents and retrogradely labeled somata of motor neurons along the sensory and motor pathways of the sciatic nerve ipsilateral to the injection, in which sensory afferents terminated on the gracile nucleus, Clarke's nucleus, and spinal dorsal horn, while motor neurons located on the spinal ventral horn. Consistently, after sciatic nerve demyelination, the activated microglial cells were observed in the same territories as CTB-labeling, showing shortened processes and enlarged cell bodies. These results support the idea that central microglia might be activated by signals from the demyelinated sciatic nerve through both sensory and motor pathways.

Region-specific response of central microglial cells to sciatic nerve demyelination through sensory and motor pathways.

Ear acupuncture, as a system for treating and preventing diseases through stimulation of points on the auricle, has been systematically introduced during the last 60 years. Although the auricular cartography was described somatotopically as an inverted fetus by Paul Nogier, MD, the underlying mechanism of auricular stimulation remains unclear. The aim of this research was to gain an understanding of the structural basis of auricular stimulation, as well as showing the distribution of the nerve fibers, and the blood and lymphatic vessels.

Networks of Nerve Fibers, and Blood and Lymphatic Vessels in the Mouse Auricle: The Structural Basis of Ear Acupuncture.

Pregabalin is a medication primarily used in the treatment of neuropathic pain and anxiety disorders, owing to its gabapentinoid properties. Pregabalin monotherapy faces limitations due to its variable efficacy and dose-dependent adverse reactions. In this study, we conducted a comprehensive investigation into the potentiation of pregabalin's analgesic effects by dexborneol, a neuroprotective bicyclic monoterpenoid compound. We performed animal experiments where pain models were induced using two methods: peripheral nerve injury, involving axotomy and ligation of the tibial and common peroneal nerves, and incisional pain through a longitudinal incision in the hind paw, while employing a multifaceted methodology that integrates behavioral pharmacology, molecular biology, neuromorphology, and lipidomics to delve into the mechanisms behind this potentiation. Dexborneol was found to enhance pregabalin's efficacy by promoting its transportation to the central nervous system, disrupting self-amplifying vicious cycles via the reduction of HMGB1 and ATP release, and exerting significant anti-oxidative effects through modulation of central lipid metabolism. This combination therapy not only boosted pregabalin's analgesic property but also notably decreased its side effects. Moreover, this therapeutic cocktail exceeded basic pain relief, effectively reducing neuroinflammation and glial cell activation-key factors contributing to persistent and chronic pain. This study paves the way for more tolerable and effective analgesic options, highlighting the potential of dexborneol as an adjuvant to pregabalin therapy.

ArticleTotal : 2	Year
Publication title Cited by 2	2022
Publication title	2024

Article	Year
Comparison of Sensory and Motor Innervation Between the Acupoints LR3 and LR8 in the Rat With Regional Anatomy and Neural Tract Tracing. Frontiers in integrative neuroscience\| PubMed ID: 34539358	2021
The immunolocalization of cluster of differentiation 31, phalloidin and alpha smooth muscle actin on vascular network of normal and ischemic rat brain. Scientific reports\| PubMed ID: 36566295	2022
Histochemistry of microinfarcts in the mouse brain after injection of fluorescent microspheres into the common carotid artery. Neural regeneration research\| PubMed ID: 34472483	2022
Alexa Fluor 488-conjugated cholera toxin subunit B optimally labels neurons 3-7 days after injection into the rat gastrocnemius muscle. Neural regeneration research\| PubMed ID: 35259856	2022
Sensory and autonomic innervation of the local tissues at traditional acupuncture point locations GB14, ST2 and ST6. Acupuncture in medicine : journal of the British Medical Acupuncture Society\| PubMed ID: 35579008	2022
Temporal alteration of microglia to microinfarcts in rat brain induced by the vascular occlusion with fluorescent microspheres. Frontiers in cellular neuroscience\| PubMed ID: 35990892	2022
Author Correction: The immunolocalization of cluster of differentiation 31, phalloidin and alpha smooth muscle actin on vascular network of normal and ischemic rat brain. Scientific reports\| PubMed ID: 36721020	2023
Three-dimensional visualization of the lymphatic, vascular and neural network in rat lung by confocal microscopy. Journal of molecular histology\| PubMed ID: 37755618	2023
Region-specific response of central microglial cells to sciatic nerve demyelination through sensory and motor pathways. Histology and histopathology\| PubMed ID: 38051019	2024
Networks of Nerve Fibers, and Blood and Lymphatic Vessels in the Mouse Auricle: The Structural Basis of Ear Acupuncture. Medical acupuncture\| PubMed ID: 38659726	2024
Dexborneol Amplifies Pregabalin's Analgesic Effect in Mouse Models of Peripheral Nerve Injury and Incisional Pain. Antioxidants (Basel, Switzerland)\| PubMed ID: 39061872	2024

Yuxin Su

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