<em>In Vivo</em> Calcium Imaging of Dorsal Root Ganglia Neurons' Response to Somatic and Visceral Stimuli

A technique is described for surgically exposing the dorsal root ganglion (DRG) of the lumbar-6 in a live, anesthetized laboratory mouse, along with the ...

China Academy Of Chinese Medical Sciences

 Scalp acupuncture is a somatic stimulation therapy that produces prominent clinical effects when used to treat cerebral diseases. However, this acupuncture's therapeutic mechanisms have not yet been well-addressed. Scalp acupoints are innervated by the trigeminal nerve, which is coincident with the intracranial sensory afferents as well as with the meningeal vessels. In recent years, cerebrospinal fluid-contacting neurons have been found and proved to transmit allergic substances between brain the parenchyma and meninges, representing a possible network between scalp acupuncture and the brain. The aim of the current study was to observe the connections between scalp acupoints and the meninges and to establish a possible mechanism for scalp acupuncture.  Twenty-five adult Sprague-Dawley rats were used for the present study. Evans Blue dye (Sigma Chemical Co, St. Louis, MO) was injected though each rat's caudal vein after trigeminal stimulation for plasma extravasation observation. Cerebral blood flow (CBF) values of the rat's brain surface were measured at different timepoints before and after electroacupuncture (EA) on GB 15 () or ST 36 ().  These preliminary studies indicated that neurogenic plasma extravasation on a rat's skin and dura mater after mechanical or electrical stimulation of the trigeminal nerves is a reliable way to show the pathologic connection between scalp acupoints and the meninges. Moreover, CBF of the rat's brain surface is increased significantly after EA stimulation at GB 15 (), which is located in the receptive field of the supraorbital nerve.  These findings suggest that the mechanism of scalp acupuncture might lie in the specific neurologic pathway that could be termed as , which is a possible shortcut to brain functional regulation and cerebral disease treatment.

A Proposed Neurologic Pathway for Scalp Acupuncture: Trigeminal Nerve-Meninges-Cerebrospinal Fluid-Contacting Neurons-Brain.

Peripheral neurostimulation within the trigeminal nerve territory has been used for pain alleviation during migraine attacks, but the mechanistic basis of this non-invasive intervention is still poorly understood. In this study, we investigated the therapeutic role of peripheral stimulation of the trigeminal nerve, which provides homosegmental innervation to intracranial structures, by assessing analgesic effects in a nitroglycerin (NTG)-induced rat model of migraine. As a result of neurogenic inflammatory responses in the trigeminal nervous system, plasma protein extravasation was induced in facial skin by applying noxious stimulation to the dura mater. Noxious chemical stimulation of the dura mater led to protein extravasation in facial cutaneous tissues and caused mechanical sensitivity. Trigeminal ganglion (TG) neurons were double-labeled via retrograde tracing to detect bifurcated axons. Extracellular recordings of wide dynamic range (WDR) neurons in the spinal trigeminal nucleus caudalis (Sp5C) demonstrated the convergence and interaction of inputs from facial tissues and the dura mater. Peripheral neurostimulation of homotopic facial tissues represented segmental pain inhibition on cephalic cutaneous allodynia in the migraine model. The results indicated that facial territories and intracranial structures were directly connected with each other through bifurcated double-labeled neurons in the TG and through second-order WDR neurons. Homotopic stimulation at the C-fiber intensity threshold resulted in much stronger inhibition of analgesia than the same intensity of heterotopic stimulation. These results provide novel evidence for the neurological bases through which peripheral neurostimulation may be effective in treating migraine in clinical practice.

Signaling Interaction between Facial and Meningeal Inputs of the Trigeminal System Mediates Peripheral Neurostimulation Analgesia in a Rat Model of Migraine.

The neurovascular structures in the cranial dura mater have been studied with various histological techniques in the past years. In order to obtain a proper approach to reveal the detailed structures, different labeling methods for the cranial vessels and nerve fibers were tested in this study. Firstly, the labeling characteristics of phalloidin, alpha smooth muscle actin (α-SMA), and CD31 were compared in rat whole-mount cranial dura mater by using fluorescent immunohistochemistry or histochemistry. Secondly, according to their properties, phalloidin and α-SMA were selected to combine with calcitonin gene-related peptide (CGRP) to further demonstrate the cranial neurovascular structure. By these approaches, a three-dimensional map of blood vessels and nerve fibers within the whole-mount rat cranial dura mater was obtained. The results showed that phalloidin, α-SMA, and CD31 were preferably expressed in the wall of cranial vessels, corresponding to the arteriors, venules, and capillaries, respectively. Additionally, CGRP + nerve fibers were clearly demonstrated together with phalloidin + or α-SMA + vessels, forming a delicate neurovascular network in the cranial dura mater. The thick nerve bundles ran closely to the phalloidin + or α-SMA + vessels in parallel pattern, while the thin nerve fibers branched off from the bundles tending to surround the phalloidin + arterioles rather than α-SMA + venules. These findings suggest that phalloidin could be an appropriate biochemical maker to be effectively used together with CGRP for experiments examining the detailed spatial correlation of cranial blood vessels and nerve fibers in a three-dimensional view, which may provide clues for understanding the underlying mechanisms of cranial neurovascular disorders.

A new approach for examining the neurovascular structure with phalloidin and calcitonin gene-related peptide in the rat cranial dura mater.

Scalp acupuncture is widely used in clinical practice, and there is still a lack of systematic studies on its nerve pathways and mechanism of action. Scalp acupoints are innervated by the trigeminal nerve which is also involved in the regulation of the leptomeninges and related arterioles nourishing the cerebral cortex. Animal experiments and human trials have shown that scalp acupuncture can exert an effect through the shortcut between the axon reflexes from the primary neurons in the trigeminal ganglion (its axon branches may separately innervate scalp acupoints and the leptomeninges directly regulating nutrient and active substances in brain tissue) and the secondary neurons of the trigeminal nucleus caudalis (head and face acupoints interact with afferents of brain tissue through presynaptic dorsal root reflex and postsynaptic neurogenic responses and convergence), which may have regulatory and therapeutic effects on intracranial tissue.

[The shortcut pathway between scalp acupuncture and brain].

To observe the effect difference of electroacupuncture (EA) at the sensitized and non-sensitized acupoint "Tianshu" (ST25) on the motility of jejunum in rats.

[Acupoint sensitization enhances inhibitory effect of electroacupuncture on jejunum mobility in rats].

The skin microecology interacts with the immune system for a lifetime, and the skin microecology is in dynamic balance in the physiological state. The flora with the same physical and chemical properties on the surface of the skin can maintain a relatively stable state in a certain period of time, and there are dynamic changes of microflora in both physiology and disease state. Combined with the study of the relationship among skin microorganisms, skin diseases and visceral diseases, the relationship between skin microecological changes and visceral pathological state is explored. On this basis, it is proposed that the sensitized acupoint is an abnormal manifestation of the body homeostasis imbalance on the body surface, and the local substance variation on the sensitization acupoint may induce microbial changes. In terms of treatment, moxibustion and other surface treatments that function through immunity may also involve microorganisms.

[Association between skin microecology and acupoint sensitization].

Myocardial ischemia (MI) causes somatic referred pain and sympathetic hyperactivity, and the role of sensory inputs from referred areas in cardiac function and sympathetic hyperactivity remain unclear. Here, in a rat model, we showed that MI not only led to referred mechanical hypersensitivity on the forelimbs and upper back, but also elicited sympathetic sprouting in the skin of the referred area and C8-T6 dorsal root ganglia, and increased cardiac sympathetic tone, indicating sympathetic-sensory coupling. Moreover, intensifying referred hyperalgesic inputs with noxious mechanical, thermal, and electro-stimulation (ES) of the forearm augmented sympathetic hyperactivity and regulated cardiac function, whereas deafferentation of the left brachial plexus diminished sympathoexcitation. Intradermal injection of the α adrenoceptor (αAR) antagonist yohimbine and agonist dexmedetomidine in the forearm attenuated the cardiac adjustment by ES. Overall, these findings suggest that sensory inputs from the referred pain area contribute to cardiac functional adjustment via peripheral αAR-mediated sympathetic-sensory coupling.

Referred Somatic Hyperalgesia Mediates Cardiac Regulation by the Activation of Sympathetic Nerves in a Rat Model of Myocardial Ischemia.

To observe the effect of electroacupuncture (EA) and pre-EA at"Taiyuan"(LU9) on histopathological changes, pulmonary ventilation function, pneodynamics, and contents of TNF-α, IL-1β and IL-10 in the bronchoalveolar lavage fluid in rats with acute lung injury (ALI), in order to explore their differences of therapeutic effects.

[Comparison of effects of routine electroacupuncture and pre-electroacupuncture in improving lung function in acute lung injury rats].

To observe the effect of mustard oil application at "Liangmen" (ST21) on gastric ulcer (GU) and gastric motility and its association with the sympathetic nerve activity in rats with GU, so as to provide experimental basis for improvement of GU by acupoint application.

[Involvement of sympathetic nerve activation in acupoint application induced improvement of gastric ulcer and motility in rats with gastric ulcer].

Caffeine consumption inhibits acupuncture analgesic effects by blocking adenosine signaling. However, existing evidence remains controversial. Hence, this study aimed to examine the adenosine A1 receptor (A1R) role in moderate-dose caffeine-induced abolishing effect on acupuncture analgesia using A1R knockout mice (A1R). We assessed the role of A1R in physiological sensory perception and its interaction with caffeine by measuring mechanical and thermal pain thresholds and administering A1R and adenosine 2A receptor antagonists in wild-type (WT) and A1R mice. Formalin- and complete Freund's adjuvant (CFA)-induced inflammatory pain models were recruited to explore moderate-dose caffeine effect on pain perception and acupuncture analgesia in WT and A1R mice. Moreover, a C-fiber reflex electromyogram in the biceps femoris was conducted to validate the role of A1R in the caffeine-induced blockade of acupuncture analgesia. We found that A1R was dispensable for physiological sensory perception and formalin- and CFA-induced hypersensitivity. However, genetic deletion of A1R impaired the antinociceptive effect of acupuncture in A1R mice under physiological or inflammatory pain conditions. Acute moderate-dose caffeine administration induced mechanical and thermal hyperalgesia under physiological conditions but not in formalin- and CFA-induced inflammatory pain. Moreover, caffeine significantly inhibited electroacupuncture (EA) analgesia in physiological and inflammatory pain in WT mice, comparable to that of A1R antagonists. Conversely, A1R deletion impaired the EA analgesic effect and decreased the caffeine-induced inhibitory effect on EA analgesia in physiological conditions and inflammatory pain. Moderate-dose caffeine administration diminished the EA-induced antinociceptive effect by blocking A1R. Overall, our study suggested that caffeine consumption should be avoided during acupuncture treatment. PERSPECTIVE: Moderate-dose caffeine injection attenuated EA-induced antinociceptive effect in formalin- and CFA-induced inflammatory pain mice models by blocking A1R. This highlights the importance of monitoring caffeine intake during acupuncture treatment.

Shuya Wang

Institute of Acupuncture and Moxibustion

China Academy Of Chinese Medical Sciences

In Vivo Calcium Imaging of Dorsal Root Ganglia Neurons' Response to Somatic and Visceral Stimuli

Shuya Wang

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Institute of Acupuncture and Moxibustion

China Academy Of Chinese Medical Sciences

In Vivo Calcium Imaging of Dorsal Root Ganglia Neurons' Response to Somatic and Visceral Stimuli

Institute of Acupuncture and Moxibustion