Temporal alterations in pericytes at the acute phase of ischemia/reperfusion in the mouse brain

Pericytes,as the mural cells surrounding the microvasculature,play a critical role in the regulation of microcirculation;however,how these cells respond to ischemic stroke remains unclear.To determine the temporal alterations in pericytes after ischemia/reperfusion,we used the 1-hour middle cerebral artery occlusion model,which was examined at 2,12,and 24 hours after reperfusion.Our results showed that in the reperfused regions,the cerebral blood flow decreased and the infarct volume increased with time.Furthermore,the pericytes in the infarct regions contracted and acted on the vascular endothelial cells within 24 hours after reperfusion.These effects may result in incomplete microcirculation reperfusion and a gradual worsening trend with time in the acute phase.These findings provide strong evidence for explaining the“no-reflow”phenomenon that occurs after recanalization in clinical practice.

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

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,45–53μm 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.

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

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.

Neural interconnection between acupoint“Chéngshān”(承山BL57)and sciatic nerve in the rat

Objective:The aim of this study is to assess the neural interconnection between the acupoint"Chéngshān(承山BL57)"and sciatic nerve from the peripheral nervous system(PNS)to the central nervous system(CNS).Methods:SD rats were systematically examined from the hip to the hind limb along the route of sciatic nerve with regional anatomical technique,and further traced from the BL57 and the trunk of sciatic nerve by the neural tracing technique with cholera toxin subunit B(CTB)and CTB conjugated Alexa Fluor 488 and 594(CTB-AF488/594).Results:After regional anatomy,it was found that the sciatic nerve ran down from the hip to the hind limb and sequentially sent out the tibial and sural branches respectively to the deep and superficial layers of BL57.The CTB labeling associated with both BL57 and sciatic nerve distributed in the same spinal segments and definite regions,including the sensory neurons in lumber 3(L3)-lumber 6(L6)dorsal root ganglia(DRGs),transganglionic axons mainly in the L3-L6 spinal dorsal horn,Clarke’s nucleus,and gracile nucleus,as well as motor neurons mainly in the L3-L6 spinal ventral horn.However,the amount of neural labeling was significantly less in the cases of BL57 than that of sciatic nerve.Conclusions:These results indicate that there is distinct sensory and motor interconnection between the BL57 and the sciatic nerve,which may potentially serve for the active role of the BL57 playing in meliorating the disorders of sciatic nerve.

Comparison of the neurovascular microstructure in local skin tissues at “PC3”“PC7” and “PC9” regions in the rat

Objective:The aim of this study was to compare the microstructure of blood vessels and nerve fibers between different acupoints in the rat skin from the perspective of histology.Methods:The skin tissue at"Quze"(曲泽PC3)""Daling(大陵PC7)"and"Zhongchong(中冲PC9)"regions was taken from the rat forelimb along the pericardium meridian,and cut sagittally with a freezing microtome.After that,the skin sections were stained by fluorescent histochemistry and immunohistochemistry with phalloidin and calcitonin gene-related peptide(CGRP)to reveal the microstructure of blood vessels and nerve fibers respectively,and then examined under a laser scanning confocal microscope.Results:The microstructure of blood vessels and nerve fibers was clearly labeled with phalloidin and CGRP,respectively.The blood vessels and nerve fibers were observed in the dermis of"PC3""PC7"and"PC9"regions,where the bundle of nerve fibers distribute in parallel to the blood vessels,and sent out thin branches to surround the wall of the blood vessels.In contrast,the density of blood vessels and nerve fibers in"PC3""PC7"and"PC9"were presented orderly in an increased tendency.Conclusion:The results of present study demonstrate the differences of the neurovascular microstructure among"PC3""PC7"and"PC9"in the rat,providing a histological view to insight into the specificity of different acupoints.

Neuroanatomical interconnection of the complementary acupoints in the rat hindlimb

Objective:The innervations in Yinlingquan(阴陵泉SP9)and Yanglingquan(阳陵泉GB34)were investigated to explore the neuroanatomical interconnection of the complementary acupoints in the rat hindlimb.Methods:A dual neural tracing technique was used,six male Sprague-Dawley rats were included in experiment.Under anesthesia with isoflurane,cholera toxin subunit B conjugated with Alexa Fluor 488 and 594(CTB-AF488/594)were separately injected into SP9 and GB34 in the same rat.Three days later,the neural labeling was examined in the dorsal root ganglion(DRG),spinal cord,and sympathetic chain at lumbar(L)spinal segments under a laser scanning confocal microscope.Results:All neural labeling were present on the ipsilateral side of tracer injection,including sensory,motor and sympathetic postganglionic neurons.The sensory neurons associated with SP9 and GB34 were distributed in the DRG from L2 to L6 and concentrated in L3-L4 and L4-L5 respectively,while most of the double labeled sensory neurons were presented in L4.On the other hand,the labeled motor neurons for SP9 and GB34 were observed in the dorsolateral part of spinal ventral horn at L2-L4 and L3-L5 respectively,and some of them were double labeled at L4.In addition,the sympathetic postganglionic neurons related to SP9 and GB34 were found in the lumbar sympathetic chain respectively.Conclusion:The similarities of the sensory,motor,and sympathetic innervation of SP9 and GB34 in the rat provide the neuroanatomical evidence to understand the synergetic effect that arise from the stimulation at the complementary acupoints.