This work was supported by S&#38;T Program of Hebei (NO.E2020100001, and NO.22372502D), High-level S &#38; T Innovation and Entrepreneurship Talent Project of Shijiazhuang (No. 07202203).

The animal experiments were conducted with the approval of the Committee on Animal Research and Ethics of the Hebei Yiling Medical Research Institute (approval number: N2022148). Male C57BL/6J mice weighing 18-22 g (see Table of Materials) were housed in the new drug evaluation center of Hebei Yiling Medical Research Institute. They were provided with normal food and clean water and exposed to artificial light for 12 h daily. The rooms maintained a controlled temperature range of 20-26 &#176;C and relative humidity of 40%-70%.
1. Establishment of a chronic hypoxia mouse model (Figure 1)
<ol>
	<li>Before commencing the experiment, prepare animal cages under normal atmospheric pressure and cages with a continuous low-oxygen environment. Establish a continuous low-oxygen environment by utilizing an automated gas control delivery system to flush the chamber with a mixture of pure oxygen and nitrogen. 
	NOTE: This system is programmed to control the electromagnetic valve switch, thereby ensuring precise delivery of gas in terms of both time and concentration.</li>
	<li>Randomly divide mice into three groups: a control group (Con), a model group (CH), and an electro-acupuncture group (EA + CH). Place control and model/electro-acupuncture mice separately in the two cages, with 10 mice per cage. Maintain the light cycle at 12 h/12 h (light/dark). 
	NOTE: No treatment or hypoxia is induced in the control group (Con). The model group (CH) consists of mice with chronic hypoxia. The electro-acupuncture group (EA + CH) comprises hypoxia-induced mice treated with electro-acupuncture.</li>
	<li>For developing chronic hypoxia, establish the parameters of the low-oxygen chamber by utilizing a digital oxygen meter to regulate the gas flow rate and maintain an oxygen concentration of 10%. Place the animals within the low-oxygen chamber at 9:00 am and remove them at 5:00 pm, resulting in a total of 8 h of uninterrupted exposure to low oxygen per day for 3 months. 
	NOTE: When setting up the delivery of nitrogen gas to reduce the oxygen concentration, it is advised to proceed slowly to prevent an excessive introduction of nitrogen gas at once, as it will lead to animal fatalities.</li>
	<li>Evaluate the chronic hypoxia-induced cognitive dysfunction model using histology examination and behavioral tests: the open field test15 and the water maze test16.</li>
</ol>
2. Anesthesia (Figure 2)
<ol>
	<li>Prepare the small animal anesthesia machine (see Table of Materials) and the constant temperature heating pad. 
	NOTE: During anesthesia, animals are susceptible to hypothermia, underscoring the necessity of employing a constant-temperature heating pad for insulation.</li>
	<li>Place the mouse in the anesthesia induction box and quickly induce with 2%-2.5% isoflurane in oxygen (see Table of Materials) for approximately 1 min. 
	NOTE: This short-term pre-treatment is a crucial step to ensure that mice can thrive under low-concentration dosage for an extended period of time.</li>
	<li>Once their excitability decreased, pinch the mouse&#39;s toe to check its reflex. Then, transfer the mouse onto the constant temperature heating pad (37 &#176;C).</li>
	<li>Adjust the anesthesia flow rate to approximately 0.5% concentration. Connect the anesthesia machine to the mouth and nose of the mouse. Proceed with electro-acupuncture treatment while ensuring the maintenance of anesthesia. 
	NOTE: The effect of anesthesia was confirmed when the mice stopped blinking. The effect of anesthesia can last for at least 30 min.</li>
</ol>
3. Electro-acupuncture treatment
<ol>
	<li>To effectively improve cognitive dysfunction, select specific acupoints, such as Baihui (GV20), Shenting (GV24), and bilateral Zusanli (ST36), based on traditional Chinese medicine theory and clinical experience (Figure 3). Administer electro-acupuncture treatment 2 weeks prior to the completion of the modeling process.
	<ol>
		<li>Locate the GV20 acupoint on the midline of the forehead, at the midpoint of a line connecting the tips of the ears7. The depth of acupuncture needle insertion must be 2 mm.</li>
		<li>Locate the GV24 acupoint 1.3 mm directly above the midpoint of the mouse&#39;s eyes on the midline of the forehead17. The depth of acupuncture needle insertion must be 2 mm.</li>
		<li>Locate the ST36 acupoint on the outside of the knee joint, about 2 mm below the head of the fibula18,19. The depth of acupuncture needle insertion must be 3-4 mm.</li>
	</ol>
	</li>
	<li>Prepare disposable acupuncture needles (see Table of Materials) and an electro-acupuncture device (see Table of Materials) for the procedure (Figure 4).</li>
	<li>Place the mouse in the prone position under mild anesthesia with 0.5% isoflurane, ensuring their heads and limbs are immobilized. Hold a stainless-steel needle (diameter: 0.18 mm; length: 7 mm) with the right hand, using the thumb, index finger, and middle finger.</li>
	<li>Perform acupuncture at GV20 and GV24 acupoints transversely for 2 mm depth, lifting the skin on the mouse&#39;s head with the left hand. Puncture the ST36 acupoint vertically for 3-4 mm depth by touching the fibular head on the lateral side of the mouse&#39;s knee joint and pressing on the skin with the left thumb. 
	NOTE: For the acupoints located on the head, it is advisable to insert the needles in the sequence of GV24 followed by GV20. This order facilitates operational convenience. Acupuncture points are discrete anatomical locations rather than stationary points. Consequently, slight deviations in the angle of needle insertion have no effect on the therapeutic effectiveness, similarly seen in patients receiving electro-acupuncture treatment in clinical settings.</li>
	<li>Connect the electronic acupuncture device to the needles, with GV20 and the left ST36 connected to one electrode set and GV24 and the right ST36 connected to another (Figure 4). Select the continuous wave mode, with an electric current intensity of 2 mA and a frequency of 2 Hz20,21. Confirm the ideal treatment by observing local mild tremors at acupoints and quiet tolerance by the mouse.
	<ol>
		<li>When connecting the electrical acupuncture instrument, connect the proximal end of the needle. This helps minimize the impact caused by the weight of the connection line and consequently improves the prevention of needle detachment. If required, utilize adhesive tape to secure the horizontally inserted needle and the connection line.</li>
	</ol>
	</li>
	<li>Administer the daily treatment for 30 min each day for 6 consecutive days, with a single day of rest between each treatment cycle.</li>
</ol>
4. Open field test (Figure 5)
NOTE: The open field test is a conventional method used to assess the autonomous behavior, exploratory behavior, cognitive abilities, and anxiety behavior of experimental animals in novel and unfamiliar environments22. It consists of an open-field reaction box and a recording device.
<ol>
	<li>To conduct the test, prepare a white-walled cube measuring 50 cm &#215; 50 cm &#215; 30 cm, with the bottom divided into 25 equal squares measuring 10 cm &#215; 10 cm.</li>
	<li>Place the mouse into the open field reaction box for acclimation. Allow the mouse to explore the testing room and familiarize with the new environment during the acclimation period. Conduct the open field test after acclimating the mouse to the experimental environment for 1 h. 
	NOTE: This guarantees the minimization of anxiety or stress induced by alterations in the environment, thus enabling more precise outcomes during the subsequent behavioral assessments.</li>
	<li>Place the mouse in the center of the box and monitor it for 10 min after allowing the mouse to adapt to the environment for 2 min.
	<ol>
		<li>Use a video tracking system (see Table of Materials) to record the mouse&#39;s movement trajectory, total distance traveled, time spent in the central area, speed of crossing the central area, and number of entries into the central area during the test.</li>
		<li>Perform the relevant operations as instructed in the product manual of the video tracking system. Each mouse undergoes a single test and begins exploration from the same location within the box.</li>
		<li>After each test, clean the open field box with 75% ethanol to prevent any false results caused by odor interference when using a mouse.</li>
	</ol>
	</li>
</ol>
5. Water maze (Figure 5)
NOTE: The water maze test is frequently employed as a behavioral assessment tool in experiments involving mice to evaluate their spatial learning and memory capabilities23.
<ol>
	<li>Prepare a circular water tank with a diameter of 120 cm and a depth of 30 cm. Divide the tank into four equal quadrants: I, II, III, and IV. If using black mice in the experiment, use a white water tank; for white mice, use a black water tank.</li>
	<li>Place curtains around the circular water tank to prevent the mouse from seeing the researchers during the test.</li>
	<li>Position different markers on the top surface of the water tank as visual cues for spatial orientation. Ensure that these markers remain stationary throughout the experiment to maintain consistency.</li>
	<li>Situate a circular platform with a diameter of 10 cm in quadrant III of the water tank as the designated target area. Ensure the platform can be easily moved and secured in any desired location.</li>
	<li>Throughout the experiment, introduce water into the tank while maintaining a temperature range of 22-24 &#176;C.
	<ol>
		<li>Ensure the water level remains consistently 1 cm above the target platform. Include a 20% concentration of non-toxic titanium dioxide in the water to achieve a distinct contrast between the black mice and the white background. This contrast facilitates the camera&#39;s recording of the mouse&#39;s movements and relevant parameters.</li>
	</ol>
	</li>
	<li>Conduct a 5-day continuous spatial exploration test by sequentially placing each mouse in quadrants I, II, III, and IV.
	<ol>
		<li>Position the mouse facing the wall. Move away from the maze to prevent the mouse from using the experimenter&#39;s position as a reference point. Record the time the mouse takes to find the platform.</li>
		<li>If the mouse fails to locate the underwater platform within 90 s, guide the mouse to the platform and provide a 30-s learning period. Additionally, record the latency period as 90 s.</li>
		<li>If the mouse locates the underwater platform within 90 s, let it remain on the platform for 10 s for learning before removing it from the water tank.</li>
		<li>Dry the mouse with a towel and return it to its cage.</li>
		<li>Rotate the placement of each mouse in each quadrant every 20 min. Record each mouse&#39;s swimming distance, speed, and time taken to find the platform (the latency period) using the video tracking system (see Table of Materials), performing the relevant operations as instructed in the product manual.</li>
		<li>Set the platform 1 cm above the water surface on day 1. Place the platform at a depth of 1 cm below the water surface on days 2-5.</li>
	</ol>
	</li>
	<li>On Day 6, remove the platform from the target quadrant and conduct a spatial exploration test.
	<ol>
		<li>Place the mouse in Quadrant I to explore freely for 90 s. The computer records the swimming trajectory of the mouse, the time spent in the target quadrant, and the number of times it crosses the platform. 
		&#8203;NOTE: In order to minimize experimental errors caused by human factors, it is important to keep the position of the reference point fixed in the water maze experiment. Additionally, the experimenter should immediately retreat after placing the mouse in the water. After the experiment is completed, the mice should be dried with a towel and placed back in their cages to maintain warmth.</li>
	</ol>
	</li>
</ol>
6. Hematoxylin and eosin (HE) staining (Figure 6)
NOTE: Histological examination of the hippocampal region assists in assessing the establishment of the hypoxia model and determining the efficacy of acupuncture treatment.
<ol>
	<li>After the behavioral experiment, anesthetize the mouse with an intraperitoneal injection of 20 mg/kg pentobarbital sodium and perfuse it with 10% paraformaldehyde solution (see Table of Materials) to ensure complete body perfusion. Isolate the brain tissue and immerse it in 10% paraformaldehyde solution at room temperature (RT) for 3 days to achieve fixation.</li>
	<li>Place the brain samples in an embedding box. Subsequently, wash the processed brain samples with running water for 6 h.</li>
	<li>Employ an automated tissue processor to dehydrate the samples using a series of alcohol solutions with increasing concentrations, namely, 60% ethanol for 1 h, 70% ethanol for 1 h, 90% ethanol for 1 h, 95% ethanol for 2 h, and finally, 100% ethanol for 2 h.</li>
	<li>Immerse the tissue specimens in xylene for 2 h to achieve transparency. Subsequently, following the completion of the dehydration process, transfer the permeabilized samples to paraffin wax heated to 60 &#176;C for 3 h. Finally, embed them in an automatic processor.</li>
	<li>Utilize a rotary slicer to obtain 4 &#181;m sections. Subsequently, subject the sections to hematoxylin staining for a duration ranging from 3-8 min, followed by eosin staining for 1-3 min.</li>
	<li>Sequentially transfer the stained sections to separate containers of pure alcohol and xylene. Then, seal and secure the stained sections with neutral gum in preparation for pathological examination under an optical microscope.</li>
	<li>Employ a slide scanner (see Table of Materials) to scan the slices. Subsequently, utilize the viewing software to obtain the HE staining results for the hippocampal region. Compare the arrangement of neurons and the condensation of neuronal nuclei.</li>
</ol>

Cognitive Improvement in Chronic Hypoxia Mice with Acupuncture

The authors have nothing to disclose.

Acupuncture, a non-pharmacological medical practice originating in China over 2,000 years ago, involves the insertion of thin needles into specific points on the body known as acupuncture points. These points are believed to be connected by channels or meridians through which the body&#39;s vital energy, or &#34;qi,&#34; flows24. By stimulating these points, acupuncture aims to restore balance and harmony to the body. It has been shown to effectively treat various conditions, including chronic pai...

The global population is currently facing a critical aging problem, resulting in a rapid increase in the prevalence of cognitive disorders. The worldwide incidence of cognitive impairment is approximately 53.97 per 1000 person-years1. Chronic cerebral hypoxia caused by vascular dysfunction or circulatory/respiratory disorders remains one of the major risk factors for age-related dementia2. Previous studies have demonstrated that cerebral hypoxia can increase amyloid-&#946; deposition by modifying BACE1 expression3. Additionally, hypoxia has been associated with glial-cell dysregulation and neuroinflammation4,5. Despite the growing magnitude of this issue, effective Western medicines for preventing chronic hypoxia-induced cognitive decline are currently lacking. Non-pharmacological traditional Chinese medicine, particularly acupuncture, has been used for thousands of years to treat cognitive disorders and has shown promising results in alleviating neurodegenerative diseases6,7. The Baihui, Shenting, and Zusanli acupoints are effective points for treating cognitive dysfunction8,9. Clinical studies have demonstrated that electro-acupuncture therapy significantly improves the Montreal Cognitive Assessment (MoCA) and Mini-Mental State Examination (MMSE) scores in patients with vascular cognitive impairment and effectively ameliorates cognitive dysfunction8. Although studies have suggested that acupuncture can significantly enhance the memory ability of rats with arterial ligation-an acute cerebral hypoxia models10, an acute cerebral hypoxia model, there is no report on the effects of acupuncture in any rodent model with chronic hypoxia-induced cognitive disorders. The lack of research into the mechanism has considerably impeded its clinical application. 
Previous research has demonstrated that subjecting rats to a hypoxic environment for a period of 8 weeks can significantly elevate levels of oxidative stress and inflammation in the brain, resulting in a decline in memory function11. The present study aims to investigate the impact of acupuncture on rodent models in order to further our understanding. It is worth noting, however, that anesthesia is typically required during acupuncture treatment in rodents due to the potential for agitation during repeated stimulation. Prolonged anesthesia can significantly impact cognitive function in mice, as most anesthetic drugs can suppress neural activity and impede information processing, leading to behavioral deficits12. Several studies have shown that administering 2.5% sevoflurane for a duration of 6 h can notably impair spatial memory, learning ability, and attention in mice13. Furthermore, evidence suggests that high doses of anesthesia may result in neuronal death or nerve damage in mice14. Therefore, it is imperative to identify a suitable approach to minimize the overall amount of anesthesia used. In this study, we introduce an effective acupuncture method for treating mice with cognitive impairment, along with behavioral tests to assess their memory abilities. Importantly, we present a modified pre-treatment anesthesia technique that can effectively reduce the total dosage of anesthesia administered during the experiment.

<table><tbody><tr><td>10% paraformaldehyde solution</td><td>Bioroyee (Beijing) Biotechnology Co., Ltd</td><td>RL3234</td><td></td></tr><tr><td>ANY-maze</td><td>Science</td><td>&amp;nbsp;SA201</td><td>Video tracking system</td></tr><tr><td>C75BL/6J mice</td><td>BEIJING HFK BIOSCIENCE CO.,LTD</td><td>No.110322220103041767</td><td>Gender: Male,&amp;nbsp; Weight: 18&amp;ndash;22 g</td></tr><tr><td>Electroacupuncture device</td><td>Great Wall</td><td>KWD-808 I</td><td></td></tr><tr><td>Hwato acupuncture&amp;nbsp; needle</td><td>Suzhou Medical Appliance Factory</td><td>2655519&amp;nbsp;</td><td></td></tr><tr><td>Isoflurane</td><td>RWD Life Science Co.,Ltd</td><td>R510-22</td><td></td></tr><tr><td>NanoZoomer Digital Pathology</td><td>Hamamatsu Photonics K. K</td><td>C9600-01</td><td></td></tr><tr><td>Small animal anesthesia machine</td><td>RWD</td><td>YL-LE-A106</td><td></td></tr></tbody></table>

acupuncture treatment in a mouse model of chronic hypoxia-induced cognitive dysfunction

The treatment of central nervous disorders has consistently posed significant challenges to the medical field. Acupuncture, a non-pharmacological practice rooted in traditional Chinese medicine, entails the insertion of fine needles into precise points on the body and is commonly employed for the management of diverse conditions. Recently, acupuncture has emerged as a promising therapeutic intervention for a range of neurological diseases, including anxiety and respiratory disorders. However, the potential of acupuncture in treating cognitive dysfunction induced by chronic hypoxia has not yet been explored. This paper presents a comprehensive protocol for establishing a mouse model of chronic hypoxia-induced cognitive impairment, administering mild anesthesia, performing acupuncture treatment, and assessing behavioral changes and memory abilities using open field tests and water mazes. The step-by-step protocol provides detailed instructions on accurately locating and positioning acupoints and needles for cognitive improvement. By employing this protocol, researchers can conduct systematic studies to thoroughly evaluate the therapeutic potential of acupuncture for cognitive dysfunction.

Characterizing mouse locomotion trajectories in the open field experiment 
The trajectory map reveals that mice in the normal group exhibit a profound inclination for exploration in unfamiliar environments. Their activity trajectories are primarily concentrated in the corners while covering the entire open field (left panel). In contrast, the long-term hypoxia model group of mice displays a significantly diminished desire to explore novel surroundings. They predominantly linger in the corners withou...

Watch this Scientific Journal Video about Acupuncture Treatment in a Mouse Model of Chronic Hypoxia-Induced Cognitive Dysfunction at JoVE.com

Author Spotlight: Improving Anesthesia Protocols for Enhanced Mouse Acupuncture Research

Here, we describe a protocol for implementing mild anesthesia and acupuncture treatment on a chronic hypoxia mouse model and conducting behavioral tests to assess the cognitive alterations post-treatment.

Acupuncture Treatment in a Mouse Model of Chronic Hypoxia-Induced Cognitive Dysfunction

The treatment of central nervous disorders has consistently posed significant challenges to the medical field. Acupuncture, a non-pharmacological practice ...

acupuncture-treatment-mouse-model-chronic-hypoxia-induced-cognitive

Research

JoVE Journal

Behavior

1.0K Views.  Heilongjiang University of Chinese Medicine. Here, we describe a protocol for implementing mild anesthesia and acupuncture treatment on a chronic hypoxia mouse model and conducting behavioral tests to assess the cognitive alterations post-treatment. 

Video: Author Spotlight: Improving Anesthesia Protocols for Enhanced Mouse Acupuncture Research

Manipulation of Epileptiform Electrocorticograms (ECoGs) and Sleep in Rats and Mice by Acupuncture

Ancient Chinese literature has documented that acupuncture possesses efficient therapeutic effects on epilepsy and insomnia. There is, however, little research to reveal the possible mechanisms behind these effects. To investigate the effect of acupuncture on epilepsy and sleep, several issues need to be addressed. The first is to identify the acupoints, which correspond between humans, rats, and mice. Furthermore, the depth of insertion of the acupuncture needle, the degree of needle twist in manual needle acupuncture, and the stimulation parameters for electroacupuncture (EA) need to be determined. To evaluate the effects of acupuncture on epilepsy and sleep, a feasible model of epilepsy in rodents is required. We administer pilocarpine into the left central nucleus of the amygdala (CeA) to simulate focal temporal lobe epilepsy (TLE) in rats. Intraperitoneal (IP) injection of pilocarpine induces generalized epilepsy and status epilepticus (SE) in rats. Five IP injections of pentylenetetrazol (PTZ) with a one-day interval between each injection successfully induces spontaneous generalized epilepsy in mice. Recordings of electrocorticograms (ECoGs), electromyograms (EMGs), brain temperature, and locomotor activity are used for sleep analysis in rats, while ECoGs, EMGs, and locomotor activity are employed for sleep analysis in mice. ECoG electrodes are implanted into the frontal, parietal, and contralateral occipital cortices, and a thermistor is implanted above the cerebral cortex by stereotactic surgery. EMG electrodes are implanted into the neck muscles, and an infrared detector determines locomotor activity. The criteria for categorizing vigilance stages, including wakefulness, rapid eye movement (REM) sleep, and non-REM (NREM) sleep are based on information from ECoGs, EMGs, brain temperature, and locomotor activity. Detailed classification criteria are stated in the text.

Manipulation of Epileptiform Electrocorticograms ECoGs and Sleep in Rats and Mice by Acupuncture

This paper demonstrates the performance of acupuncture, epilepsy models, and the analysis of sleep in rodents. The acupuncture procedure and the identification of acupoints are described. Pilocarpine or pentylenetetrazol (PTZ) is used to induce epilepsy. Electrocorticogram (ECoG), electromyogram (EMG), brain temperature, and locomotor activity recordings are employed for sleep analysis.

Ancient Chinese literature has documented that acupuncture possesses efficient therapeutic effects on epilepsy and insomnia. There is, however, little ...

The Unpredictable Chronic Mild Stress Protocol for Inducing Anhedonia in Mice

Depression is a highly prevalent and debilitating condition, only partially addressed by current pharmacotherapies. The lack of response to treatment by many patients prompts the need to develop new therapeutic alternatives and to better understand the etiology of the disorder. Pre-clinical models with translational merits are rudimentary for this task. Here we present a protocol for the unpredictable chronic mild stress (UCMS) method in mice. In this protocol, adolescent mice are chronically exposed to interchanging unpredictable mild stressors. Resembling the pathogenesis of depression in humans, stress exposure during the sensitive period of mice adolescence instigates a depressive-like phenotype evident in adulthood. UCMS can be used for screenings of antidepressants on the variety of depressive-like behaviors and neuromolecular indices. Among the more prominent tests to assess depressive-like behavior in rodents is the sucrose preference test (SPT), which reflects anhedonia (core symptom of depression). The SPT will also be presented in this protocol. The ability of UCMS to induce anhedonia, instigate long-term behavioral deficits and enable reversal of these deficits via chronic (but not acute) treatment with antidepressants strengthens the protocol&#39;s validity compared to other animal protocols for inducing depressive-like behaviors.

Here we present the unpredictable chronic mild stress protocol in mice. This protocol induces a long-term depressive-like phenotype and enables to assess the efficacy of putative antidepressants in reversing the behavioral and neuromolecular depressive-like deficits.

Depression is a highly prevalent and debilitating condition, only partially addressed by current pharmacotherapies. The lack of response to treatment by ...

Analysis of Learning and Memory Ability in an Alzheimer's Disease Mouse Model using the Morris Water Maze

A Morris water maze (MWM) experiment forces experimental animals to swim and learn to find a platform hidden in the water. It is widely used in scientific research to assess the learning and memory of animals. Due to the extensive use of the MWM test, visual experimental protocols are essential for researchers. This manuscript uses the latest studies to introduce the protocol of the MWM test. Alzheimer&#8217; Disease (AD) is characterized by a progressive loss of memory and cognitive function. An alternative and complementary treatment used for AD is Manual Acupuncture (MA). To assess the learning and memory ability of AD model mice, the MWM test was conducted. The visible platform trial, hidden platform trial, probe trial, and reversal trial of MWM were used to evaluate spatial learning and memory ability. In the visible platform trial, the swimming speed and escape latency of mice in different groups was not significantly different. In the hidden platform and reversal trials, the AD group showed a long escape latency. The escape latency decreased significantly after the MA treatment. Low platform crossover number and the proportion of time in the SW quadrant in the probe trial increased after the MA treatment (p &#60; 0.05 or p &#60; 0.01). The results of the MWM tests suggest that MA can effectively improve the spatial learning and memory abilities of AD model mice. Rigorous experimental operations provided assurance of the reliability of the results.

Herein, a protocol to conduct the Morris water maze tests to evaluate the ability of learning and memory of Alzheimer&#8217;s Disease model mice and to assess the effect of manual acupuncture for treating them is described.

A Morris water maze (MWM) experiment forces experimental animals to swim and learn to find a platform hidden in the water. It is widely used in scientific ...

Quantification of Neurovascular Protection Following Repetitive Hypoxic Preconditioning and Transient Middle Cerebral Artery Occlusion in Mice

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week protocol for repetitive hypoxic preconditioning (RHP) induces long-term protection against central nervous system (CNS) injury in a mouse model of focal ischemic stroke. RHP consists of 9 stochastic exposures to hypoxia that vary in both duration (2 or 4 hr) and intensity (8% and 11% O2). RHP reduces infarct volumes, blood-brain barrier (BBB) disruption, and the post-stroke inflammatory response for weeks following the last exposure to hypoxia, suggesting a long-term induction of an endogenous CNS-protective phenotype. The methodology for the dual quantification of infarct volume and BBB disruption is effective in assessing neurovascular protection in mice with RHP or other putative neuroprotectants. Adult male Swiss Webster mice were preconditioned by RHP or duration-equivalent exposures to 21% O2 (i.e. room air). A 60 min transient middle cerebral artery occlusion (tMCAo) was induced 2 weeks following the last hypoxic exposure. Both the occlusion and reperfusion were confirmed by transcranial laser Doppler flowmetry. Twenty-two hr after reperfusion, Evans Blue (EB) was intravenously administered through a tail vein injection. 2 hr later, animals were sacrificed by isoflurane overdose and brain sections were stained with 2,3,5- triphenyltetrazolium chloride (TTC). Infarcts volumes were then quantified. Next, EB was extracted from the tissue over 48 hr to determine BBB disruption after tMCAo. In summary, RHP is a simple protocol that can be replicated, with minimal cost, to induce long-term endogenous neurovascular protection from stroke injury in mice, with the translational potential for other CNS-based and systemic pro-inflammatory disease states.

This protocol describes repetitive hypoxic preconditioning, or brief exposures to systemic hypoxia that reduce infarct volumes and blood-brain barrier disruption following transient middle cerebral artery occlusion in mice. It also details dual quantification of infarct volume and blood-brain barrier disruption after stroke to assess the efficacy of neurovascular protection.

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week ...

Medicine

Neurobehavioral Assessments in a Mouse Model of Neonatal Hypoxic-ischemic Brain Injury

We performed unilateral carotid artery occlusion on CD-1 mice to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of neonatal HI brain injury by studying neurobehavioral functions in these mice compared to non-operated (i.e., normal) mice. During the study, Rice-Vannucci&#39;s method was used to induce neonatal HI brain damage in postnatal day 7-10 (P7-10) mice. The HI operation was performed on the pups by unilateral carotid artery ligation and exposure to hypoxia (8% O2 and 92% N2 for 90 min). One week after the operation, the damaged brains were evaluated with the naked eye through the semi-transparent skull and were categorized into subgroups based on the absence (&#34;no cortical injury&#34; group) or presence (&#34;cortical injury&#34; group) of cortical injury, such as a lesion in the right hemisphere. On week 6, the following neurobehavioral tests were performed to evaluate the cognitive and motor functions: passive avoidance task (PAT), ladder walking test, and grip strength test. These behavioral tests are helpful in determining the effects of neonatal HI brain injury and are used in other mouse models of neurodegenerative diseases. In this study, neonatal HI brain injury mice showed motor deficits that corresponded to right hemisphere damage. The behavioral test results are relevant to the deficits observed in human neonatal HI patients, such as cerebral palsy or neonatal stroke patients. In this study, a mouse model of neonatal HI brain injury was established and showed different degrees of motor deficits and cognitive impairment compared to non-operated mice. This work provides basic information on the HI mouse model. MRI images demonstrate the different phenotypes, separated according to the severity of brain damage by motor and cognitive tests.

We performed unilateral carotid artery occlusion on postnatal day 7-10 CD-1 mouse pups to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of HI brain injury. We studied neurobehavioral functions in these mice compared to non-operated normal mice.

We performed unilateral carotid artery occlusion on CD-1 mice to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of neonatal HI ...

Neuroscience

Acupuncture in a Rat Model of Asthma

A high platform can fix rats without restriction and completely expose the acupoints on the back during acupuncture manipulation. This article describes methods for the fabrication of the high platform, establishes a rat model of asthma and measures changes in respiratory function using a noninvasive and real-time whole-body plethysmography (WBP) system.

A high platform can fix rats without restriction and completely expose the acupoints on the back during acupuncture manipulation. This article describes ...

Multiple Intravenous Bolus Dosing and Invasive Hemodynamic Assessment in a Hypoxia-Induced Mouse Pulmonary Artery Hypertension Model

Pulmonary arterial hypertension (PAH) is a progressive life-threatening disease, primarily affecting small pulmonary arterioles of the lung. Currently, there is no cure for PAH. It is important to discover new compounds that can be used to treat PAH. The mouse hypoxia-induced PAH model is a widely used model for PAH research. This model recapitulates human clinical manifestations of PAH Group 3 disease and is an important research tool to evaluate the effectiveness of new experimental therapies for PAH. Research using this model often requires the administration of compounds in mice. For a compound that needs to be given directly into the bloodstream, optimizing intravenous (IV) administration is a key part of the experimental procedures. Ideally, the IV injection system should permit multiple injections over a set time course. Although the mouse hypoxia-induced PAH model is very popular in many laboratories, it is technically challenging to perform multiple IV bolus dosing and invasive hemodynamic assessment in this model. In this protocol, we present step-by-step instructions on how to carry out multiple IV bolus dosing via mouse jugular vein and perform arterial and right ventricle catheterization for hemodynamic assessment in mouse hypoxia-induced PAH model.

This protocol provides a step-by-step procedure for executing multiple intravenous bolus dose administration and invasive hemodynamic monitoring in mice. Investigators can use this protocol for future therapeutic compound screening for pulmonary artery hypertension.

Pulmonary arterial hypertension (PAH) is a progressive life-threatening disease, primarily affecting small pulmonary arterioles of the lung. Currently, ...

Improving Cognitive Functions in Rats via Thread-Embedding Acupuncture Therapy

Operation Procedure and Precautions of Thread-Embedding Acupuncture Therapy in Alzheimer

Thread-embedding therapy (TEAT) is a treatment that prevents and manages diseases by inserting a biodegradable suture into an acupoint, providing long-lasting stimulation. TEAT is a simple approach that avoids the discomfort of regular acupuncture and provides sustained therapeutic effects. This article discusses the potential impact of TEAT on the learning and memory abilities of rats with Alzheimer&#39;s disease-like symptoms. Since chemically induced neuronal degeneration and cognitive impairments in rats does not entirely reflect the true pathological changes observed in Alzheimer&#39;s disease. Consequently, our research group has designated these manifestations as Alzheimer&#39;s disease-like symptoms. A protocol has been established to outline the selection of acupoints, the operation process, and necessary precautions for the head and lower back. The experiment was conducted on three groups: a control group, a model group, and a TEAT group, each containing 6 rats. To induce Alzheimer&#39;s disease-like symptoms, rats were intraperitoneally injected with D-galactose for 7 weeks (49 days). The rats in the TEAT group received acupoint catgut embedding treatment. Following the intervention period, a Morris Water Maze (MWM) was conducted to evaluate the rats&#39; learning and memory. Subsequently, the rats were sacrificed, and their brain tissue was examined. A histological examination was performed to understand the effects of TEAT on the pathology of rats exhibiting symptoms of Alzheimer&#39;s disease. This study suggests that TEAT may improve learning and memory in rats with Alzheimer&#39;s disease-like symptoms, indicating a potentially promising new treatment approach for this neurodegenerative condition.

Author Spotlight: Exploring Acupuncture in Alzheimer's Research from Thread-Embedding Techniques to Clinical Trials

This study presents a protocol for thread-embedding acupuncture therapy in Alzheimer's disease-like rats.

Thread-embedding therapy (TEAT) is a treatment that prevents and manages diseases by inserting a biodegradable suture into an acupoint, providing ...

A Needle Model for Vascular Cognitive Impairment Studies in Mice

A Mouse Model for Vascular Cognitive Impairment and Dementia Based on Needle-guided Asymmetric Bilateral Common Carotid Artery Stenosis

Vascular cognitive impairment and dementia (VCID) results from vascular brain injury. Given VCID&#39;s high incidence, which is expected to continue rising as the population ages, it is critical to establish a robust animal model for the disease. This paper presents a novel method of creating a mouse model of VCID that is based on asymmetric bilateral common carotid artery stenosis, which mimics human chronic cerebral hypoperfusion caused by carotid atherosclerosis.
Briefly, common carotid arteries (CCAs) are ligated to different gauge needles (32 G for the right CCA and 34 G for the left CCA) using 7-0 silk sutures followed by immediate needle removal. The remaining suture rings cause persistent blood flow reduction and long-term cognitive impairment associated with white matter injury, microinfarcts, and reactive gliosis, thus closely mimicking the pathogenesis of VCID. Importantly, in this needle model, the clinical representations do not revert with time, providing reliable long-term cognitive impairment. Moreover, the survival rate 24 weeks post surgery was 81.6%, which is higher compared to the other established models of VCID with a similar level of blood flow reduction.
Additional advantages include low material cost and compatibility with MRI to monitor brain injury in live animals since no metal is implanted. The main challenge in employing the needle model of VCID is the requirement for developing advanced surgical skills since mouse CCAs are less than 0.6 mm in diameter and are very fragile. High-quality visual representation of the surgery will thus help researchers to master this technique and advance our understanding of VCID, potentially leading to the development of novel therapeutic modalities to decrease the devastating cognitive decline associated with VCID.

The needle method of asymmetric bilateral common carotid artery stenosis is proposed to create a mouse model for vascular cognitive impairment and dementia. It results in longer-term outcomes compared to previously established models and is compatible with live MRI. Visual representation demonstrating the procedure provides guidance for mastering the surgery.

Vascular cognitive impairment and dementia (VCID) results from vascular brain injury. Given VCID&#39;s high incidence, which is expected to continue ...

Using a 3D Printed Holder for Bulk Electroacupuncture in Small Animals

Bulk Electroacupuncture Operation for Mice or Young Rats

Electroacupuncture (EA) is widely used to treat various health conditions. However, the underlying mechanism of EA treatment remains unclear, hindering its promotion. The mechanistic study requires mouse or rat models to address this issue. However, these animals are not obedient to the experimental process, which is time-consuming. To solve these problems, we designed a 3D-printed small animal body bulk fixator to improve the efficiency of EA's animal experiments. This video shows in detail how to use the fixator to perform bulk EA on mice or young rats. For the selection of acupoints, the anterior oblique line of vertex temporal (MS6 head) and Tianshu point (ST25 belly) were chosen to verify the effect of the fixation device with prone positioning and supine positioning. Using the 3D-printed small animal holder allows multiple rodents to be immobilized and treated simultaneously, reducing the time and resources required for the experiment. This technique could be applied to other animal models by 3D printing different sizes and could potentially be used for various fixing conditions. The device is beneficial for the promotion of experimental scientific research in EA.

Author Spotlight: Using a 3D-Printed Holder for Simultaneous Multiple Electroacupuncture Operation

Here, we present a protocol for efficient and rapid electroacupuncture (EA) in mice or young rats using a three-dimensional (3D) printed holder. This technique allows simultaneous operation on multiple animals, saving time and increasing experimental efficiency.

Electroacupuncture (EA) is widely used to treat various health conditions. However, the underlying mechanism of EA treatment remains unclear, hindering ...