This work was supported with funds from the National Natural Science Foundation of China (NSFC) Nos. 82374564 (to YJD) and Nos. 82074566 (to YJD) and from Sun Guojie Inheritance Base for TCM Acupuncture-Moxibustion of World Federation of Acupuncture-Moxibustion Societies in Wuhan, China (World Federation of Acupuncture-Moxibustion Societies [2019] No. 26) (to YJD) and the National Famous TCM Inheritance Studio construction project of the National Administration of Traditional Chinese Medicine, China (the National Administration of Traditional Chinese Medicine [2022] No. 5) (to YJD).

All experiments were carried out following the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Hubei Provincial Center for Disease Control and Prevention Laboratory Animal Management and Use Committee. A total of 18 3-month-old female Sprague-Dawley (SD) rats weighing 220-250 g were used for the test. All rats were maintained on a 12 h light/dark cycle at 23 &#177; 1 &#176;C and 40%-50% humidity, with free access to food and water. Following a one-week acclimatization period, during which the rats were allowed to habituate to the laboratory conditions, the experiments were initiated.
1. Model development
<ol>
	<li>The experimental design is shown in Figure 1. Randomly divide 18 Sprague-Dawley 3-month-old female rats weighing 220-250 g into control, model, and TEAT groups. Each group consists of 6 rats.</li>
	<li>Inject the model group and TEAT group daily with D-galactose (D-gal; 120 mg/kg/day), while the control group receives saline injections (120 mg/kg/day), for 7 weeks (49 days) in the lower ventral area22. The injection should be administered daily between 8:00 and 9:00 a.m.</li>
	<li>Weigh the rats on the 7th day of each week between 9:00 and 10:00 a.m. The experiment lasted for 49 days. Bury the threads every 14 days on the 14th, 28th, and 42nd days. Fix the time for each thread embedding between 9:00-12:00 am.</li>
	<li>Continue intraperitoneal administration of D-galactose for 7-weeks to induce Alzheimer&#39;s disease-related pathological manifestations in rats22.</li>
</ol>
2. Preparation for TEAT
<ol>
	<li>Sterilize surgical instruments such as ophthalmic scissors, trays, and tweezers and disinfect. Clean the surfaces in the operating room environment thoroughly and disinfect.</li>
	<li>Prepare the surgical platform by laying a small animal heating pad flat on the surface, followed by draping with a sterile towel. Subsequently, place the cleaned and disinfected operating board smoothly on top, with the rough side facing upwards. Then, switch on the heating pad and set the temperature to 32 &#176;C.</li>
	<li>For the procedure, select a No. 6 disposable thread-embedding needle with a diameter of 0.6 mm and a 5-0 absorbable surgical suture (collagen suture) with a length of 2 cm. Cut the 2 cm 5-0 absorbable surgical suture into 0.5 cm and 0.2 cm pieces using ophthalmic scissors. Soak the cut thread in 0.9% saline and set aside.</li>
	<li>Wash hands with soap and water for 20 s, and then dry them. Afterward, wear sterile gloves.</li>
	<li>Anesthetize the rats with an IP injection of chloral hydrate (300 mg/kg) in the lower ventral area, ensuring that anesthesia is confirmed when animals are unresponsive in a surgical plane, based on the lack of response from a toe pinch test, and maintaining a stable physiological state. Additionally, to prevent corneal dryness and potential damage while the rats are under anesthesia, apply a generous amount of veterinary ointment to the eyes, ensuring they remain lubricated throughout the procedure. 
	NOTE: It is recommended to anesthetize the rat, as it is painful for them to experience the piercing.</li>
</ol>
3. Thread-embedding operation for Baihui (GV 20)
<ol>
	<li>Locate the acupoint Baihui (GV 20) in rats at the right midpoint of the parietal bone (Figure 2A). Position the rats to fully expose the head, with the ears pulled forward to approximate the method used in humans for connecting the ear apex lines. Identify the Baihui (GV 20) point by palpating the central longitudinal ridge of the skull and pinpointing the location roughly at its midpoint. Confirm this acupoint further at the level where the ears would make contact with the head if laid flat, aligning with the intersection of the sagittal suture and the parietal bone. Use a marker to mark Baihui (GV 20).</li>
	<li>Shave the head area around the acupoint of the rat using a trimmer (usually an electric men&#39;s beard trimmer).</li>
	<li>Prior to surgery, disinfect the acupoints and surrounding skin by applying a 0.5% iodine solution in circular motions, starting from the center, and moving outwards. Subsequently, perform a triple disinfection process: use the iodophor 3x, each followed by an application of alcohol to ensure proper decontamination and prepare the site for aseptic technique.</li>
	<li>Use forceps to clamp out the 0.2 cm absorbable surgical suture soaked in saline and place it in the front end of the needle tube. Then, connect it to the needle core.</li>
	<li>Pinch the area around Baihui (GV 20) with thumb and index finger on one hand, while holding the needle with the other.</li>
	<li>Insert the needle horizontally and quickly at a 15&#176; angle to the skin surface, 0.1 cm below the Baihui (GV 20) point, while pinching the skin (Figure 3A).</li>
	<li>After the needle tip reaches 0.1 cm above the Baihui (GV 20) point, push the needle core while retracting the needle tube to fully implant the 0.2 cm absorbable surgical suture into the subcutaneous tissue of the acupoint (Figure 3B).</li>
	<li>Slowly remove the needle, making sure no suture is exposed. Press the needle hole with sterile dry cotton for 10 s to prevent bleeding. If the thread is outside the body after embedding the line at acupoints, extract it and re-operate.</li>
</ol>
4. Thread-embedding operation for Shenshu (BL 23)
<ol>
	<li>The acupoint Shenshu (BL 23) in rats is located 5 mm lateral to the 2nd lumbar vertebra (Figure 2B). First, locate the 6th lumbar vertebra based on the hip tubercle of the rat. Then locate the 2nd lumbar vertebra by counting upward four vertebral bodies. Shenshu (BL 23) will be 5 mm lateral to it23. 
	NOTE: The combination of the Baihui (GV 20) and Shenshu (BL 23) acupoints is a frequently utilized set in Traditional Chinese Medicine (TCM) theory for the prevention and treatment of cerebral disorders. As confirmed by previous research20,21, this acupoint pairing has been established for its efficacy.</li>
	<li>Use a marker to mark Shenshu (BL 23). Shave the back and waist area around the acupoint of the rat using a trimmer (usually an electric beard trimmer).</li>
	<li>Prior to surgery, disinfect the acupoints and surrounding skin by applying 0.5% iodine solution in a circular motion, starting from the center and moving outwards. Subsequently, perform a triple disinfection process: use the iodophor three times, each followed by an application of alcohol to ensure proper decontamination and preparation of the site for aseptic technique.</li>
	<li>Use forceps to clamp out the 0.5 cm absorbable surgical suture soaked in saline and place it in the front end of the needle tube. Then, connect it to the needle core.</li>
	<li>Stretch the skin with the index and middle fingers of the pressing hand or the thumb and index fingers. Hold the needle with the puncturing hand.</li>
	<li>Quickly insert the needle perpendicularly into the point 0.25 cm below the Shenshu (BL 23). See Figure 4A.</li>
	<li>After the needle tip reaches 0.5 cm under the skin, adjust the needle tip, making the direction of insertion centripetal, forming a 45&#176; angle with the skin surface and obliquely insert 0.5 cm (Figure 4B). 
	NOTE: It is important to exercise caution when needling the Shenshu (BL 23), which is located near the kidney. Deep insertion or repeated lifting and thrusting of the needle can cause damage. Therefore, we recommend an insertion depth of around 0.3-0.6 cm.</li>
	<li>Push the needle core while retracting the tube and fully implant the 0.5 cm absorbable surgical suture into the acupoint&#39;s muscle tissue of the acupoint (Figure 4C).</li>
	<li>Slowly pull out the needle, ensuring no suture is exposed. Then, press the needle hole with sterile dry cotton for 10 s to prevent bleeding. If the thread is outside the body after embedding the line at acupoints, extract it and re-operate.</li>
</ol>
5. Post-operative care
<ol>
	<li>Postoperatively, place the rat on a small animal heating pad set at a temperature of 32&#176;C to facilitate recovery from anesthesia.Ensure the animal is fully conscious and its condition is satisfactory before returning it to its housing enclosure.</li>
	<li>After surgery, observe signs of sterile inflammatory responses in the affected area within a week. These signs may include redness, swelling, or a pus-like discharge. Typically, these symptoms do not require any treatment. However, if there is an excessive amount of exudate, drain and disinfect the affected area. In such cases, apply erythromycin ointment until the wound fully recovers.</li>
</ol>
6. Behavioral testing and histological examination
<ol>
	<li>Following the intervention, at week 8, perform the Morris Water Maze (MWM) experiment2,24 to evaluate their learning and memory capabilities. Perform the learning trials over 5 days, with four trials per day. Then administer a probe trial after 24 h from the last acquisition day.</li>
	<li>Perform statistical comparisons using statistical analysis software. Analyze the behavioral data of learning ability by one-way analysis of variance (ANOVA) and post-hoc Tukey&#39;s test. Assess the significance of escape latency data by two-way repeated measure analysis of variance (ANOVA) followed by the post-hoc Bonferroni test. A value of p &#60;0.05 is considered significant in all tests.</li>
	<li>After the behavioral assessment, anesthetize the rats as above and euthanize with intraperitoneal injections of 150 mg/kg body weight of pentobarbital sodium.</li>
	<li>Fix the rat&#39;s brain through perfusion using 4% paraformaldehyde perfused via the vascular system25.
	<ol>
		<li>Insertion and fixation of the gavage needle
		<ol>
			<li>Prepare a 12G needle and ensure it is straight and suitable for passing through the ventricle of the rat. Locate the ventricle of the rat, which is typically the left ventricle, and insert the needle through it into the ascending aorta. This step requires precision and a good understanding of rat anatomy.</li>
			<li>Once the needle is in place, use a hemostat to clamp the gavage needle near the point where it enters the aorta. This will help to secure the needle and prevent it from moving.</li>
			<li>Ensure that the clamp is tight enough to hold the needle securely but not so tight as to damage the aorta or impede blood flow. The gavage needle should remain in the ascending aorta throughout the procedure to maintain proper perfusion.</li>
		</ol>
		</li>
		<li>Perfusion with normal saline
		<ol>
			<li>Prepare a 50cc syringe without a needle for the perfusion. Attach the other end of the gavage needle, which is outside the rat&#39;s body, to the syringe filled with normal saline.</li>
			<li>Make a small incision to cut the right atrial appendage to allow blood to flow out of the heart. This step ensures that the blood is displaced by the saline during perfusion.</li>
			<li>Begin injecting the normal saline into the ascending aorta at a steady and constant rate. This will flush out the blood and prepare the tissues for fixation.</li>
			<li>Monitor the fluid coming out of the right atrial appendage and be prepared to change the syringe as needed to maintain a continuous perfusion with fresh saline. 
			&#8203;placed, prepare to perfuse with the fixative.</li>
			<li>Replace the syringe containing saline with one containing 4% paraformaldehyde, a commonly used fixative in histology. Proceed with the perfusion using the paraformaldehyde to fix the tissues in place for subsequent processing and analysis. 
			NOTE: Throughout this procedure, it is crucial to maintain aseptic techniques to prevent infection and to handle the rat gently to minimize injury. Additionally, all personnel involved should be trained in the technique to ensure consistency and effectiveness.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Remove the rat brain and place it in a specimen bottle containing 4% paraformaldehyde for 24-48 h at 4 &#176;C25,26. Perform ethanol dehydration and xylene treatment step by step, followed by conventional continuous coronal sections (4-5 &#181;m) for measurement. Evaluate the morphology and loss of hippocampal neurons in each group using Hematoxylin and Eosin (H&#38;E) staining (200x). 
	NOTE: A necropsy examination to assess for gastrointestinal (GI) lesions was not conducted on the rodents based on the absence of clinical signs indicative of GI disturbances throughout the experimental period. The animals exhibited no overt adverse reactions that would suggest the presence of such pathologies.</li>
</ol>

Improving Cognitive Functions in Rats via Thread-Embedding Acupuncture Therapy

The authors declare that they have nothing to disclose.

Intervention time 
The etiology of AD is complex, and its pathogenesis remains uncertain. Currently, there is no effective treatment for the disease, and its course is irreversible. Therefore, the academic community has reached a consensus on the importance of focusing on the prevention and treatment of AD. The prevention and treatment strategies for AD emphasize early intervention, following the TCM concept of treating conditions before they occur, emphasizing treatment before its onset, transmissi...

Thread-embedding acupuncture (TEA) involves embedding catgut or absorbable sutures into acupoints using a special needle. This technique has a prolonged effect until the sutures are absorbed and degraded. It provides continuous and sustained stimulation to the acupoints for a week or longer1, achieving a comparable outcome without the necessity for daily repetitive procedures2. This reduces the number of patients seeking medical treatment, thus conserving medical resources to some extent. In recent years, TEAT has been increasingly used in various medical fields in China, including internal medicine, external medicine, dermatology, facial features, gynecology, and pediatrics3. Studies have shown that it is effective in producing long-term and reliable effects for certain chronic and complex diseases, such as epilepsy4 and facial nerve paralysis5. It has the advantages of minimal trauma and simple operation6. Additionally, the treatment costs of TEAT have been reported to be lower than those of electroacupuncture (EA)7. Therefore, TEAT is increasingly valued in clinical and scientific research.
Alzheimer's disease (AD) is a neurodegenerative condition that progresses over time. Its pathophysiological process begins to accumulate approximately 20 years before clinical symptoms appear, but its mechanism is still not fully understood8. Given the limited availability of effective drugs for AD, research is increasingly focusing on the prodromal and preclinical stages of the disease9. Prevention is a crucial strategy for addressing the development and progression of AD9,10. In recent years, traditional Chinese medicine (TCM) has made significant progress, and it is expected to offer new possibilities for preventing and treating AD11,12. As a subtype of acupuncture, TEAT is preferred over other therapies due to its wide treatment range, minimal trauma, simple operation, low treatment frequency, and long-lasting efficacy6. Emerging literature suggests that TEAT may benefit individuals with obesity13,14, diabetes15, insomnia16, postmenopausal osteoporosis17, and depression18, all of which are risk factors for AD. It also improves senescence in aging rats by regulating mitophagy2, improves spatial learning and memory impairment, alleviates pathological damage of the hippocampus, and inhibits inflammation response in vascular dementia (VD) rats19. It appeared attractive to figure out whether TEAT could also act on pathological processes during AD.
The current research lacks uniformity and standardization in several critical elements of TEAT operation, including the embedding method, embedding tool, thread, and embedding interval period. This lack of standardization affects the evaluation of the therapy's effect and the exploration of related mechanisms and restricts external promotion and communication. Prior research20,21 has demonstrated that intraperitoneal administration of D-galactose (D-gal) can induce Alzheimer's disease-like pathological changes in rats, including neuronal degeneration and cognitive impairments. Furthermore, electroacupuncture at the Baihui(GV20) and Shenshu(BL23) has been shown to effectively ameliorate cognitive decline, mitigate neuroinflammation, and reduce neuronal damage. This study takes Alzheimer's disease-like model rats as an example to introduce the selection of acupoints and the operation process and precautions of TEAT under this model. It discusses the critical operational elements of TEAT in experimental research to provide a reference for future studies.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.9% saline, 4% paraformaldehyde, Hematoxylin, Eosin Y (water-soluble), absolute ethanol, 0.01M PBS powder, xylene, hydrochloric acid, embedded paraffin, neutral gum</td>
			<td>sinopharm (www.sinopharm.com)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5-0 absorbable surgical suture</td>
			<td>bodamedical (www.bodamedical.com)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>70% alcohol, iodophor, cotton ball</td>
			<td></td>
			<td></td>
			<td>Used for disinfection.</td>
		</tr>
		<tr>
			<td>chloral hydrate</td>
			<td>sigma</td>
			<td>C8383</td>
			<td></td>
		</tr>
		<tr>
			<td>coverslips, glass slides</td>
			<td>Guangzhou Wuqiang Experimental Equipment Co., Ltd</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dehydrator</td>
			<td>Wuhan Junjie JT-12J Computerized Biological Tissue Dehydrator</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DELUXE THERMOSTAT HEAT MAT</td>
			<td>Zhenhua Instruments</td>
			<td></td>
			<td>For maintaining the body temperature of rats.</td>
		</tr>
		<tr>
			<td>D-galactose</td>
			<td>sigma</td>
			<td>G0750</td>
			<td></td>
		</tr>
		<tr>
			<td>electric men&#39;s beard trimmer</td>
			<td></td>
			<td></td>
			<td>Used for shaving rats.</td>
		</tr>
		<tr>
			<td>Electronic scale for small animals (ZK-DST)</td>
			<td>Henan Zhike Information Technology Co., Ltd</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>erythromycin ointment</td>
			<td>frontpharm</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>glass dish</td>
			<td></td>
			<td></td>
			<td>Used to place cut absorbable sutures.</td>
		</tr>
		<tr>
			<td>High-Pressure Steam Sterilization Pot (YM100Z)</td>
			<td>Shanghai Sanshen medical equipment factory</td>
			<td></td>
			<td>For sterilization of surgical instruments.</td>
		</tr>
		<tr>
			<td>Ice Maker (CMB40)</td>
			<td>Shanghai Sheyan Instrument Co., Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope (BX-53)</td>
			<td>OLYMPUS</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>milk powder</td>
			<td></td>
			<td></td>
			<td>Used for the MWM test.</td>
		</tr>
		<tr>
			<td>Morris Water Maze Video Tracking System &#38; Data Analysis Software (Version: Watermaze 2.0)</td>
			<td>Chengdu Taimeng Technology Co., Ltd</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>No.6 thread-embedding needle</td>
			<td>bodamedical (www.bodamedical.com)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ophthalmic scissors, trays, tweezers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pathology Microtome&#160; (RM 2016)</td>
			<td>LEICA, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pentobarbital</td>
			<td>Germany</td>
			<td>P11011</td>
			<td></td>
		</tr>
		<tr>
			<td>permanent marker</td>
			<td></td>
			<td></td>
			<td>Used to mark the acupoint.</td>
		</tr>
		<tr>
			<td>Slicing knives (R35)</td>
			<td>Feathers, Japanese</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>soap</td>
			<td></td>
			<td></td>
			<td>Used to wash hands.</td>
		</tr>
		<tr>
			<td>sterile gloves</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

This article introduces the specific operation method of TEAT in Alzheimer&#39;s disease-like rats. It discusses the critical operational elements of TEAT in experimental research to provide a reference for future studies.
TEAT improves learning in Alzheimer&#39;s disease-like rats 
As illustrated in the flowchart (Figure 5), during the adaptive training period, it was observed that rats in each group were observing the surrounding environmen...

Read this Scientific Article Protocol about Author Spotlight: Exploring Acupuncture in Alzheimer's Research from Thread-Embedding Techniques to Clinical Trials at JoVE.com

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.

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 ...

operation-procedure-precautions-thread-embedding-acupuncture-therapy

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Neuroscience

472 Views.  Hubei University of Chinese Medicine. This study presents a protocol for thread-embedding acupuncture therapy in Alzheimer's disease-like rats.

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

Consensus Brain-derived Protein, Extraction Protocol for the Study of Human and Murine Brain Proteome Using Both 2D-DIGE and Mini 2DE Immunoblotting

Two-dimensional gel electrophoresis (2DE) is a powerful tool to uncover proteome modifications potentially related to different physiological or pathological conditions. Basically, this technique is based on the separation of proteins according to their isoelectric point in a first step, and secondly according to their molecular weights by SDS polyacrylamide gel electrophoresis (SDS-PAGE). In this report an optimized sample preparation protocol for little amount of human post-mortem and mouse brain tissue is described. This method enables to perform both two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and mini 2DE immunoblotting. The combination of these approaches allows one to not only find new proteins and/or protein modifications in their expression thanks to its compatibility with mass spectrometry detection, but also a new insight into markers validation. Thus, mini-2DE coupled to western blotting permits to identify and validate post-translational modifications, proteins catabolism and provides a qualitative comparison among different conditions and/or treatments. Herein, we provide a method to study components of protein aggregates found in AD and Lewy body dementia such as the amyloid-beta peptide and the alpha-synuclein. Our method can thus be adapted for the analysis of the proteome and insoluble proteins extract from human brain tissue and mice models too. In parallel, it may provide useful information for the study of molecular and cellular pathways involved in neurodegenerative diseases as well as potential novel biomarkers and therapeutic targets.

A common protein extraction protocol using urea/thiourea/SDS buffer for human and mice brain tissue allows indentification of proteins by 2D-DIGE and their subsequent characterization by mini 2DE immunoblotting. This method enables one to obtain more reproducible and reliable results from human biopsies and experimental models.

Two-dimensional gel electrophoresis (2DE) is a powerful tool to uncover proteome modifications potentially related to different physiological or ...

Assessment of Dendritic Arborization in the Dentate Gyrus of the Hippocampal Region in Mice

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity and extent of dendritic arborization correlates well with the synaptic plasticity in these cells. A reliable method for assessment of dendritic arborization is needed to characterize the deleterious effects of neurological disorders on these structures and to determine the effects of therapeutic interventions. However, quantification of these structures has proven to be a formidable task given their complex and dynamic nature. Fortunately, sophisticated imaging techniques can be paired with conventional staining methods to assess the state of dendritic arborization, providing a more reliable and expeditious means of assessment. Below is an example of how these imaging techniques were paired with staining methods to characterize the dendritic arborization in wild type mice. These complementary imaging methods can be used to qualitatively and quantitatively assess dendritic arborization that span a rather wide area within the hippocampal region.

We describe two methods for visualization and quantification of dendritic arborization in the hippocampus of mouse models: real-time and extended depth of field imaging. While the former method allows sophisticated topographical tracing and quantification of the extent of branching, the latter allows speedy visualization of the dendritic tree.

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity ...

Intracerebroventricular Injection of Amyloid-&#946; Peptides in Normal Mice to Acutely Induce Alzheimer-like Cognitive Deficits

Amyloid-&#946; (A&#946;) is a major pathological mediator of both familial and sporadic Alzheimer's disease (AD). In the brains of AD patients, progressive accumulation of A&#946; oligomers and plaques is observed. Such A&#946; abnormalities are believed to block long-term potentiation, impair synaptic function, and induce cognitive deficits. Clinical and experimental evidences have revealed that the acute increase of A&#946; levels in the brain allows development of Alzheimer-like phenotypes. Hence, a detailed protocol describing how to acutely generate an AD mouse model via the intracerebroventricular (ICV) injection of A&#946; is necessary in many cases. In this protocol, the steps of the experiment with an A&#946;-injected mouse are included, from the preparation of peptides to the testing of behavioral abnormalities. The process of preparing the tools and animal subjects before the injection, of injecting the A&#946; into the mouse brain via ICV injection, and of assessing the degree of cognitive impairment are easily explained throughout the protocol, with an emphasis on tips for effective ICV injection of A&#946;. By mimicking certain aspects of AD with a designated injection of A&#946;, researchers can bypass the aging process and focus on the downstream pathology of A&#946; abnormalities.

The amyloid-&#946; (A&#946;)-injected animal model enables the administration of a defined quantity and species of A&#946; fragments and reduces individual differences within each study group. This protocol describes the intracerebroventricular (ICV) injection of A&#946; without stereotactic instruments, enabling the production of Alzheimer-like behavioral abnormalities in normal mice.

Amyloid-&#946; (A&#946;) is a major pathological mediator of both familial and sporadic Alzheimer's disease (AD). In the brains of AD patients, ...

The Optical Fractionator Technique to Estimate Cell Numbers in a Rat Model of Electroconvulsive Therapy

Stereological methods are designed to describe quantitative parameters without making assumptions about size, shape, orientation and distribution of cells or structures. These methods have been revolutionary for quantitative analysis of the mammalian brain, in which volumetric cell populations are too high to count manually, and stereology is now the technique of choice whenever estimates of three-dimensional quantities need to be extracted from measurements on two-dimensional sections. All stereological methods are in principle unbiased; however, they rely on proper knowledge about the structure of interest and the characteristics of the tissue. Stereology is based on Systematic Uniformly Random Sampling (SURS), with adjustment of sampling to the most efficient level in respect to precision, providing reliable, quantitative information about the whole structure of interest. Here we present the optical fractionator in conjunction with BrdU immunohistochemistry to estimate the production and survival of newly-formed neurons in the granule cell layer (including the sub-granular zone) of the rat hippocampus following electroconvulsive stimulation, which is among the most potent stimulators of neurogenesis. The optical fractionator technique is designed to provide estimates of the total number of cells from thick sections sampled from the full structure. Thick sections provide the opportunity to observe cells in their full 3-D extent and thus, allow for easy and robust cell classification based on morphological criteria. When correctly implemented, the sensitivity and efficiency of the optical fractionator provides accurate estimates with a fixed and predetermined precision.

Here, we present a stereological method, the optical fractionator, used to quantify the formation of new neurons, and their survival, in the rat hippocampus following electroconvulsive stimulation. When correctly implemented, the sensitivity and efficiency of stereological methods ensures accurate estimates with a fixed and predetermined precision.

Stereological methods are designed to describe quantitative parameters without making assumptions about size, shape, orientation and distribution of cells ...

Improving the Application of High Molecular Weight Biotinylated Dextran Amine for Thalamocortical Projection Tracing in the Rat

High molecular weight biotinylated dextran amine (BDA) has been used as a highly sensitive neuroanatomical tracer for many decades. Since the quality of its labeling was affected by various factors, here, we provide a refined protocol for the application of high molecular weight BDA for studying optimal neural labeling in the central nervous system. After stereotactic injection of BDA into the ventral posteromedial nucleus (VPM) of the thalamus in the rat through a delicate glass pipette, BDA was stained with fluorescent streptavidin-Alexa (AF) 594 and counterstained with fluorescent Nissl stain AF500/525. On the background of green Nissl staining, the red BDA labeling, including neuronal cell bodies and axonal terminals, was more distinctly demonstrated in the somatosensory cortex. Furthermore, double fluorescent staining for BDA and the calcium-binding protein parvalbumin (PV) was carried out to observe the correlation of BDA labeling and PV-positive interneurons in the cortical target, providing the opportunity to study the local neural circuits and their chemical characteristics. Thus, this refined method is not only suitable for visualizing high quality neural labeling with the high molecular weight BDA through reciprocal neural pathways between the thalamus and cerebral cortex, but also will permit the simultaneous demonstration of other neural markers with fluorescent histochemistry or immunochemistry.

Here, we present a refined protocol to effectively reveal biotinylated dextran amine (BDA) labeling with a fluorescent staining method through a reciprocal neural pathway. It is suitable for analyzing the fine structure of BDA labeling and distinguishing it from other neural elements under a confocal laser scanning microscope.

High molecular weight biotinylated dextran amine (BDA) has been used as a highly sensitive neuroanatomical tracer for many decades. Since the quality of ...

A Protocol for Transcranial Photobiomodulation Therapy in Mice

Transcranial photobiomodulation is a potential innovative noninvasive therapeutic approach for improving brain bioenergetics, brain function in a wide range of neurological and psychiatric disorders, and memory enhancement in age-related cognitive decline and neurodegenerative diseases. We describe a laboratory protocol for transcranial photobiomodulation therapy (PBMT) in mice. Aged BALB/c mice (18 months old) are treated with a 660 nm laser transcranially, once daily for 2 weeks. Laser transmittance data shows that approximately 1% of the incident red light on the scalp reaches a 1 mm depth from the cortical surface, penetrating the dorsal hippocampus. Treatment outcomes are assessed by two methods: a Barnes maze test, which is a hippocampus-dependent spatial learning and memory task evaluation, and measuring hippocampal ATP levels, which is used as a bioenergetics index. The results from the Barnes task show an enhancement of the spatial memory in laser-treated aged mice when compared with age-matched controls. Biochemical analysis after laser treatment indicates increased hippocampal ATP levels. We postulate that the enhancement of memory performance is potentially due to an improvement in hippocampal energy metabolism induced by the red laser treatment. The observations in mice could be extended to other animal models since this protocol could potentially be adapted to other species frequently used in translational neuroscience, such as rabbit, cat, dog, or monkey. Transcranial photobiomodulation is a safe and cost-effective modality which may be a promising therapeutic approach in age-related cognitive impairment.

Photobiomodulation therapy is an innovative noninvasive modality for the treatment of a wide range of neurological and psychiatric disorders and can also improve healthy brain function. This protocol includes a step-by-step guide to performing brain photobiomodulation in mice by transcranial light delivery, which can be adapted for use in other laboratory rodents.

Transcranial photobiomodulation is a potential innovative noninvasive therapeutic approach for improving brain bioenergetics, brain function in a wide ...

Visualizing the Calcitonin Gene-Related Peptide Immunoreactive Innervation of the Rat Cranial Dura Mater with Immunofluorescence and Neural Tracing

The aim of this study was to examine the distribution and origin of the calcitonin gene-related peptide (CGRP)-immunoreactive sensory nerve fibers of the cranial dura mater using immunofluorescence, three-dimensional (3D) reconstruction and retrograde tracing technique. Here, the nerve fibers and blood vessels were stained using immunofluorescence and histochemistry techniques with CGRP and fluorescent phalloidin, respectively. The spatial correlation of dural CGRP-immuoreactive nerve fibers and blood vessels were demonstrated by 3D reconstruction. Meanwhile, the origin of the CGRP-immunoreactive nerve fibers were detected by neural tracing technique with fluorogold (FG) from the area around middle meningeal artery (MMA) in the cranial dura mater to the trigeminal ganglion (TG) and cervical (C) dorsal root ganglia (DRGs). In addition, the chemical characteristics of FG-labeled neurons in the TG and DRGs were also examined together with CGRP using double immunofluorescences. Taking advantage of the transparent whole-mount sample and 3D reconstruction, it was shown that CGRP-immunoreactive nerve fibers and phalloidin-labeled arterioles run together or separately forming a dural neurovascular network in a 3D view, while the FG-labeled neurons were found in the ophthalmic, maxillary, and mandibular branches of TG, as well as the C2-3 DRGs ipsilateral to the side of tracer application in which some of FG-labeled neurons presented with CGRP-immunoreactive expression. With these approaches, we demonstrated the distributional characteristics of CGRP-immunoreactive nerve fibers around the blood vessels in the cranial dura mater, as well as the origin of these nerve fibers from TG and DRGs. From the perspective of methodology, it may provide a valuable reference for understanding the complicated neurovascular structure of the cranial dura mater under the physiological or pathological condition.

Here we present a protocol to visualize spatial correlation of calcitonin gene-related peptide (CGRP)-immunoreactive nerve fibers and blood vessels in the cranial dura mater using immunofluorescence and fluorescent histochemistry with CGRP and phalloidin, respectively. In addition, the origin of these nerve fibers was retrograde traced with a fluorescent neural tracer.

The aim of this study was to examine the distribution and origin of the calcitonin gene-related peptide (CGRP)-immunoreactive sensory nerve fibers of the ...

Conventional and Threshold-Tracking Transcranial Magnetic Stimulation Tests for Single-handed Operation

Most single-pulse transcranial magnetic stimulation (TMS) parameters (e.g., motor threshold, stimulus-response function, cortical silent period) are used to examine corticospinal excitability. Paired-pulse TMS paradigms (e.g., short- and long-interval intracortical inhibition (SICI/LICI), short-interval intracortical facilitation (SICF), and short- and long-latency afferent inhibition (SAI/LAI)) provide information about intracortical inhibitory and facilitatory networks. This has long been done by the conventional TMS method of measuring changes in the size of the motor-evoked potentials (MEPs) in response to stimuli of constant intensity. An alternative threshold-tracking approach has recently been introduced whereby the stimulus intensity for a target amplitude is tracked. The diagnostic utility of threshold-tracking SICI in amyotrophic lateral sclerosis (ALS) has been shown in previous studies. However, threshold-tracking TMS has only been used in a few centers, in part due to the lack of readily available software but also perhaps due to uncertainty over its relationship to conventional single- and paired-pulse TMS measurements.
A menu-driven suite of semi-automatic programs has been developed to facilitate the broader use of threshold-tracking TMS techniques and to enable direct comparisons with conventional amplitude measurements. These have been designed to control three types of magnetic stimulators and allow recording by a single operator of the common single- and paired-pulse TMS protocols.
This paper shows how to record a number of single- and paired-pulse TMS protocols on healthy subjects and analyze the recordings. These TMS protocols are fast and easy to perform and can provide useful biomarkers in different neurological disorders, particularly neurodegenerative diseases such as ALS.

We present a suite of standardized single- and paired-pulse transcranial magnetic stimulation (TMS) recording protocols, with options for conventional amplitude measurements and threshold-tracking. This program can control three different types of magnetic stimulators and is designed to enable all tests to be performed conveniently by a single operator.

Most single-pulse transcranial magnetic stimulation (TMS) parameters (e.g., motor threshold, stimulus-response function, cortical silent period) are used ...

Demonstrating Hairy and Glabrous Skin Innervation in a 3D Pattern Using Multiple Fluorescent Staining and Tissue Clearing Approaches

Skin innervation is an important part of the peripheral nervous system. Although the study of the cutaneous nerve fibers has progressed rapidly, most of the understanding of their distributional and chemical characteristics comes from conventional histochemical and immunohistochemical staining on thin tissue sections. With the development of the tissue clearing technique, it has become possible to view the cutaneous nerve fibers on thicker tissue sections. The present protocol describes multiple fluorescent staining on tissue sections at a thickness of 300 &#181;m from the plantar and dorsal skin of rat hindfoot, the two typical hairy and glabrous skin sites. Here, the calcitonin gene-related peptide labels the sensory nerve fibers, while phalloidin and lymphatic vessel endothelial hyaluronan receptor 1 label the blood and lymphatic vessels, respectively. Under a confocal microscope, the labeled sensory nerve fibers were followed completely at a longer distance, running in bundles in the deep cutaneous layer and freestyle in the superficial layer. These nerve fibers ran in parallel to or surrounded the blood vessels, and lymphatic vessels formed a three-dimensional (3D) network in the hairy and glabrous skin. The current protocol provides a more effective approach to studying skin innervation than the existing conventional methods from the methodology perspective.

The thickness of tissue sections limited the morphological study of the skin innervation. The present protocol describes a unique tissue clearing technique to visualize cutaneous nerve fibers in thick 300 &#181;m tissue sections under confocal microscopy.

Skin innervation is an important part of the peripheral nervous system. Although the study of the cutaneous nerve fibers has progressed rapidly, most of ...

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 three essential histological components: the pre-synaptic motor axon terminals, post-synaptic nicotinic acetylcholine receptors (AchRs), and peri-synaptic Schwann cells (PSCs). In order to demonstrate the morphological characteristics of NMJ, the rat medial gastrocnemius muscle was selected as the target-tissue and examined by using multiple fluorescent staining with various kinds of biomarkers, including neurofilament 200 (NF200) and vesicular acetylcholine transporter (VAChT) for the motor nerve fibers and their pre-synaptic terminals, alpha-bungarotoxin (&#945;-BTX) for the post-synaptic nicotinic AchRs, and S100 for the PSCs. In this study, staining was performed in two groups: in the first group, samples were stained with NF200, VAChT, and &#945;-BTX, and in the second group, samples were stained with NF200, &#945;-BTX, and S100. It was shown that both protocols can effectively demonstrate the detailed structure of NMJ. Using the confocal microscope, morphological characteristics of the pre-synaptic terminals, post-synaptic receptors, and PSC were seen, and their Z-stacks images were reconstructed in a three-dimensional pattern to further analyze the spatial correlation among the different labeling. From the perspective of methodology, these protocols provide a valuable reference for investigating the morphological characteristics of NMJ under physiological conditions, which may also be suitable to evaluate the pathological alteration of NMJ, such as peripheral nerve injury and regeneration.

The protocol shows a method to examine spatial correlation among the pre-synaptic terminals, post-synaptic receptors, and peri-synaptic Schwann cells in the rat medial gastrocnemius muscle using fluorescent immunohistochemistry with different biomarkers, namely, neurofilament 200, vesicular acetylcholine transporter, alpha-bungarotoxin, and S100.

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