This work was supported by the National Natural Science Foundation (No. 81971042) and the Key Support Specialist Projects of Shanghai Hongkou District Health Commission (No. HKZK2020A06).

Intrathecal catheterization was carried out in strict accordance with the recommendations in the Guidelines for the Care and Use of Laboratory Animals of the National Institutes of Health, and the protocol was approved by the Ethics Committee of Experimental Animals, China (No. TJBH15523201). Male Sprague-Dawley (SD) rats were used in the experiment. Care was taken to minimize the pain and discomfort of the animals.
1. Material and instrument preparation
NOTE: The preparation of materials and instruments is very important for successful intrathecal catheterization.
<ol>
	<li>Prepare a 15 cm long PE10 tube (the length is determined according to the distance between the rat head and the end of the tail), insert a 20 cm long stainless-steel wire (0.2 mm in diameter) with two polished ends into the PE10 tube as a support, and mark the tube 2 cm from one end to indicate the insertion depth (as marked by black crosses in Figure 1A,B).</li>
	<li>Cut the sharp tip of a 22 G needle and seal the distal end (Figure 1C).</li>
	<li>Cut an epidural catheter (1.0 mm in external diameter) into 1 cm fragments. Then, insert a fragment into a sharp-tip-free 22 G needle (Figure 1D), and seal the distal end of the fragment with a pair of heated straight forceps. This apparatus is called the tube sealing cap (Figure 1E) .</li>
	<li>Prepare a 0.3 cm &#215; 0.5 cm antiallergic band by cutting a silk tape (1.25 cm &#215; 9.1 m) with a pair of scissors (Figure 1F).</li>
</ol>
2. Preparation for surgery
<ol>
	<li>Prepare the instruments for intrathecal catheterization by sterilizing them before surgery. The instruments used for the surgery are toothed forceps, scissors, a gavage apparatus, a scalpel handle and #10 blades. (Figure 2).</li>
	<li>Immerse the PE10 tube and guide wire in 75% ethanol for sterilization for approximately 2 h.</li>
</ol>
3. Surgery
<ol>
	<li>Anesthetize the rat&#160;with 3% isoflurane at a flow rate of 3 L/min.</li>
	<li>Place the rat on the operating table and observe the withdrawal reflex when pinching the hind paw with forceps. The absence of hind paw movement in response to stimulation confirmed successful anesthesia. Administer adequate analgesia by intramuscular injection of 1 mg/kg meloxicam before intrathecal catheterization.</li>
	<li>Remove hair from the lumbar spine region of the back and the area between two ears with a shaver.</li>
	<li>Place a centrifuge tube (3 cm in diameter) under the abdomen of the&#160;rat at the waist-hip junction to increase the flexion in the lumbar spine, giving more room for the needle and the catheter to pass through.</li>
	<li>Sterilize the surgical sites (the area over the lumbar spine region and the area between two ears) with povidone-iodine solution&#160;and then with ethanol solution three times. Cover the rat with an aseptic dressing, and expose the surgical sites. Then wash the PE10 tube and guide wire with normal saline before surgery. 
	NOTE: The tail was not covered so that tail movement could be observed during intrathecal catheterization.</li>
	<li>Determine the location of the intervertebral space between L5 and L6 by locating the spinous process of L6 at the midpoint between the left and right bilateral iliac crests. Fix the skin with the left thumb and the left index finger of the operator, and then make a 3-4 cm long midline incision just above the spinous process between L4 and S1.</li>
	<li>Bluntly separate the subcutaneous tissues with a pair of scissors. Locate the intervertebral space between L5 and L6 again and make a small incision (0.3 - 0.5 cm) on both sides of the L5 and L6 dorsal processes.</li>
	<li>Clamp and lift the L5 dorsal process with a pair of toothed forceps to expand the intervertebral space. Then, bluntly separate the muscles around the vertebral body with a pair of scissors until the top of the L6 dorsal process is completely exposed. 
	NOTE: Removal of any part of the vertebral body and muscles should be avoided, with the aim of minimizing damage to surrounding tissues.</li>
	<li>When the L5 dorsal process is lifted with a pair of toothed forceps and the intervertebral space is expanded with another pair of forceps, clean the L5-6 intervertebral space with a cotton ball until the inverted &#34;V&#34; area is completely exposed.</li>
	<li>Puncture the spine with a 23 G needle in the inverted &#34;V&#34; area just under the top of the L6 dorsal process. 
	NOTE: A tail flick is observed, and/or colorless transparent fluid flows out from the subarachnoid space, indicating a successful puncture into the subarachnoid space.</li>
	<li>Carefully insert the PE10 tube containing stainless-steel wire into the spinal canal at the puncture site, being tilted 30&#176; toward the tail. Adjust the insertion angle until the PE10 tube can be successfully inserted without resistance (a tail flick was observed during this process).</li>
	<li>When the marked area of the PE10 tube reaches the posterior muscle, catheterization is stopped.</li>
	<li>Slowly remove the stainless wire from the PE10 tube. A tail flick may be observed. 
	NOTE: A tail flick may be observed, and after the wire is removed, transparent fluid (or light red fluid) may flow out of the tube.</li>
	<li>Then, connect the PE10 tube to a 1 mL syringe, through which 20 &#181;L of normal saline is injected. After the syringe is removed, saline will flow continuously out of the PE10 tube, indicating that it has been successfully inserted into the subarachnoid space.</li>
	<li>Once the PE10 tube is confirmed to be unobstructed, suture the muscles on one side of the vertebral body with a 4-0 suture and make a knot. Then, tie the suture around the PE10 tube and make another knot. Do not cut the suture; suture the muscles on the other side; tie the suture to the PE10 tube again, make a third knot, and cut the suture. 
	NOTE: This process fixes PE10 tube with a figure-8 suture to reduce the possibility of displacement and retraction of the tube.</li>
	<li>Make a 0.5 cm long incision 1 cm below the midpoint between the ears. Bluntly separate the subcutaneous tissues with scissors, and insert a metal gavage tube toward the tail until the tip is visible in the lumbar incision.</li>
	<li>Insert the distal end of the PE10 tube into the gavage tube until the PE10 tube exits the other end of the gavage tube; then gently withdraw the gavage.</li>
	<li>When the PE10 tube is confirmed to be unobstructed again, suture the remaining muscles around the lumbar incision with a 4-0 suture, tie the suture around the PE10 tube, and make another knot to fix the PE10 tube again.</li>
	<li>Suture the skin, avoiding damage to the PE10 tube. Then, suture the neck skin with a 4-0 suture, tie the suture around the PE10 tube, and make a knot to fix the PE10 tube.</li>
	<li>When the PE10 tube is confirmed to be unobstructed again, seal the extracorporeal end of the PE10 tube with a sealing cap.</li>
	<li>Dry the PE10 tube with a piece of tissue and then tie the antiallergic band around the PE10 tube several times to prevent retraction of the PE10 tube during rat movement.</li>
</ol>
4. Lidocaine validation experiment
<ol>
	<li>After the surgery, return the rat to its cage (one per cage) and closely monitor it during the recovery from anesthesia until the rat gains consciousness.</li>
	<li>After the rat is fully awake, remove the sealing cap and inject 20 &#181;L of 2% lidocaine into PE10 tube at a rate of 0.02 mL/s via a Hamilton syringe, followed by the injection of 10 &#181;L of normal saline.</li>
	<li>Seal the PE10 tube with the sealing cap.</li>
	<li>Place the rat on a table and observe carefully. The presence of hind limb paralysis after an intrathecal injection of lidocaine (from PE10 tube) indicates successful catheterization (Figure 3). Hind limb paralysis usually lasts approximately 30 min10. 
	NOTE: Allow the rat to recover for 5-7 days before the following experiments.</li>
	<li>Closely monitor the rat during the recovery period until complete recovery of limb function.</li>
</ol>

<ol>
	<li>Kong, G., Huang, Z., Zhu, Q., Wan, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+two+modified+methods+of+intrathecal+catheterization+in+rats.">Comparison of two modified methods of intrathecal catheterization in rats.</a> Exp Anim. 69 (2), 219-223 (2020).</li><li>Xu, C. S., Sun, P., Lin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=a+new+design+puncture+needle+and+a+device+of+microcatheter+protection+for+lumbar+intrathecal+catheterization+in+rats.">a new design puncture needle and a device of microcatheter protection for lumbar intrathecal catheterization in rats.</a> Zhongguo Ying Yong Sheng Li Xue Za Zhi. 36 (3), 283-288 (2020).</li><li>Yamamoto, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurosteroid+dehydroepiandrosterone+sulphate+enhances+pain+transmission+in+rat+spinal+cord+dorsal+horn.">Neurosteroid dehydroepiandrosterone sulphate enhances pain transmission in rat spinal cord dorsal horn.</a> Br J Anaesth. 123 (2), e215-e225 (2019).</li><li>Mattioli, T. A., Sutak, M., Milne, B., Jhamandas, K., Cahill, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrathecal+catheterization+influences+tolerance+to+chronic+morphine+in+rats.">Intrathecal catheterization influences tolerance to chronic morphine in rats.</a> Anesth Analg. 114 (3), 690-693 (2012).</li><li>Wang, B. C., Hillman, D. E., Li, D., Turndorf, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lumbar+subarachnoid+catheterization+in+rats.">Lumbar subarachnoid catheterization in rats.</a> Pharmacol Biochem Behav. 38 (3), 685-688 (1991).</li><li>Yaksh, T. L., Rudy, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+catheterization+of+the+spinal+subarachnoid+space.">Chronic catheterization of the spinal subarachnoid space.</a> Physiol Behav. 17 (6), 1031-1036 (1976).</li><li>Martin, H., Kocher, L., Chery-Croze, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+lumbar+intrathecal+catheterization+in+the+rat+with+reduced-length+spinal+compression.">Chronic lumbar intrathecal catheterization in the rat with reduced-length spinal compression.</a> Physiol Behav. 33 (1), 159-161 (1984).</li><li>Mazur, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+simple,+rapid,+and+robust+intrathecal+catheterization+method+in+the+rat.">Development of a simple, rapid, and robust intrathecal catheterization method in the rat.</a> J Neurosci Methods. 280, 36-46 (2017).</li><li>Zhang, S. X., Huang, F., Gates, M., White, J., Holmberg, E. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extensive+scarring+induced+by+chronic+intrathecal+tubing+augmented+cord+tissue+damage+and+worsened+functional+recovery+after+rat+spinal+cord+injury.">Extensive scarring induced by chronic intrathecal tubing augmented cord tissue damage and worsened functional recovery after rat spinal cord injury.</a> J Neurosci Methods. 191 (2), 201-207 (2010).</li><li>Ohara, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+intrathecal+catheterization+in+the+rat.">Long-term intrathecal catheterization in the rat.</a> J Neurosci Methods. 110 (1-2), 81-89 (2001).</li><li>St&#216;rkson, R. V., Kj&#216;rsvik, A., Tj&#216;lsen, A., Hole, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lumbar+catheterization+of+the+spinal+subarachnoid+space+in+the+rat.">Lumbar catheterization of the spinal subarachnoid space in the rat.</a> J Neurosci Methods. 65 (2), 167-172 (1996).</li><li>Poon, Y. Y., Chang, A. Y., Ko, S. F., Chan, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+procedure+for+catheterization+of+the+thoracic+spinal+subarachnoid+space+in+the+rat.">An improved procedure for catheterization of the thoracic spinal subarachnoid space in the rat.</a> Anesth Analg. 101 (1), 155-160 (2005).</li><li>Xu, F., Li, T., Zhang, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+method+for+protecting+and+fixing+the+lumbar+catheters+placed+in+the+spinal+subarachnoid+space+of+rats.">An improved method for protecting and fixing the lumbar catheters placed in the spinal subarachnoid space of rats.</a> J Neurosci Methods. 183 (2), 114-118 (2009).</li><li>Igawa, Y., Andersson, K. E., Post, C., Uvelius, B., Mattiasson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rat+model+for+investigation+of+spinal+mechanisms+in+detrusor+instability+associated+with+infravesical+outflow+obstruction.">A rat model for investigation of spinal mechanisms in detrusor instability associated with infravesical outflow obstruction.</a> Urol Res. 21 (4), 239-244 (1993).</li><li>Hou, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modified+procedure+for+lumbar+intrathecal+catheterization+in+rats.">A modified procedure for lumbar intrathecal catheterization in rats.</a> Neurol Res. 38 (8), 725-732 (2016).</li><li>Farhadi, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+open+and+ultrasound-guided+placement+of+central+venous+catheter+in+children+weighing+less+than+five+kilograms;+a+randomized+clinical+trial.">Comparison of open and ultrasound-guided placement of central venous catheter in children weighing less than five kilograms; a randomized clinical trial.</a> Acad Radiol. 30 (7), 1419-1425 (2023).</li></ol>

The authors of this manuscript declare that there are no conflicts of interest.

There are several critical tips for this modified method to maximize the success rate of intrathecal catheterization. First, a 20 cm long stainless-steel wire with two polished ends should be prepared and inserted into PE10 tube as a support. Second, the operator should completely expose the inverted &#34;V&#34;&#160;area after cleaning the L5-6 intervertebral space with a cotton ball, and the intervertebral space should be expanded with another forceps while lifting the L5 dorsal process with toothed forceps. Third, PE10 tube should be fixed with a figure-8 suture four times. Finally, the extracorporeal end of PE10 tube should be tied with a band and sealed with a self-made cap.
The success rate of intrathecal catheterization and damage to tissues around vertebral bodies may significantly influence the reliability of the experimental results15. Thus, improving the success rate as much as possible and reducing the damage to the surrounding tissues are&#160;crucial in establishing animal models and relevant experiments1. In this modified method, a stainless-steel wire is inserted into PE10 tube for guidance, which increases the elasticity of the tube and improves the success rate of intrathecal catheterization. Moreover, the amount of space needed for operation is reduced with this modified method, and the damage to the tissues around the lumbar spine is minimized because the surrounding tissues are bluntly separated, but not cut. In comparison, in the previously reported method11, a 20 G guide cannula is used to reduce resistance during puncturing, and repeated puncturing is often needed, which may injure the tissues. In addition, in the previously reported method, to reduce the diameter of PE10 tube, it is immersed in warm water (60 &#176;C) and then stretched at one end to approximately 150% of the original length, which may not ensure the consistency of tube diameter and therefore may cause leakage of cerebrospinal fluid because the diameter of 20G guide cannula is approximately two times greater than or equal to that of the stretched PE10 tube. Moreover, in our method, lumbar function is preserved to the greatest extent, which avoids the influence of surgery on the results of subsequent experiments. These results are consistent with those reported by Xu et al2.
In previously reported method11, the length of PE10 tube is approximately 14 cm if the tube is fixed at the site around the puncture site, but the catheter indwelling time is often shorter than 7 days under these conditions (or the tube is removed from the body by the rat). The length of PE10 tube is approximately 28 cm if the tube is fixed at the back of the neck, which is significantly longer than the PE10 tube used in our method (15 cm). Although beads were formed following the protocol reported by St&#248;rkson et al.11, some tubes were removed from the body, and only approximately 65% of the tubes were still fixed in place at 7 days after surgery, which significantly affected the outcomes of subsequent experiments. In our method, PE10 tube is fixed with a figure-8 suture 4 times, and the extracorporeal end of PE10 tube is tied with a band to reduce the possibility of displacement and retraction. Following our method, approximately 85% of the tubes remained in place at 7 days after surgery, and approximately 80% of the tubes remained in place at 28 days after surgery.
In the previously reported method11, the extracorporeal tip of the intrathecal catheter should be cut off for each drug administration. However, the repeated intrathecal administration of drugs may shorten the catheter indwelling time, which makes the intrathecal administration of drugs inconvenient. Therefore, in our method, a self-made cap is used to seal PE10 tube, which is sterilized with ethanol once daily. This not only prevents the leakage of cerebrospinal fluid but also reduces the need for repeated cutting of PE10 tube for intrathecal administration of drugs, ensuring the effective delivery of the drugs.
The advantages and disadvantages of the modified method and previously reported method are summarized in Table 1. First, for the modified method, the use of stainless-steel wire in PE10 tube increases the elasticity of the tube and improves the success rate of intrathecal catheterization, the amount of space needed for the operation is reduced, and the damage to the tissues around the lumbar spine is minimized. In previously reported method, a 20G guide cannula is inserted until resistance is felt, and repeated puncture is often needed, which may result in damage to tissues. In addition, the PE10 tube at one end is stretched until its length reaches approximately 150% of original length, which may cause cerebrospinal fluid leakage because the diameter of the 20G guide cannula is 2 times greater than or equal to that of stretched PE10 tube. Second, in the modified method, the length of PE10 tube is determined before surgery, and the catheter indwelling time can be longer than one week. In previously reported method, the length of PE10 tube is approximately 14 cm if it is fixed at the puncture site, but the catheter indwelling time is often shorter than 7 days because the tube is susceptible to being pulled out of the body by the rat; the length of PE10 tube is approximately 28 cm if it is fixed at the back of the neck, which is significantly longer than the length of the tube used in our method. Third, in the modified method, the PE10 tube is fixed with a figure-8 suture 4 times to prevent tube movement and retraction; a self-made cap is used to seal PE10 tube, which not only prevents leakage of cerebrospinal fluid but also prevents the need for repeated cutting of PE10 tube. In the previously reported method, it is difficult to obtain beads with a consistent diameter, the displacement of PE10 tube is common when beads are formed, and repeated cutting of PE10 tube is often needed. Finally, in the modified method, the extracorporeal end of PE10 tube is tied with a band, which prevents the tube from retracting during movement. However, in the prior method, the beads cannot reliably prevent PE10 tube retraction because it is difficult to obtain beads with a consistent diameter.
Overall, this modified method for intrathecal catheterization has the following advantages. First, the use of stainless-steel wire in PE10 tube increases the elasticity of the tube and improves the success rate of intrathecal catheterization, the amount space needed for the operation is reduced, and damage to the tissues around the lumbar spine is minimized, which preserves the lumbar function to the greatest extent possible and avoids the influence of surgery on the results of subsequent experiments. Second, PE10 tube is fixed with a figure-8 suture 4 times, which prevents tube movement and retraction during movement. Third, a self-made sealing cap is used to seal PE10 tube, which not only prevents cerebrospinal fluid leakage but also prevents the need for repeated cutting of PE10 tube. Repeated cutting of the catheter may shorten the catheter, which makes the delivery of drugs inconvenient. Finally, the extracorporeal end of PE10 tube is tied with an antiallergic band, which prevents the tube from retracting during movement.
However, there are several limitations in this modified intrathecal catheterization technique. First, after surgery, the rats need to be housed separately (one per cage) to avoid damage to the extracorporeal end of PE10 tube. Second, recovery for 5-7 days after intrathecal injection of lidocaine is needed before subsequent experiments.
In conclusion, this modified method for intrathecal catheterization may serve as a useful tool for the repetitive intrathecal administration of drugs and represent&#160;a simple, convenient, and reliable way to shorten the duration of experiments.

Intrathecal catheterization (also known as subarachnoid catheterization) in rats is a method that involves inserting a catheter into the subarachnoid space through the intervertebral space1. Drugs are directly injected into the subarachnoid space through the catheter, which helps researchers investigate the effects of drugs on the spinal cord without considering the effects of drugs that penetrate the blood-brain barrier2,3. Moreover, cerebrospinal fluid can be collected following intrathecal catheterization to investigate the microenvironment of the central nervous system4,5. The currently used method for intrathecal catheterization was first established by Yaksh and Rudy6 in 1976, and since then, it has been widely applied in animal experiments in the fields of neuroscience, anesthesia and analgesia, spinal cord-mediated cardiovascular regulation, and especially neuropathic pain2,7. However, this method still has several limitations, such as a high incidence of spinal cord damage, subarachnoid hemorrhage, postoperative sensory and motor dysfunction, high postoperative mortality, and a high risk of neurological impairment4,5,8,9,10. In an attempt to overcome these limitations, catheterization of the subarachnoid space through lumbar interspaces was proposed by St&#248;rkson et al. in 199611, and a higher postsurgical success rate was reported. Notably, the fixation of indwelling catheter is still a challenge in this method, and catheter retraction is common due to animal movement, which makes intrathecal drug administration inconvenient.
Because of the above limitations, some investigators12,13,14,15 have attempted to improve the tools for puncturing, the methods of catheterization, and the methods of catheter fixation, but the available methods still need to be modified due to the difficulty&#160;in quantifying the diameter of the beads used, the need for repeated punctures and the short length of the catheter, etc.11
According to the lumbar approach for intrathecal catheterization1 and the Seldinger technique for central vein catheterization,16 we developed a method for intrathecal catheterization in rats that uses a stainless-steel wire, a self-made sealing cap, and an antiallergic band to simplify the existing method. Through this method, the catheter can be easily inserted into the subarachnoid space and stably fixed on the back of the rat, and the need for repeated punctures for repeated intrathecal drug administration is avoided.
Herein, we introduce a modified method that may improve the success rate of intrathecal catheterization in rats and represent&#160;a simple, convenient, and reliable approach for repetitive intrathecal administration of drugs.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 cc syringe</td>
			<td></td>
			<td>Jiangxi Hongda Medical Equipment Co., Ltd</td>
			<td>1 cc</td>
		</tr>
		<tr>
			<td>22 gauge &#215; 1&#8221; needles</td>
			<td></td>
			<td>Jiangxi Hongda Medical Equipment Co., Ltd</td>
			<td>22G</td>
		</tr>
		<tr>
			<td>23 gauge &#215; 1&#8221; needles</td>
			<td></td>
			<td>Jiangxi Hongda Medical Equipment Co., Ltd</td>
			<td>23G</td>
		</tr>
		<tr>
			<td>25 &#956;L Hamilton Syringes</td>
			<td></td>
			<td>Shanghai Bolige Co.,Ltd</td>
			<td>0.31mm 25 &#956;L&#160;</td>
		</tr>
		<tr>
			<td>4-O MERSILK NON-ABSORBABLE SUTURE</td>
			<td></td>
			<td>ETHICON</td>
			<td>SA83G</td>
		</tr>
		<tr>
			<td>50 mL corning centrifuge tubes 3 cm diameter</td>
			<td>430820</td>
			<td>CORNING</td>
			<td></td>
		</tr>
		<tr>
			<td>Epidural catheter and connector</td>
			<td></td>
			<td>Henan Tuoren Medical Device Co., Ltd</td>
			<td>regular type</td>
		</tr>
		<tr>
			<td>Gavage apparatus</td>
			<td></td>
			<td>Shanghai Bolige Co.,Ltd</td>
			<td>8#</td>
		</tr>
		<tr>
			<td>PE-10 Mirco Medical Tubing</td>
			<td>BB31695-PE/1</td>
			<td>Scientific Commodities, Inc</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel handle and #10 blades</td>
			<td></td>
			<td>Jiangsu Songxin Medical Equipment Co., Ltd</td>
			<td>125mm</td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td></td>
			<td>Jiangsu Songxin Medical Equipment Co., Ltd</td>
			<td>100mm</td>
		</tr>
		<tr>
			<td>Sprague-Dawley (SD) rats</td>
			<td></td>
			<td>Shanghai BK/KY Biotechnology Co., Ltd</td>
			<td>Male</td>
		</tr>
		<tr>
			<td>Stainless steel wire 0.2 mm diameter</td>
			<td></td>
			<td>Dongguan Jiazhi Metal Products Technology Co., Ltd.</td>
			<td>0.2mm&#160; &#215;&#160; 1m</td>
		</tr>
		<tr>
			<td>Toothed forceps</td>
			<td></td>
			<td>Jiangsu Songxin Medical Equipment Co., Ltd</td>
			<td>18cm</td>
		</tr>
		<tr>
			<td>URGO silk tape</td>
			<td></td>
			<td>URGO</td>
			<td>1.25cm &#215; 9.1m</td>
		</tr>
	</tbody>
</table>

a modified method for intrathecal catheterization in rats

Intrathecal catheterization has been widely applied in animal experiments, especially those on neuropathic pain. However, the traditional methods still have&#160;several limitations. Although some investigators have attempted to improve the traditional methods, the available methods still need to be modified. Herein, we introduce a modified method for intrathecal catheterization in rats.
This method uses a 20 cm long stainless-steel wire (0.2 mm in diameter), a 15 cm long plastic PE10 tube, a self-made sealing cap, and a 0.3 cm &#215; 0.5 cm anti-allergic band. Our modified method for intrathecal catheterization has several advantages. First, introducing a stainless-steel wire to PE10 tube increases the elasticity of the tube, improves the success rate of intrathecal catheterization, reduces the amount of space required for the operation, and minimizes the damage to the tissues around the lumbar spine. Second, the length of PE10 tube is determined before the surgery, and catheter indwelling time can be longer than one week. Third, the PE10 tube is fixed by a figure-8 suture, 4 times, which prevents tube movement and retraction when the animal moves. Fourth, a&#160;self-made sealing cap is used to seal the PE10 tube, which not only prevents cerebrospinal fluid leakage but also reduces the need for repeated cutting of PE10 tube. Finally, the extracorporeal end of PE10 tube is tied with a band, which prevents tube retraction when the animal moves.
This method can increase the catheterization success rate in rats, as approximately 80% of PE10 tubes remained in place even 28 days after surgery. Thus, this modified method may represent a simple, convenient, and reliable approach for repetitive intrathecal drug administration.

For intrathecal injection, the extracorporeal tip of PE10 tube was cut off, and PE10 tube was sealed with a sealing cap between two drug injections. In our pilot study, the success rate of intrathecal catheterization was approximately 95% (19 out of 20 rats); success was indicated by a tail flick and/or the release of colorless transparent fluid during the procedure. Approximately 85% of the tubes remained in place 7&#160;days after surgery, and approximately 80% remained in place 28 days after surgery. The rats recovered soon after the operation, and no complications were observed within 7 days after surgery. The daily movement was normal, and behavioral abnormality was not observed. These results indicate our method is superior to those previously reported in terms of the success rate and long-indwelling rate.
Complete paralysis of the lower limbs upon lidocaine injection via catheters indicates successful intrathecal catheterization15. The success rate of intrathecal catheterization is calculated by dividing the total number of rats by the number of rats with successful catheterization. With our modified method, the success rate was 95%, which was greater than the rate achieved with the method reported by Hou et al. (88%)15. This is shown in Figure 4.
The intrathecal tube was monitored at 2, 5, and 7 days after intrathecal catheterization and the rate of successful indwelling catheter was calculated as number of rats with successful indwelling catheter/total number of rats &#215; 100%. At&#160;2, 5, and 7 days after intrathecal catheterization, the rate of successful indwelling catheter was 94%, 81%, and 65%, respectively, in the study of St&#216;rkson et al.11. The rate of successful indwelling catheter at 2, 5 and 7 days after intrathecal catheterization was 95%, 90% and 85%, respectively, with our technique (Figure 5).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66487/66487fig1.jpg" /> 
Figure 1. Materials and instruments used for intrathecal catheterization. (A)&#160;A 15-cm long PE10 tube was prepared, and the tube was marked 2 cm from one end to indicate the insertion depth. (B)&#160;A 20-cm long stainless-steel wire with two polished ends was inserted into the PE10 tube as a support. (C)&#160;The sharp tip of the 22G needle was cut with a pair of scissors, and the distal end was sealed with a pair of forceps. (D) An epidural catheter (1.0 mm in external diameter) was cut into 1-cm fragments, which were then inserted into the sharp-tip-free 22G needle. (E)&#160;The distal end of the epidural catheter was sealed with a pair of heated straight forceps; this apparatus was called the tube sealing cap. (F)&#160;A 0.3 cm &#215; 0.5 cm anti-allergic band (silk tape, 1.25 cm &#215; 9.1 m) was prepared with a pair of scissors. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/66487/66487fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66487/66487fig2.jpg" /> 
Figure 2. Preparation of instruments for intrathecal catheterization. Instruments (such as toothed forceps, scissors, a gavage apparatus, a scalpel handle, and #10 blades) were sterilized with ethanol for approximately 2 h and then washed with normal saline approximately 30 min before surgery. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/66487/66487fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66487/66487fig3.jpg" /> 
Figure 3. Results of the lidocaine validation experiment after intrathecal catheterization. After intrathecal injection of 20 &#181;L&#160;of 2% lidocaine followed by injection of 10 &#181;L of normal saline, the rat was&#160;temporarily paralyzed: lower limb paralysis occurred within 30 s and disappeared 30 min later, indicating successful intrathecal catheterization. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/66487/66487fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66487/66487fig4.jpg" /> 
Figure 4. Comparison of the rate of successful catheterization between our modified method and a previously reported method. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/66487/66487fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/66487/66487fig5.jpg" /> 
Figure 5. Comparison of the long-indwelling catheter rate between our modified method and a previously reported method. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/66487/66487fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#12288;</td>
			<td>Modified method</td>
			<td>Prior method</td>
			<td>Advantages of modified method</td>
			<td>Disadvantages of prior method</td>
		</tr>
		<tr>
			<td rowspan="4">Guiding method for insertion</td>
			<td rowspan="4">a stainless-steel wire</td>
			<td rowspan="4">Guide-canula (20G 0.9&#215; 38 mm)</td>
			<td>Increases the elasticity of the tube,</td>
			<td>The resistance is difficult to feel, increasing the difficulty of operation</td>
		</tr>
		<tr>
			<td>Improves the success rate of intrathecal catheterization</td>
			<td>Damage to the tissues due to repeated puncture</td>
		</tr>
		<tr>
			<td>Reduces the requirement of operative space</td>
			<td>One end of the tube is stretched to 1.5 times original length, making the diameter of both ends different</td>
		</tr>
		<tr>
			<td>Minimizes the damage to tissues around the lumbar spine</td>
			<td>Susceptibility to cerebrospinal fluid leakage because the diameter of 20G guide-canula is 2 times or more that of stretched PE10 tube</td>
		</tr>
		<tr>
			<td>Length of PE10 tube</td>
			<td>15 cm</td>
			<td>14 or 28 cm</td>
			<td>Easy to determine the length of PE10 tube regardless of the duration of catheter indwelling</td>
			<td>Duration of catheter indwelling is shorter for shorter PE10 tube; susceptibility to falling out of the body for long PE10 tube</td>
		</tr>
		<tr>
			<td>Fixation method</td>
			<td>&#34;8&#34; suture and 4 times</td>
			<td>1 or 2 beads</td>
			<td>Avoids the tube movement and retraction during the animal activities</td>
			<td>Difference in the diameter of the tube at both ends, and susceptibility to displacement of PE10 tube during the bead-making </td>
		</tr>
		<tr>
			<td rowspan="2">Method for tube sealing</td>
			<td rowspan="2">Self-made cap</td>
			<td rowspan="2">No</td>
			<td>Prevents the leakage of cerebrospinal fluid </td>
			<td rowspan="2">Requirement of repeated cutting of PE10 tube </td>
		</tr>
		<tr>
			<td>Avoids the repeated cutting of PE10 tube </td>
		</tr>
		<tr>
			<td>Method for prevention of retraction</td>
			<td>Anti-allergic band</td>
			<td>1 or 2 beads</td>
			<td>Prevents the tube from retraction during the animal activities </td>
			<td>Susceptibility to retraction</td>
		</tr>
	</tbody>
</table>
Table 1.&#160;Advantages and disadvantages of the modified method and a previously reported method.

Herein, we introduce a modified method for intrathecal catheterization in rats that represents a simple, convenient, and reliable approach for repetitive intrathecal drug administration.

A Modified Method for Intrathecal Catheterization in Rats

Intrathecal catheterization has been widely applied in animal experiments, especially those on neuropathic pain. However, the traditional methods still ...

a-modified-method-for-intrathecal-catheterization-in-rats

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

403 Views.  Tongji University. Herein, we introduce a modified method for intrathecal catheterization in rats that represents a simple, convenient, and reliable approach for repetitive intrathecal drug administration.

Video: A Modified Method for Intrathecal Catheterization in Rats

Basic Surgical Techniques in the G&ouml;ttingen Minipig: Intubation, Bladder Catheterization, Femoral Vessel Catheterization, and Transcardial Perfusion

The emergence of the G&ouml;ttingen minipig in research of topics such as neuroscience, toxicology, diabetes, obesity, and experimental surgery reflects the close resemblance of these animals to human anatomy and physiology 1-6.The size of the G&ouml;ttingen minipig permits the use of surgical equipment and advanced imaging modalities similar to those used in humans 6-8. The aim of this instructional video is to increase the awareness on the value of minipigs in biomedical research, by demonstrating how to perform tracheal intubation, transurethral bladder catheterization, femoral artery and vein catheterization, as well as transcardial perfusion. 
Endotracheal Intubation should be performed whenever a minipig undergoes general anesthesia, because it maintains a patent airway, permits assisted ventilation and protects the airways from aspirates. Transurethral bladder catheterization can provide useful information about about hydration state as well as renal and cardiovascular function during long surgical procedures. Furthermore, urinary catheterization can prevent contamination of delicate medico-technical equipment and painful bladder extension which may harm the animal and unnecessarily influence the experiment due to increased vagal tone and altered physiological parameters. Arterial and venous catheterization is useful for obtaining repeated blood samples and monitoring various physiological parameters. Catheterization of femoral vessels is preferable to catheterization of the neck vessels for ease of access, when performing experiments involving frame-based stereotaxic neurosurgery and brain imaging. When performing vessel catheterization in survival studies, strict aseptic technique must be employed to avoid infections6. Transcardial perfusion is the most effective fixation method, and yields preeminent results when preparing minipig organs for histology and histochemistry2,9. For more information about anesthesia, surgery and experimental techniques in swine in general we refer to Swindle 2007. Supplementary information about premedication and induction of anesthesia, assisted ventilation, analgesia, pre- and postoperative care of G&ouml;ttingen minipigs are available via the internet at <a href="http://www.minipigs.com">http://www.minipigs.com</a>10. For extensive information about porcine anatomy we refer to Nickel et al. Vol. 1-511.

Basic Surgical Techniques in the G&#246;ttingen Minipig: Intubation, Bladder Catheterization, Femoral Vessel Catheterization, and Transcardial Perfusion

The use of domestic and miniature pigs in science has increased significantly in recent years. By demonstrating how to perform intubation, transurethral bladder catheterization, femoral artery and vein catheterization, as well as transcardial perfusion, we aim to further increase the value of G&ouml;ttingen minipigs in biomedical research.

The emergence of the G&ouml;ttingen minipig in research of topics such as neuroscience, toxicology, diabetes, obesity, and experimental surgery reflects ...

Femoral Arterial and Venous Catheterization for Blood Sampling, Drug Administration and Conscious Blood Pressure and Heart Rate Measurements

In multiple fields of study, access to the circulatory system in laboratory studies is necessary. Pharmacological studies in rats using chronically implanted catheters permit a researcher to effectively and humanely administer substances, perform repeated blood sampling and assists in conscious direct measurements of blood pressure and heart rate. Once the catheter is implanted long-term sampling is possible. Patency and catheter life depends on multiple factors including the lock solution used, flushing regimen and catheter material. This video will demonstrate the methodology of femoral artery and venous catheterization of the rat. In addition the video will demonstrate the use of the femoral venous and arterial catheters for blood sampling, drug administration and use of the arterial catheter in taking measurements of blood pressure and heart rate in a conscious freely-moving rat. A tether and harness attached to a swivel system will allow the animal to be housed and have samples taken by the researcher with minimal disruption to the animal. To maintain patency of the catheter, careful daily maintenance of the catheter is required using lock solution (100 U/ml heparinized saline), machine-ground blunt tip syringe needles and the use of syringe filters to minimize potential contamination. With careful aseptic surgical techniques, proper catheter materials and careful catheter maintenance techniques, it is possible to sustain patent catheters and healthy animals for long periods of time (several weeks).

Chronic catheterization of blood vessels in the rat is often required for administration of substances, obtain blood sample over a period of time or for direct conscious blood pressure measurements. Femoral arterial catheterization of the rat and corresponding measurements of blood pressure in the conscious animal will be demonstrated.

In multiple fields of study, access to the circulatory system in laboratory studies is necessary. Pharmacological studies in rats using chronically ...

Roux-en-Y Gastric Bypass Operation in Rats

Currently, the most effective therapy for the treatment of morbid obesity to induce significant and maintained body weight loss with a proven mortality benefit is bariatric surgery1,2. Consequently, there has been a steady rise in the number of bariatric operations done worldwide in recent years with the Roux-en-Y gastric bypass (gastric bypass) being the most commonly performed operation3. Against this background, it is important to understand the physiological mechanisms by which gastric bypass induces and maintains body weight loss. These mechanisms are yet not fully understood, but may include reduced hunger and increased satiation4,5, increased energy expenditure6,7, altered preference for food high in fat and sugar8,9, altered salt and water handling of the kidney10 as well as alterations in gut microbiota11. Such changes seen after gastric bypass may at least partly stem from how the surgery alters the hormonal milieu because gastric bypass increases the postprandial release of peptide-YY (PYY) and glucagon-like-peptide-1 (GLP-1), hormones that are released by the gut in the presence of nutrients and that reduce eating12.
During the last two decades numerous studies using rats have been carried out to further investigate physiological changes after gastric bypass. The gastric bypass rat model has proven to be a valuable experimental tool not least as it closely mimics the time profile and magnitude of human weight loss, but also allows researchers to control and manipulate critical anatomic and physiologic factors including the use of appropriate controls. Consequently, there is a wide array of rat gastric bypass models available in the literature reviewed elsewhere in more detail 13-15. The description of the exact surgical technique of these models varies widely and differs e.g. in terms of pouch size, limb lengths, and the preservation of the vagal nerve. If reported, mortality rates seem to range from 0 to 35%15. Furthermore, surgery has been carried out almost exclusively in male rats of different strains and ages. Pre- and postoperative diets also varied significantly.
Technical and experimental variations in published gastric bypass rat models complicate the comparison and identification of potential physiological mechanisms involved in gastric bypass. There is no clear evidence that any of these models is superior, but there is an emerging need for standardization of the procedure to achieve consistent and comparable data. This article therefore aims to summarize and discuss technical and experimental details of our previously validated and published gastric bypass rat model.

Numerous studies using gastric bypass rat models have been recently conducted to uncover the underlying physiological mechanisms of Roux-en-Y gastric bypass operations. This article aims to demonstrate and discuss the technical and experimental details of our published gastric bypass rat model to understand advantages and limitations of this experimental tool.

Currently, the most effective therapy for the treatment of morbid obesity to induce significant and maintained body weight loss with a proven mortality ...

A Modified Precipitation Method to Isolate Urinary Exosomes

Identification of biomarkers that allow early detection of kidney diseases in urine and plasma has been an area of active interest for several years. Urinary exosome vesicles, 40-100 nm in size, are released into the urine under normal conditions by cells from all nephron segments and may contain protein, mRNA and microRNA representative of their cell type of origin. Under conditions of renal dysfunction or injury, exosomes may contain altered proportions of these components, which may serve as biomarkers for disease. There are currently several methods available for isolation of urinary exosomes, and we have previously conducted an experimental comparison of each of these approaches, including three based on ultracentrifugation, one using a nanomembrane ultrafiltration concentrator, one using a commercial precipitation reagent and one using a modification of the precipitation technique using ExoQuick reagent that we developed in our laboratory. We found the modified precipitation method produced the highest yield of exosome particles, miRNA, and mRNA, making this approach suitable for the isolation of exosomes for subsequent RNA profiling. We conclude that the modified exosome precipitation method offers a quick, scalable, and effective alternative for the isolation of exosomes from urine. In this report, we describe our modified precipitation technique using ExoQuick reagent for isolating exosomes from human urine.

This manuscript describes a protocol for isolating exosomes from human urine using a modified precipitation technique.

Identification of biomarkers that allow early detection of kidney diseases in urine and plasma has been an area of active interest for several years. ...

A Modified Method for Heterotopic Mouse Heart Transplantion

Mice are often used as heart transplant donors and recipients in studies of transplant immunology due to the wide range of transgenic mice and reagents available. A difficulty is presented due to the small size of the animal and the considerable technical challenges of the microsurgery involved in heart transplantation. In particular, a high rate of technical failure early after transplantation may result from recipient death and post-operative complications such as hind limb paralysis or a non-beating heart. Here, the complete technique for heterotopic mouse heart transplantation is demonstrated, involving harvesting the donor heart and its subsequent implantation into a recipient mouse. The donor heart is harvested immediately following in situ perfusion with cold heparinized saline and transection of the ascending aorta and pulmonary artery. The recipient operation involves preparation of the abdominal aorta and inferior vena cava (IVC), followed by end-to-side anastomosis of the donor aorta with the recipient aorta using a single running 10-0 microsuture and a similar anastomosis of the donor pulmonary artery with the recipient IVC. Following the operation the animal is injected with 0.6 ml normal saline subcutaneously and allowed to recover on a 37&#160;&#176;C heating pad. The results from 227 mouse heart transplants are summarized with a success rate at 48 hr of 86.8%. Of the 13.2% failures within 48 hr, 5 (2.2%) experienced hind limb paralysis, 10 (4.4%) had a non-beating heart due to graft ischemic injury and/or thrombosis, while 15 (6.6%) died within 48 hr.

A technique is demonstrated for the microsurgical procedure for heterotopic transplantation of hearts in mice, including simplified methods for donor harvesting and recipient vessel anastomosis.

Mice are often used as heart transplant donors and recipients in studies of transplant immunology due to the wide range of transgenic mice and reagents ...

A Method for Quantifying Upper Limb Performance in Daily Life Using Accelerometers

A key reason for referral to rehabilitation services after stroke and other neurological conditions is to improve one's ability to function in daily life. It has become important to measure a person's activities in daily life, and not just measure their capacity for activity in the structured environment of a clinic or laboratory. A wearable sensor that is now enabling measurement of daily movement is the accelerometer. Accelerometers are commercially-available devices resembling large wrist watches that can be worn throughout the day. Data from accelerometers can quantify how the limbs are engaged to perform activities in peoples' homes and communities. This report describes a methodology to collect accelerometry data and turn it into clinically-relevant information. First, data are collected by having the participant wear two accelerometers (one on each wrist) for 24 h or longer. The accelerometry data are then downloaded and processed to produce four different variables that describe key aspects of upper limb activity in daily life: hours of use, use ratio, magnitude ratio, and the bilateral magnitude. Density plots can be constructed that visually represent the data from the 24 h wearing period. The variables and their resultant density plots are highly consistent in neurologically-intact, community-dwelling adults. This striking consistency makes them a useful tool for determining if upper limb daily performance is different from normal. This methodology is appropriate for research studies investigating upper limb dysfunction and interventions designed to improve upper limb performance in daily life in people with stroke and other patient populations. Because of its relative simplicity, it may not be long before it is also incorporated in clinical neurorehabilitation practice.

This protocol describes a method to quantify upper limb performance in daily life using wrist-worn accelerometers.

A key reason for referral to rehabilitation services after stroke and other neurological conditions is to improve one's ability to function in daily life. ...

Brachial Artery Catheterization in Swine

The video describes in detail the catheterization of the distal brachial artery in swine. This technique enables researchers to measure arterial blood pressure continuously and collect arterial blood samples to assess arterial blood gas measurements. Arterial blood pressures and arterial blood gases are important physiological parameters to monitor during experimental procedures. In swine, four common methods of arterial catheterization have been described, including catheterization of the carotid, femoral, auricular, and medial saphenous arteries. Each of these techniques have advantages, such as ease of access for the auricular artery, and disadvantages that include deep tissue dissection for carotid artery catheterization. The described alternative method of arterial catheterization in swine, the catheterization of the distal aspect of the brachial artery, is a rapid procedure that requires relatively minimal tissue dissection and provides information that is in line with data collected from other arterial catheterization sites. The procedure uses a medial approach along an oblique plane of the lower brachium, positioned between the olecranon and the flexor aspect of the elbow joint, and this approach allows researchers the major advantage of unimpeded freedom for procedures that involve the caudoventral, caudodorsal back, or hind limbs of the pig. Due to the location of the upper forelimb of the catheterized vessel and potential challenges of effective homeostasis following catheter removal from the artery, this technique may be limited to non-recovery procedures.

The video describes in detail the catheterization of the distal brachial artery in swine. This procedure accurately measures arterial blood pressure and is a simple and fast method to collect samples for arterial blood gas measurements.

The video describes in detail the catheterization of the distal brachial artery in swine. This technique enables researchers to measure arterial blood ...

Intrathecal Application of a Fluorescent Dye for the Identification of Cerebrospinal Fluid Leaks in Cochlear Malformation

In cases of cerebrospinal fluid (CSF) leaks, reliable detection of their origins is needed to seal the leak sufficiently and prevent complications, such as meningitis. A method is presented here using intrathecal administered fluorescein in a clinical case of bilateral congenital ear malformation. A fluorescent dye is administered intrathecally to achieve intraoperative visualization of CSF leaks. The dye is applied 20 min before surgery, and concentration of 5% is used. Per every 10 kg of body weight, 0.1 mL of the fluid is applied intrathecally. The fluorescein is visualized using a fully digital microscope. The origin of the fluid leak is identified in the stapes footplate. During primary surgery, it is sealed, and cochlea implantation is performed for hearing restoration. In this specific case, 6 weeks later, the implant was explanted due to acute meningitis, and the electrode array was left as a spacer. Postoperatively, in the aural smear, &#946;-transferrin was detected. During a revision mastoidectomy, dislocated coverage of the leak was found. The stapes was removed and oval window sealed. Five days after revision surgery, no &#946;-transferrin was detected in the aural smear. During the revision of cochlea implantation 6 months later, intact coverage of the oval niche was observed. Thus, intrathecal fluorescein application proves to be a reliable tool for the detection of CSF leaks. It facilitates the orientation in malformations and complicated or unknown surgical situs. In the literature, its use is described for CSF fistulas in endonasal surgery but is rarely described in skull base and mastoid surgeries. The method has been used successfully in several cases with CSF leaks, and the results confirm the feasibility of safely accessing the origin of the leak.

Intrathecally applied fluorescein is used to achieve intraoperative visualization of CSF leaks. This protocol describes a lumbar puncture, the application of 5% fluorescein, and intraoperative visualization using a fully digital microscope.

In cases of cerebrospinal fluid (CSF) leaks, reliable detection of their origins is needed to seal the leak sufficiently and prevent complications, such ...

Semi-Minimal Invasive Method to Induce Myocardial Infarction in Rats and the Assessment of Cardiac Function by an Isolated Working Heart System

Myocardial infarction (MI) remains the main contributor to morbidity and mortality worldwide. Therefore, research on this topic is mandatory. An easily and highly reproducible MI induction procedure is required to obtain further insight and better understanding of the underlying pathological changes. This procedure can also be used to evaluate the effects or potency of new and promising treatments (as drugs or interventions) in acute MI, subsequent remodeling and heart failure (HF). After intubation and pre-operative preparation of the animal, an anesthetic protocol with isoflurane was performed, and the surgical procedure was conducted&#160;quickly. Using a minimally invasive approach, the left anterior descending artery (LAD) was located and occluded by a ligature. The occlusion can be performed acutely for subsequent reperfusion (ischemia/reperfusion injury). Alternatively, the vessel can be ligated permanently to investigate the development of chronic MI, remodeling or HF. Despite common pitfalls, the drop-out rates are minimal. Various treatments such as remote ischemic conditioning can be examined for their cardioprotective potential pre-, peri- and post-operatively. The post-operative recovery was quick as the anesthesia was precisely controlled and the duration of the operation was short. Post-operative analgesia was administered for three days. The minimally invasive procedure reduces the risk of infection and inflammation. Furthermore, it facilitates rapid recovery. The &#8220;working heart&#8221; measurements were performed ex vivo and enabled precise control of preload, afterload and flow. This procedure requires specific equipment and training for adequate performance. This manuscript provides a detailed step-by-step introduction for conducting these measurements.

This article presents an efficient method to perform myocardial ischemia and subsequent chronic reperfusion in rats using a minimally invasive approach. In addition, left ventricular hemodynamic function of rats is assessed by echocardiography and isolated working heart methods.&#160;

Myocardial infarction (MI) remains the main contributor to morbidity and mortality worldwide. Therefore, research on this topic is mandatory. An easily ...

A Modified Simple Method for Induction of Myocardial Infarction in Mice

Myocardial infarction (MI) represents one of the leading causes of death. MI models are widely used for investigating the pathomechanisms of post-MI remodeling and evaluation of novel therapeutics. Different methods (e.g., isoproterenol treatment, cryoinjury, coronary artery ligation, etc.) have been used to induce MI. Compared with isoproterenol treatment and cryoinjury, coronary artery ligation may better reflect the ischemic response and chronic remodeling after MI. However, traditional methods for coronary ligation in mice are technically challenging. The current study describes a simple and efficient process for induction of MI in mice with readily available materials. The mouse chest skin was cut open under stable anesthesia. The heart was immediately externalized through the intercostal space after blunt separation of the pectoralis major and pectoralis minor. The left anterior descending branch (LAD) was ligated with a 6-0 suture 3 mm from its origin. Following LAD ligation, staining with 2,3,5-Triphenyltetrazolium chloride (TTC) indicated successful induction of MI and temporal changes of post-MI scar size. Meanwhile, survival analysis results showed overt mortality within 7 days after MI, mainly due to cardiac rupture. Moreover, post-MI echocardiographic assessment demonstrated successful induction of contractile dysfunction and ventricular remodeling. Once mastered, an MI model can be established in mice within 2-3 min with readily available materials.

Under adequate anesthesia, the mouse heart was externalized through the intercostal space, and myocardial infarction was successfully induced by ligating the left anterior descending artery (LAD) using materials readily available in most laboratories.

Myocardial infarction (MI) represents one of the leading causes of death. MI models are widely used for investigating the pathomechanisms of post-MI ...