Venous catheterization, endotracheal intubation, and mechanical ventilation are necessary for performing total intravenous general anesthesia in rats. Intubation and IV cannulation of the rat is challenging because of the animals’ small size and the lack of equipment specifically designed for the restricted anatomical dimensions. Here, we present methods for tail venous catheterization and intubation that are quickly learned by the provider with clinical operating room experience but lack experience in the lab. For tail venous catheterization, each rat (n = 20) was gently restrained in a rat chamber; its tail was placed in warm water for 10 minutes, and a 24 gauge intravenous catheter was inserted into the lateral tail vein. The catheter was fixed in place using tape and attached to a T-connector for drug administration. A bolus of propofol (n = 10), ketamine (n = 7), or etomidate (n = 3) was administered to achieve rapid deep anesthesia. Once anesthetized, rats were intubated with the aid of a modified pediatric laryngoscope. The standard miller blade 0 was cut on each side for approximately 2/3 of the total blade length to remove a total of half the width. After the ventilator was properly set, the rats’ vital signs and metabolic status were monitored. Throughout the one-hour infusion, the rats’ physiologic parameters were maintained within normal range. These results indicate that intravenous general anesthesia can be performed effectively and safely in small animals using the refined catheterization and intubation methods tested in this study. These techniques are easily reproducible and learned as they mimic the tools and strategies commonly used in the OR.
Intravenous (IV) administration of anesthetic drugs is a well-established practice in anesthetic delivery and a popular alternative to inhalational anesthesia [
Rats lack readily accessible veins large enough for venipuncture. Many methods for vascular access in rats have been described in the literature, but these techniques are difficult to perform in a standard laboratory that lacks specialized equipment. Similarly, rats’ small oral cavities make intubation difficult, and no standard equipment is available for rat intubation. Different rat intubation methods described in the literature include blind oral tracheal intubation and direct visualization of the trachea with or without the use of an intubation wedge [
When inserting a blade into the rat’s oral cavity, visualization which is of the oral cavity is grossly obscured, making surveillance of the complete vocal cord difficult. As a result, investigators, especially those without experience with these techniques, may require multiple intubation attempts on the same animals. Repeated attempts on the same animal can increase the frequency of complications, such as laryngospasm, mucus plugging, airway edema, and glottic or local tissue trauma [
Sprague-Dawley (SD) rats (Taconic Farm, Germantown, NY, USA), weighing 230 - 450 g at the start of the experiment, were housed in ventilated Plexiglas cages in a climate-controlled room (20˚C - 22˚C). Animals received food and water ad libitum. The room was illuminated on a 12-h light/dark schedule with lights on at 7:00 a.m. All experiments were performed in accordance with the guidelines of the National Institutes of Health for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of Rutgers, New Jersey Medical School, Newark, New Jersey.
At the initiation of the experiment, each rat was gently restrained in a clear plastic rat holder (
To facilitate successful catheterization, the tail was bent slightly so the approach angle of the needle was almost parallel to the vein (
was being inserted. The catheter needle was inserted through the skin at a flat angle and advanced for several millimeters. When the needle was successfully in the vein, blood entered the catheter (
Several pieces of equipment were modified to simplify the rat intubation procedure. An intubation stand was assembled using an adjustable metal book holder (
The small animal ventilator (Harvard Apparatus, Holliston, MA) was used to ventilate the rats by maintaining
the guideline parameters set by the manufacturer for tidal volume, respiratory mode and rate, inspiration to expiration ratio, and inspiratory pressure. End title CO2 (EtCO2) was monitored by Capnomac Ultima CO2 monitor (Datex Ohmeda, Louisville, KY). Vital signs (e.g., continuous arterial oxygen saturation, pulse distension, heart rate, and breath rate) were monitored using a pulse oximeter (Harvard Apparatus, Holliston, MA). A temperature controller (Harvard Apparatus, Holliston, MA) was used to maintain the body temperature of rats at 37.5˚C.
Prior to intubation, general anesthesia was induced with a bolus of propofol, ketamine, or etomidate. Rats lost righting reflex several seconds after receiving a bolus of anesthetic. Subject rats were then immediately positioned in a 45-degree plane and suspended by a regular ribbon hooked around the upper incisors (
To facilitate intubation, the animal’s mouth was opened using forceps and the tongue was displaced to the side using forceps. The modified laryngoscope blade was inserted into the oral cavity (
The ventilator settings for respiration rate were 80 - 90 respirations per minute and for tidal volume 3.0 - 4.5 cc. The ventilator was connected to an oxygen tank and supplied with 1.0 L/min of oxygen to maintain a satisfactory pulse oxygen saturation (>97.0%). The respirator settings above were tested and optimized based on the rat’s vital signs and metabolic studies. A temperature probe was inserted into rat’s rectum. The probe was connected to a temperature controller (
After administering a bolus of propofol, ketamine, or etomidate, the venous catheter was immediately connected to a 10 ml syringe driven by a syringe pump (Harvard Instruments, South Natick, Mass., USA). A continuous infusion was administered to maintain an intermediate surgical plane of anesthesia (
changed in heart rate in response to profound mosquito-clamp pinch at hand and foot performed at 5 minute intervals (response defined as greater than 10% increase from baseline heart rate). After one hour of anesthesia, the infusion was discontinued and the rats weaned from mechanical ventilation through the assisted ventilation mode. When the rats showed signs of adequate spontaneous respiration the mechanical ventilation was stopped and the endotracheal tube was gently removed.
Physiologic parameters were analyzed using repeated measures two-way ANOVA. Statistical significance was declared at p < 0.05.
The described procedures were tested with a total of 20 adult SD rats. After a catheter was placed in the rats’ tail veins, they were allowed to move freely in their home cages (
The 20 study rats were randomized to one of three medication groups. Regardless of their group, none of the rats showed any significant change in physical parameters during anesthesia. Vital signs obtained via pulse oximeter showed that the arterial oxygen saturation was maintained at greater than 97% and heart rate and pulse distension were stable over 1 hour anesthesia with propofol, ketamine, or etomidate (
Agent | Drug Dosage | |
---|---|---|
Bolus Dose (mg/kg) | Maintenance Infusion (mg/kg/hr) | |
Propofol (n = 10) | 10 | 60 - 90 |
Ketamine (n = 7) | 30 | 90 |
Etomidate (n = 3) | 3 | 10 |
This study demonstrates a successful, safe intravenous anesthesia rat model using refined venous catheterization and intubation techniques. Moreover, these techniques are easily learned by those with clinical anesthesia experience but with minimal knowledge of advanced laboratory techniques. In this study, one of the keys to successfully catheterizing the conscious rat’s tail vein was to dilate the vein by warming the tail. In rats, the tail is a major thermoregulation organ with a large surface area available for heat loss. Therefore, warming prior to injection results in vein dilation and enhanced blood flow, which support successful venipuncture. Previous studies suggest that a rat’s whole body should be warmed to approximately 40˚C by placing the animal into a ther- mostatically warmed “hot-box” [
Although commercial vascular catheters for rats and mice are available, such as restrained tail vein infusion model (Braintree Scientific Inc., Braintree, MA), our method provides similar advantages with readily available materials. The method described in the Braintree model was a nonsurgical approach where a “finder needle” punctured the vein and once free flow of blood was confirmed, a guide wire was inserted and a catheterr was then placed over this into the vein. However, it can be difficult to identify the insertion site for the guide wire after withdrawing the finder needle, especially for the inexperienced operator. In the current study, we noted that 24 gauge catheter (3/4" length) has two distinct advantages. First, the needle and catheter are combined, thereby eliminating the need to remove the needle once the vein is accessed. Once the needle is inserted into the tail vein through the intact skin, the catheter is simultaneously guided into the vein minimizing accidental dislodgement of catheter outside the vein. Second, it provides a visual check that the vein has been entered: blood fills the needle chamber (
In the current experiment, a refined method of endotracheal intubation in rats was also described. Various authors indicate that visualizing the laryngeal opening is important in order to perform successful intubation in the rat and reduce the time of the procedure [
The intubation procedure becomes simple and quick with the help of the modified pediatric laryngoscope blade presented in this article. The time required to complete the intubation was less than 30 seconds on average. The technique was quickly learned. By scaling the size versus creating a new device, we retain the familiarity of the airway tool and the associated intubation technique. Other methods may require additional steps that the novice is unfamiliar with therefore increasing rate of initial complication when learning the technique. Moreover, many of the techniques may be considered “blind” or “partially blind” techniques that rely on operator expertise and again produce a steep learning curve.
In our model the combination of the adjustable stand which allows rapid positioning which minimizes hypoxia and apnea time and the use of the modified blade allows for a reliably unobstructed view of the glottis opening and placement of the endotracheal catheter under direct visualization. In particular, the insertion of the
During 1 hour Propofol infusion | ||||
---|---|---|---|---|
15 min | 30 min | 45 min | 60 min | |
pH (arterial) | 7.37 ± 0.00 | 7.38 ± 0.02 | 7.37 ± 0.02 | 7.39 ± 0.03 |
PACO2 (mmHg) | 44.7 ± 2.1 | 46.7 ± 1.4 | 45.6 ± 2.9 | 46.1 ± 3.4 |
PAO2 (mmHg) | 282 ± 51 | 298 ± 62 | 299 ± 51 | 283 ± 23 |
HCO3 | 29.9 ± 2.8 | 31.3 ± 1.5 | 30.7 ± 1.1 | 28.3 ± 0.3 |
Na+ | 141.2 ± 0.9 | 140.3 ± 0.7 | 140.0 ± 0.6 | 141.3 ± 0.9 |
K+ | 4.7 ± 0.2 | 5.1 ± 0.3 | 5.2 ± 0.3 | 4.9 ± 0.2 |
Glucose (mM) | 136.3 ± 10.4 | 135.0 ± 10.2 | 133.7 ± 3.3 | 141.3 ± 8.7 |
Hb (mg/dl) | 13.4 ± 0.0 | 13.3 ± 0.3 | 13.6 ± 0.5 | 13.5 ± 0.7 |
Data are presented as mean ± S.M.E.; PACO2 = arterial carbon dioxide tension; PAO2 = arterial oxygen tension; Hb = hemoglobin; n = 4.
endotracheal catheter into the trachea became much easier because the modified blade allowed for refined manipulations of the upper airway while the LED light source allowed a clear, direct view of the epiglottis and vocal cords. This greatly reduced the potential for airway complications like esophageal intubation and direct trauma to oropharynx and trachea.
In the current study, we set up an intravenous anesthesia rat model using the refined venous catheterization and intubation techniques. It is well known that there are differences in anesthetic sensitivity and pharmacokinetics in different species and aging young animals. Accordingly, rodents require a higher dosage of anesthetics per body weight to achieve general anesthesia than humans and the dose of anesthetics varies slightly from animal to animal [
In summary, we have created an intuitive user-friendly method of tail vein catheterization and orotracheal intubation that allows for safe performance of intravenous anesthesia in the rat. The development of these methods affords the benefit of minimizing confounding variables such as hypotension, hypoxia, and acidosis. Additionally, this technique is particularly useful in specific situations that may not allow the use of more than one anesthetic agent. In our lab, these methods have served as the foundation for multiple studies investigating the neurotoxic effects on offspring exposed to propofol via the mother during the gestational period [