Laboratory Animals
Volume 41, Number 3, July 2007

WORKING GROUP REPORT

Rulicke et al.  FELASA guidelines for the production and nomenclature of transgenic rodents, pp. 301-311

Primary species: transgenic rodents (i.e. mice and rats)

SUMMARY: The use of transgenic rodents in recent years has been substantially increased, particularly because the genomes of man, mouse and rat have now been completely sequenced. This situation has stimulated the development of models for investigating the functional roles of newly discovered genes and prompted basic studies of gene function and regulation. In contrast with standard strains of laboratory rodents, the development and maintenance of transgenic mice and rats has several additional issues that need to be addressed. It is not only important to thoroughly understand the general biology, genetics and methods appropriate for breeding transgenic rodents, but also to assess the phenotypic changes likely to arise from the genetic modification. In particular, careful consideration needs to be given to special needs for housing, husbandry, handling and the maintenance of records. In this context, it is essential to use an effective, standardized nomenclature system. Rigorous identification of genetically engineered rodent lines is indispensable if investigators are to understand and accurately interpret findings in the animals they are working with. It enables precise communication of scientific findings arising from research involving transgenic animals, and facilitates incorporation of information concerning the animal model into databases and its subsequent retrieval to avoid unnecessary repetitions of models already available. Therefore, the aim of a standardized nomenclature of rodent strains, genes and mutations is to provide proper designation of laboratory animals used in research projects and to convey as much information on each strain as possible. Since the development of different techniques to mutate the genome of laboratory rodents on a large scale, the correct application of current nomenclature systems is of increased significance. It facilitates not only the accurate communication of scientific results but is indispensable in controlling the dramatically increased number of transgenic animal models in experimental units, archives and databases. It is regrettable that many publications, especially on transgenic rodents, use vague and inappropriate strain designation. This situation should definitely be improved, particularly considering the increasingly emphasized importance of genetic background on the phenotype of mutations. These guidelines raise awareness about specific features of production and current nomenclature systems used for transgenic rodents. Their limitations are also highlighted in order to encourage the development of a more robust classification scheme. In addition to all that, the guidelines also include some data on different types of mutations (spontaneous, chemically and physically induced, transgenic, conditional mutagenesis), genetic background of transgenic rodents (which background is most appropriate with respect to the trait to be studied?, changing and monitoring the genetic background), nomenclature of transgenic rodents (reference sources for nomenclature guidelines, basic principles in nomenclature of transgenic rodents, nomenclature of standard mice and rat strains, designation of engineered mutations, genotypes and transgenic strains, specific rules for conditional transgenes) and strain data sheet of transgenic strains.

QUESTIONS -TRUE or FALSE:
(1) Mutant rodents have served as models for the study of genetically determined human and animal diseases.
(2) An effective nomenclature system enables precise communication of scientific data generated from research involving transgenic animals.
(3) At present, only mice have yielded ES cells, which can be successfully isolated, cultured, modified and shown to contribute to germline, and these from only a very few inbred backgrounds, mostly of C57/BL/6J origin.
(4) Currently, about 90% of the mouse’s genes have been modified experimentally and incorporated into genetically engineered organisms.
(5) Rapid and unrestricted access to unambiguous, comprehensive information is important to ensure that best practice prevails and welfare standards are not compromised, both of which are principles of good science.

ANSWERS:
(1)	True.
(2)	True.
(3)	False (true: 129 origin).
(4)	False (true: only 10% of mouse’s genes have been modified).
(5)	True.

PAPERS

Gaines Das and North.  Implications of experimental technique for analysis and interpretation of data from animal experiments: outliers and increased variability resulting from failure of intraperitoneal injection procedures, pp. 312-320

SUMMARY: In laboratory animal experiments, intraperitoneal (i.p.) injections are widely used, but the failures of this technique and their consequences are not so widely reported as the use of the technique. So, in this analysis, they illustrate the consequences of i.p. injection failures and they suggest approaches to data analysis that should be considered, and emphasize the need to recognize and allow for the possibility of i.p. injection failure in the analysis and interpretation of laboratory animal experiments involving this technique.

QUESTIONS
1.	Which is the immediate consequence of the increased variability of the observed qualitative responses of the animals to the failures in i.p. injections?
2.	Tell us the main risk of a failure in i.p. injection in an animal.
3.	Describe in few words a number of possible complications of a failure in i.p. injection
4.	Which is the possible solution that in this study they propose?

ANSWERS
1.	The number of animals required to achieve valid experimental conclusions has to be increased.
2.	An inadvertent administration into the urinary bladder, intestine, caecum or other organ, or into muscle or fat.
3.	Apart from incorrect dosing, the discomfort due to the material or the volume administered, peritonitis and adhesions
4.	The add “measurable markers” to the substances for administration by i.p. injection 

Wiles et al.  Use of biophotonic imaging as a training aid for administration of substances in laboratory rodents, pp. 321-328

SUMMARY: Misadministration of test compounds can both negatively affect the outcome of an experiment and the health and welfare of the animals involved.
 
This paper outlines the possible use of BLI as a method for assessing competence in the mostly common used routes of administration of substances to laboratory animals.
 
Bioluminescence imaging (BLI) is based on the detection of visible light produced by luciferase-catalysed reactions which require energy, oxygen and a specific substrate.
 
Bioluminescence was rapidly detected after dosing of animals with a bioluminescent substance. However, living animals were required for a signal to be generated.
 
The results indicate that the use of bioluminescence to assess injections technique is a more refined method for assessing competence when compared with techniques such as the use of dye or tracer substances. BLI is less invasive, requires fewer animals and is less time consuming.  These results also indicate that BLI is an ideal tool for following the administration of substances to rodents, via the routes tested.
 
QUESTIONS (true of false):
1.	BLI could be use as a method for assessing competence in administration of substances to laboratory animals.
2.	The use of this method is less refined for assessing competence than the use of dye or tracer substances.
3.	The use of BLI to assess injections technique requires living animals.
 
ANSWERS:
1.	V
2.	F
3.	V

Alves et al.  Intraperitoneal propofol and propofol fentanyl, sufentanil and remifentanil combinations for mouse anaesthesia, pp. 329-336

Task 2, K6.  Prevent, Alleviate, and Minimize Pain and Distress.
Primary Species – Mouse

SUMMARY: The purpose of this study was to test the ability of various concentrations of propofol alone and propofol:fentanyl, sufentanil, and remifentanil combinations delivered by the intraperitoneal route to provide surgical anesthesia in mice.  Animals were restrained, and IP injections were performed lateral to the midline next to the umbilicus and into the lower left quadrant with the needle at a 45o angle to the abdominal wall.  Maximum volume administered was 1.1ml.  After injections of drugs, loss of righting reflex (RR) and pedal withdrawal reflex (PWR) were used to determine the depth of anesthesia. 

A total of 67 animals were used to test different combinations of propofol and the mu-agonist opioids listed above.  (NOTE: 13 of mice used in the propofol only group were used again in the propofol:sufentanil group.  There were 10 days between the 2 trials.).  Initially 16 male mice were divided into 4 groups, each group receiving a different dose of propofol alone.  None of the animals receiving propofol alone reached a level of surgical anesthesia; some lost their RR; only 1 mouse (in the 75mg/kg group) lost PWR as well, but this mouse did not recover from anesthesia.  With the propofol:fentanyl groups 13/20 animals lost their RR, while only 2/20 lost their PWR.  In the propofol:sufentanil groups 13/20 mice lost their RR, but only 6/20 PWR.  With propofol:remifentanil combinations 20/24 mice (at least 1 from each group) displayed loss of RR, but only 5/24 mice (from the 50/1, and all 75mg/kg groups) displayed PWR.  The loss of PWR was spread across 4 of the groups.  No animals died in the propofol:remifentanil groups.

The conclusion from this study was that none of the propofol:opioid combinations provided consistent outcomes.  Higher doses did not induce a surgical plane of anesthesia and often resulted in higher mortality rates.  The authors suggested that the intra- and inter-group inconsistencies might be attributed intraperitoneal administration of the drugs.  They further stated that the propofol:opioid combinations in this study provided no advantages over other more standard injectable anesthesia combinations, including ketamine:xylazine, fentanyl:fluanisone, etc.

QUESTIONS:
1.	Fentanyl alone: 
a.	Produces analgesia and sedation in mice 
b.	Produces excitation and mydriasis in mice 
c.	produces analgesia and sedation in humans 
d.	Produces balanced anesthesia in mice 
e.	b and c
2.	Fentanyl, sufentanil, and remifentanil: 
f.	Are mu-antagonists 
g.	Are mu-agonists 
h.	Produce an acceptable surgical anesthesia plane in combination with propofol in mice 
i.	Do not provide satisfactory post-operative analgesia 

ANSWERS:
1.	e. 
2.	b. 

Avsaroglu et al.  Differences in response to anaesthetics and analgesics between inbred rat strains, pp. 337-344

Primary species: rat

SUMMARY: Differences in response to analgesics and anaesthetic drugs can partly be attributed to variations in the genetic background of experimental animals. This study was carried out to determine differences in the response of inbred rat strains to a selection of analgesics (buprenorphine and nalbuphine) and drugs used in anaesthetics protocols (propofol, medetomidine and ketamine). Eight inbred strains (6 rats/strain) were studied: the pigmented ACI, BN and COP, and the albino F344, LEW, SHR, WAG and WK strains.

Findings from this study of particular interest are:
1	Six out of eight strains responded significantly different from each other to the analgesic effect of buprenorphine with the ACI strain as hyper-responder
2	All strains were non-responsive to the analgesic effects of nalbuphine
3	The response to propofol, medetomidine and ketamine drugs used in the anaesthetic protocols differed significantly among the strains
4	The sleep time induced by propofol, medetomidine and ketamine drugs in albino rats was significantly longer compared with that of the pigmented ones
5	The F344 and BN strains were relatively resistant to the sedative effects of medetomidine
6	The use of ketamine was abandoned in the ACI and BN strains when the first two animals of both strains died soon after induction

QUESTIONS: True or False
1.	F344, LEW, SHR, WAG and WK are all albino rat strains
2.	Nalbuphine may not be a good analgesic agent in rats
3.	Pigmented rats are more susceptible to the sleeping effects of propofol, medetomidine and ketamine than albinos
4.	All rat strains, but especially albino, are sensitive to the sedative effects of medetomidine
5.	Ketamine may cause mortality in some rat strains

ANSWERS:
1.	True
2.	True
3.	False (true: albino are more susceptible to the sleeping effects of propofol, medetomidine and ketamine than pigmented rats).
4.	False (true: some rat strains are resistant to the sedative effects of medetomidine, e.g. F344 (albino) and BN (pigmented) strains).
5.	True

Abu-Serriah et al.  Pain assessment following experimental maxillofacial surgical procedures in sheep, pp. 345-352

Task 2 - Prevent, Alleviate, and Minimize Pain and Distress
Secondary Species - Sheep (Ovis aries)

SUMMARY: Assessment of postoperative pain and general animal health following experimental jaw surgery is lacking. Thus, the severity and duration of postoperative pain and hyperalgesia in sheep undergoing mandibular reconstructive surgery was investigated. Objective indicators of altered pain processing in female sheep were measured before surgery, one, two, three, seven, and fourteen days after. Stimulus-evoked sensitivity at the surgical site as well as the ipsilateral and contralateral forelimbs was assessed. Also measured were concentrations of the acute-phase protein haptoglobin as a marker of inflammation on days one and seven after surgery.  

Secondary hyperalgesia was seen as a significant decrease in forelimb 
mechanical withdrawal thresholds on day one after surgery and 
persisted for at least three days.

Allodynia was seen as decreased response thresholds to punctuate stimulation of the area surrounding the surgical incision again on day one after surgery and persisting for at least three days.

Haptoglobin concentrations were significantly increased one day post-surgery and returned to pre-surgical concentrations by seven days.

QUESTIONS:
1. 	Secondary hyperalgesia is defined as:
2. 	Allodynia is defined as:
3. 	Indicator(s) of pain in sheep include:
a. 	Lip curling
b.	Separation from the group
c. 	Head held low
d. 	Tooth grinding
e. 	Aggressive behavior
4. 	Increased haptoglobins are an indication of:
5. 	The duration of action of buprenorphine in sheep is reported to be:

ANSWERS:
1. 	An increased responsiveness to a stimulus that is normally painful and occurring remote from the site of injury.
2. 	Pain caused by a stimulus that does not normally provoke pain.
3. 	All of the answers are indicators of pain in sheep
4. 	Significant ongoing inflammatory reaction
5. 	4 hours

Xanthos et al.  Cardiopulmonary arrest and resuscitation in Landrace/Large White swine: a research model, pp. 353-362

Primary Species: Pig
Task # 9: Collaborate on the Selection and Development of Animal Models 

SUMMARY:  The purpose of this article was to describe the experimental protocols of inducing cardiac arrest in a swine model. This model was successfully resuscitated using the International Guidelines. Cardiac arrest was achieved by using an ordinary rechargeable lithium battery through a pacemaker wire inserted into the right ventricle.  Among the different ways of inducing cardiac arrest, the ordinary lithium battery is a simple, safe and valuable technique.  

Animals Used: 15 Landrace/Large White swine

Sedation: Ketamine HCl 10mg/kg, Midazolam 0.5mg/kg and Atropine Sulfate 0.05mg/kg

Anesthesia Induction: Propofol 2mg/kg IV, additional propofol at 1mg/kg, cis-atracurium 0.15mg/kg and fentanyl 4 mcg/kg.  

Anesthesia Maintenance: 150 mcg/kg/min

Monitoring: End-tidal PETCO2, ECG (leads I, II, III, aVR, aVL, aVF), Cerebral oxygenation, spO2, aortic pressure, mean arterial pressure, right atrial pressure, cardiac output, coronary perfusion pressure, 

Protocol: Ventricular fibrillation was induced with a 9V lithium battery. Resuscitation was performed with chest compressions followed with up to 3 shocks with initial voltages of 2, 2 and 4 joules/kg followed by 2, 2, and 4 joules/kg.  Successful resuscitation was defined as the return of spontaneous rhythm with a MAP of at least 60mmHg for a minimum of 5 minutes.

Endpoints: Systole or return of spontaneous circulation.

Results: 7 of 15 animals were successfully resuscitated

Reasons the porcine model is most frequently used for cardiac arrest:
1. 	Swine cardiac anatomy and physiology closely resemble humans.
2. 	Myocardial histology is similar in swine and humans
3. 	After induction of myocardial ischemia, the porcine myocardium responds biochemical and metabolically in the same way as the human heart.
4.  	The swine model has a resting heart rate which averages that of the human.

In addition Landrace/Large White swine closely resemble the human homodynamic as far as the pressures of the left ventricle and atrium are concerned.

At the time this article was published there were no experimental studies that induced ventricular fibrillation with an ordinary rechargeable lithium battery. 

QUESTIONS:
1.	What two international organizations were founded as a result of the efforts associated with the resuscitation of animals?
2.	What is the most common cause of death in Coronary Artery Disease?
a.	Atrial fibrillation
b.	Ventricular fibrillation
c.	Ventricular tachycardia
d.	Hyperkalemia
3.	Which of the following is not a pathophysiological effect of coronary artery occlusion?
a.	Regional Ischemia
b.	Intracellular and extracellular acidosis
c.	Loss of cellular membrane integrity
d.	Atrial fibrillation
4.	What two therapeutic recommendations are recommended by the international organizations in the treatment of cardiac arrest?
a.	Defibrillation
b.	Epinephrine
c.	Atropine
d.	Open heart massage

ANSWERS:
1. 	International Liaison Committee on Resuscitation (ILCOR) European Resuscitation Council (ERC)
2.	b
3.	d
4.	a and b

Kolkert et al.  The gradual onset brain death model: a relevant model to study organ donation and its consequences on the outcome after transplantation, pp. 363-371

SUMMARY: Organs used for transplantation are derived from heart-beating brain dead donors. Most of these donors suffer subarachnoid hemorrhage or traumatic brain injury with different negative effects on donor organ quality. Two types of brain death models have been described: "sudden onset" and "gradual onset".

Sudden onset models are generated by increasing intracranial pressure within 30-60 s., and gradual onset models are generated by slowly increasing the intracranial pressure. Up to now sudden onset models carrying with haemodynamic stability and congestive lung failure so ionotropic support is used. Ionotropic support have a damaging side-effect on several organs therefore sudden onset models many animals died because cardiac arrest or can not be included due to haemodynamic stability.

Authors have developed in rats a reproducible and clinical relevant animal brain death model. This model mimics intracranial hemorrhage without ionotropic support. They inserted a balloon catheter into the extradural space. Brain death was induced by gradually increasing the intracranial pressure by inflating the balloon with saline. During balloon inflation, a hypotension period occurred follow by short peak and a subsequent drop in blood pressure. After that, blood pressure was maintained at normotensive levels up to 4 h. without ionotropic support. The presence of polymorph nuclear cells in liver was studied to compare with previous models.

QUESTIONS:
1.	What are the lesions founded in heart beating brain dead donors?
2.	What are the most important problems in sudden onset models?
3.	What are the problems with ionotropic support?
4 	What are the problems without ionotropic support?

ANSWERS:
1.	Subarachnoid hemorrhage or traumatic brain injury
2.	Haemodynamic stability and congestive lung failure.
3.	Ionotropic support has a damaging side-effect on several organs
4.	Many animals died because cardiac arrest or can not be included due to haemodynamic stability.

Lepschy et al.  Non-invasive measurement of adrenocortical activity in male and female rats, pp. 372-387

SUMMARY: Due to the small body size of rats, blood sampling can be complicated and may therefore interfere with endocrine function and compromise welfare. A non invasive technique to measure stress hormones such as corticosterone would therefore be desirable. The aims of the study were to gain information about corticosterone metabolism in rats and to validate an enzyme immunoassay (5α- pregnane- 3β, 11βtriol-20-one) to measure faecal corticosterone metabolites in laboratory rats. Eighteen rats were administered radiolabelled corticosterone via the IV or PO routes. All excreta were then collected for 5 days. About 75+/- 9 % of recovered corticosterone metabolites were found in faeces. The time course and delay for both routes of administration were the same except for a delay of peak concentrations in urine in per os administered males. 
Using high performance liquid chromatography the radiolabelled metabolites were characterized and differences found between the sexes. In both sexes corticosterone was extensively metabolized but whilst males showed only minor variations in metabolite patterns, females differed largely between individuals. The diurnal variation of glucocorticoids as well as effects of the injection procedure itself were measured to validate the EIA. An ACTH challenge test and a dexamethasone suppression test were also performed. The results suggested that corticosterone metabolite measurement in faeces accurately reflected stimulation, suppression and diurnal variation of adrenocortical activity. The suitability of this assay will have applications in animal welfare science and biomedical and pharmacological investigations. 

QUESTIONS:
1.	True/False- Diurnal variation in corticosterone occurs in rats.
2.	True/False- Measurement of corticosterone metabolites in faeces is a suitable way of evaluating stress in rats according to this paper.  
3.	True/False- There is no sex difference in metabolism of corticosterone in rats. 

ANSWERS:
1.	True
2.	True
3.	False

Kaidi et al.  Comparison of two methods (left carotid artery and abdominal aorta) for surgical implantation of radiotelemetry devices in CD-1 mice, pp. 388-402

This study compared, for efficacy, two surgical methods for implantation of mouse telemetry devices (a. left carotid artery-LC and b. abdominal aorta-AA).  The cardiovascular (CV) parameters to be measured and compared between the two techniques, in freely moving mice, were: surgery success rate, postsurgical recovery rate, clinical parameters, CV data (baseline and response to nicotine) and circadian rhythm measurements.
 
31 CD-1 male mice were instrumented with PA C20 devices (10 with LC and 21 with AA). The brains of the LC-implanted mice were evaluated for potential ischemia due to interference with the Circle of Willis (via histopathology).

The device accurately measures systolic, diastolic and mean blood pressure, heart rate, and physical activity.
 
Telemetry implantation vs. conventional cardiovascular measurement methods:
Advantages: 
1	Elimination of confounding stress effects induced by handling, restraint, and anesthesia.
2	Reduction of animal usage by 60-70% (single-use studies) and 90% (multiple-use studies)        
3	Refinement of procedure, without the need for special animal care

Disadvantages: 
1	Invasive surgical technique for transmitter implantation.
2	Cost of equipment
3	Large amounts of data generated
4	Necessity of dedicated animal facility space for study conduction.
 
Traditional methods for CV measurements:
Indirect and noninvasive tail-cuff plethysmography (disadvantage: req. extensive animal training, heating or restraint; advantage: non-invasive) OR fluid-filled catheters (disadvantage: requires animal tethering and has patency limitation for long-term studies).
 
Discussion:
Results from this study showed implantation of the pressure-sensing catheter in the thoracic aorta via the LC artery and SQ placement of the transmitter body along the right flank is less invasive, faster, and yielded higher percentage of surviving mice; 80% survival rate with LC technique vs. 57% survival rate with AA technique.

The AA method presented several practical problems intraoperatively and post-op from anesthesia, dehiscence and hemorrhage, hind limb paralysis, and respiratory depression. LC-implanted mice presented with non-lethal transient lesions; AA-implanted mice presented with lethal complications.
 
Effect on body weight (BW)
The BW loss observed in AA-implanted mice was more pronounced vs. LC-implanted mice. Both groups lost up to 10% of initial BW immediately after surgery. LC-implanted mice showed a stabilization of their BW from the second to the fourth day post-surgery. AA mice continued to lose weight until the fourth day.
 
Effect on food consumption
Food consumption decreased dramatically by more than 50% during the two first days after surgery in both groups of mice. Both groups (from day 3) slowly regained the level of approximately 80% of the initial consumption and water intake followed the same pattern.
 
Effect on circadian rhythm
Mice implanted with both techniques showed a return of normal circadian rhythm from the 6th day after surgery as evidenced from mean blood pressure (MBP) and heart rate (HR) profiles. Simultaneous measurements of MBP from the carotid artery and the aorta during recovery and 4-day baseline periods indicated that there were no significant differences in the MBP or HR recorded from the two sites.
 
Effect of nicotine administration
Both groups receiving 1 mg/kg of nicotine showed a typical CV response with an increase in blood pressure (BP)-both systolic and diastolic, following a 2h post-dose and biphasic effect on HR with a significant decrease within the 1st 30 minutes after nicotine administration.
 
The major concern associated with carotid placement of the telemetry catheter is the induction of cerebral ischemia due to ligation of the carotid artery. No histopathological changes suggestive of ischemia were noted in the brain of mice after necropsy.

The choice between LC and AA implantation techniques must take into consideration the developmental differences between strains when considering the anatomy of the Circle of Willis (at the base of the brain; formed by the union between internal carotids, anterior and posterior arteries, and anterior and posterior communicating arteries). In case of an incomplete circle, the carotid approach is not advisable for these strains (namely, transgenics). The method for visualizing the Circle of Willis consisted of an intracardiac perfusion under deep anesthesia of carbon black ink.
 
Conclusion
The findings indicate that the LC and AA implantation techniques did not present a significant difference in terms of CV profile, and both groups of mice showed a similar CV response to nicotine. The LC implantation method presents several advantages; the main advantages are that surgery is faster and easier to learn, less invasive and yields a better survival success rate. LC implantation techniques also seemed to be more suitable for long-term telemetry studies and could be used for several successive studies.

The LC implantation was perceived to be more efficient, reliable, and a less labor-intensive method, especially for transgenic mice, per study conductors.
  
QUESTIONS:
1) 	List the following:
a)	The location of the Circle of Willis:
b)	The vessels which form the Circle of Willis 
2) 	Describe the method for visualizing the Circle of Willis
3) 	List the lethal complications associated with the AA implantation technique:
 
ANSWERS:
1)	a. 	At the base of the brain
	b. 	Formed by the union between internal carotids, anterior and posterior arteries, and anterior and posterior communicating arteries.
2)	 Intracardiac perfusion under deep anesthesia of carbon black ink.
3) 	Dehiscence and hemorrhage, hind limb paralysis, and respiratory depression.

Palm and Hollander.  A procedure for intravenous injection using external jugular vein in Mongolian gerbil (Meriones unguiculatus), pp. 403-405

SUMMARY: The purpose of this article was to discuss a method for intravenous administration into the jugular vein in the Mongolian gerbil by using an 'over-the-needle' catheter under anesthesia. The method described is simple and avoids extensive surgery in the animals. The jugular vein is preferred over the femoral vein because it allows for the administration of a larger volume and ensures that the intended dose and volume enter the circulatory system without the risk of perivascular administration.

QUESTIONS:
1. 	T/F Intravenous administration of compounds is more difficult in contrast to rats and mice, because tail vein injection sites are not visible in gerbils?
2. 	In rodents the most common dosing routes is intraperitoneal and orally.  Name two alternative routes for compound administration in rodents?
3. 	The jugular vein is preferred over the femoral vein for compound administration because:
a. 	The jugular vein is easily visible as compared to the femoral vein
b. 	The femoral vein can cause a severe hematoma formation which can affect ambulation
c. 	A larger injection volume can be administered
d. 	None of the above
4. 	What muscle is penetrated when the 'over the needle catheter' is inserted into the jugular vein?
a. 	Sternohyoideus
b. 	Sternothyroideus
c. 	Pectoral
d. 	No muscle is penetrated
5. 	What size catheter is used in the 'over the needle technique for jugular vein administration?
a. 	22 gauge
b. 	24 gauge
c. 	26 gauge
d. 	28 gauge
6. 	What technique was used to avoid potential bleeding during removal of the plastic tube in which the needle sits?
a. 	Applying pressure at the site for 5 minutes
b. 	Applying heparin around the site to constrict the jugular vein
c.	Ligating the jugular vein
d. 	The use of tweezers to clamp around the plastic tube
7. 	What is the genus and species name of Mongolian gerbils?
a. 	Meriones unguiculatus
b. 	Dasypus novemcinctus
c. 	Mesocricetus auratus
d. 	Sigmodon hispidus
8. 	How much volume can be safely administered into the jugular vein using the technique described in this article?
a.	 0.05 to 0.1 ml
b. 	0.10 to 0.2 ml
c. 	0.25 ml to 0.5 ml
d. 	Up to 0.5 ml

ANSWERS:
1. 	True
2. 	Jugular and Femoral veins
3. 	c
4. 	c
5. 	b
6. 	d
7. 	a
8. 	d