Laboratory Animals
Volume 41, Number 4, October 2007

Gil et al.  Pituitary-adrenocortical axis, serum serotonin and biochemical response after halothane or isoflurane anaesthesia in rabbits, pp. 411-419.
 
Introduction: Anaesthetic protocols may modify biochemical and hormonal markers. It is very important to avoid erroneous interpretation of laboratory data in anaesthetized rabbits. This study was undertaken to determine whether the administration of halothane or isoflurane causes alterations in serum serotonin, adrenocorticotrophic hormone (ACTH), corticosterone levels and select biochemical parameters in response to inhalant anaesthesia, and so to avoid misinterpretation of laboratory results in anaesthetized rabbits.

Material and Methods: Animals: New Zealand White rabbits. Experimental design: two treatment groups with halothane and isoflurane. Variables measured: Serum serotonin and corticosterone levels were measured by competitive enzyme immunoassay, ACTH by radioimmunoassay. Serum glucose, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), blood urea nitrogen (BUN) and creatinine levels were measured using an automated analyzer. Statistics: Data was presented as mean SEM and Kolmogorov-Smirnov test was used to determine id data were normally distributed. Each treatment group was analyzed by one-way analysis of variance followed by Duncan post hoc test when significant difference was indicated.

Results: Body weight and Rectal Temperature: No significant differences between groups. Heart and respiratory rates (HR and RR): No changes in HR were observed in the isoflurane group but significantly decreased in halothane group. RR was significantly decreased in both groups. Reflexes: All measured reflexes were negative during the maintenance phase of anaesthesia. Serum ACTH and corticosterone levels: Significant increases in serum ACTH and corticosterone levels occurred after halothane administration while serum serotonin levels did not change. Serum Serotonin levels: An increase in serum corticosterone and serotonin levels occurred in the isoflurane group but no changes in ACTH concentrations were detected. Serum glucose levels: Administration of halothane and isoflurane significantly increased serum glucose. Serum ALT, AST and ALP levels: Administration of halothane significantly increased serum glucose, ALT, AST. After isoflurane administration there was a significant increase in AST levels. Serum BUN and Creatinine levels: Administration of halothane and isoflurane significantly increased BUN and creatinine levels.

Discussion: There is a lack of information regarding the effects of volatile anaesthetics on the HPA axis, serum serotonin and some biochemical parameters in rabbits. Halothane stimulates the hypothalamic-pituitary-adrenal axis to a greater extent than isoflurane, but isoflurane increases serum serotonin levels. Both anaesthetic agents alter select biochemical parameters so these results should be taken into account when blood samples are evaluated in treated isoflurane or halothane anaesthetized rabbits.
 
QUESTIONS:
1. 	Should procedures causing actual or potential suffering to animals be carried out under anaesthesia by FELASA? Yes/No
2. 	Intravenous anaesthetics could modify selected biochemical parameters in rabbits. True or False.
3. 	Pain and distress may also influence some biochemical parameters. True or False.
4. 	An increase in serum creatinine and BUN levels was not also found in humans.
5. 	The relationship between serotonin and the HPA axis is of great importance in normal physiology and pathophysiological disorders such as…..
a. 	Circadian rhythm and stress.
b. 	Depression.
c. 	Anxiety and eating disorders.
d. 	All of them.
 
ANSWERS:
1. 	Yes. (FELASA 1994)
2. 	True. (Bito and Eains 1969; González Gil et al. 2003).
3. 	True.
4. 	False. (Mazze et al. 2000).
5. 	d. All of them. (Hanley and Van Kar 2003).

Burger et al.  The sheep as a knee osteoarthritis model: early cartilage changes after meniscus injury and repair, pp. 420-431

SUMMARY: The purpose of this study was to analyze cartilage changes after traumatic meniscal lesions and to provide a detailed description of the sheep model. The sheep’s knee was chosen as ovine biomechanics are similar to that of humans.

In two groups of 10 sheep each, a radial tear in the medial meniscus was either sutured with polydioxanone (PDS) of left untreated (sham-operated). In each group, half of the animals were sacrificed after six months, and the other half after one year. The time periods to achieve weight bearing, meniscus healing, joint effusion (magnetic resonance imaging scan) and knee cartilage in the medial, lateral and patellofemoral compartments were evaluated in comparison to the opposite knee (control). Osteoarthritis (OA) was assessed by a modified Outerbridge classification and confirmed by scanning electron microscopy.

Only one sutured meniscus remained completely adapted. In all other meniscus lesions, the rupture healed with a scar. In the PDS and sham-operated groups, OA was significantly higher in the medial knee compartment than in the lateral compartment and in controls (P<0.001). In the operated groups, joint effusion was higher in the right hind limb knee than in the normal left hind limb knee (control) after six and twelve months (P<0.001). Non-treated, displaced and even adapted sutured radial ovine meniscus tears produced intense OA within less than six months.

The findings of this study showed that the sheep is a suitable animal model for knee osteoarthritis and to monitor cartilage changes after meniscus injury and repair. The sheep model may prove valuable for the assessment of new therapeutic regimens in meniscal surgery.

QUESTIONS- True or false:
1.	The meniscal injury as an animal model is rarely used despite there are various in vivo models dealing with this type of tear.
2.	The distribution of compression and shearing forces of the sheep’s meniscus are different than in humans because the sheep load knees usually in flexion while human knees are habitually loaded in extension.
3.	Hyaluronan enhances meniscal healing after repair in rabbits, while this is not the case with fibrin clots and marrow cells in goats.
4.	The ovine knee is a good OA model, and the cartilage degeneration occurs even more rapidly than in human’s knee.

ANSWERS:
1.	False
2.	True
3.	True
4.	True

Nevalainen et al.  Pair housing of rabbits reduces variances in growth rates and serum alkaline phosphatase (APHOS) levels, pp. 432-440

SUMMARY: In this study they try to see changes in growth and selected serum chemistry parameters due to pair housing or single housing of rabbits (six sets of four female siblings of Crl:KBL(NZW)BR rabbits, and seven weeks old or arrival).  The rabbits were weighed and blood samples were taken at four different times during the study. Blood sera were assayed for a set of routinely assayed clinical chemistry parameters: ALT, AST, APHOS, BUN, CHOL and PROT. During the 21-week study, pair housing appears to have a decreasing effect on growth and APHOS variance, but fighting remains a serious problem.

QUESTIONS:
1.	Which sentence is true and which one is false?
a. 	Group housing is considered as social enrichment and thus represents the refinement alternative on the 3Rs concept to the use of laboratory animals.
b. 	Rabbits, as species, are very easy to house in groups.
c.  	To enhance social enrichment in rabbits, the European regulations doesn’t make it possible to house two rabbits in a cage space previously considered adequate for one single rabbit.
2.	Female rabbits should be housed in harmonious social groups unless there are good veterinary or scientific reasons for not doing so; which are these both reasons?
3.	Although anti-social behavior as fighting exits, even increasing to intolerable levels, pair-housing could be viewed as solution of choice for housing of female rabbits; do you think is that true?

ANSWERS:
1.	a. TRUE; b. FALSE; c. FALSE
2.	The veterinary reason could be aggression among incompatible animals, and the scientific reason could be potential interference with the experimental protocol.
3.	I am sure that is FALSE

Loo et al.  Impact of "living apart together" on postoperative recovery of mice, pp. 441-455.

SUMMARY: Mouse is gregarious specie, so individual housing has a negative impact on animal wellbeing. This suggests socially housed mice (SOC) need less time to recover from abdominal surgery (telemetry implant) than individually mice (IND). However, some times, individual housing is required in some experimental designs.

Living apart together (LAT), housing condition with a metal grid partition inserted in the middle of the cage, could be an alternative to individual housing.

They hypothesized that SOC female mice and familiar female mice LAT would be less stressed and show more rapid postoperative recovery than IND female mice.

Heart rate (HR), body temperature (BT) and activity were measured by telemetry (every 3’/24h). Body weight (BW) was measured for a period of three weeks. A score was made of wound. Food and water intake, nest site and complexity, and score ethogram, were data collected too.

In general wounds closed within three days after surgery. HR was stable around day 3. Pattern of HT was LAT>IND>SOC. BT was stable at day 7, but it didn’t show any differences between housing conditions.

BW was decreased until two days post and returned to presurgical levels after 12 days (IND), 14 days (SOC) and 17 days (LAT). IND mice ate more than SOC and LAT. Water intake didn’t show differences.

Nest complexity showed a decrease at day 1 post surgery. From this point nest complexity increased to presurgical levels at day 5.

Only a few small overall housing conditions could be detected in behavior, although animals showed an increased amount of pain behavior until 5-7 days after surgery.

In conclusion most parameters returned to presurgical levels 5-7 days, while body weight needed two weeks. SOC animals showed less pain behavior and HR levels were lower during experiment. LAT did not promote animal welfare in this sense.

QUESTIONS:
1. 	Mouse is a gregarious specie, so individual housing is recommended (T/F)
2. 	HR measured was stable at day 3, what does housing condition have HR highest?
3. 	How many days need BW to return to presurgical levels?
4. 	In this abdominal surgery animals showed an increased amount of pain behavior until 5-7 days after surgery. (T/F)
5. 	LAT apparently is a better option than IND (T/F)

ANSWERS:
1.	F
2.	LAT
3.	12 days (IND), 14 days (SOC) and 17 days (LAT)
4.	T
5.	F

Fante et al.  Refinement of a transplantation project in the non-human primate by the use of a humane endpoint, pp. 456-469

SUMMARY: The researchers involved developed an assessment scheme to establish a
humane endpoint in a pig to primate renal xenotransplantation model. The transplant research objective was to identify new immunosuppressive strategies for the long term survival of pig organs transplanted to cynomolgus macaques. After surgery, animals were monitored by clinical system which included daily physical examination and recording of parameters such as temperature, body weight and heart rate. A daily therapy sheet recorded all drugs administered, clinical observations and blood samples taken. Objective and subjective humane endpoints were established. Objective parameters included renal function as measured by serum creatinine, body weight changes (increase/decrease), temperature changes, pronounced anaemia, and electrolyte balance. Subjective parameters included responsiveness, clinically evident disease, and behavioral signs of pain. Animals were euthanized on presentation of one or more of the clinical criteria defining the humane endpoint. The authors believe that their assessment scheme provided a reduction in animal suffering without compromising validity of the scientific results. Continuous clinical, laboratory and instrumental monitoring and the application of detailed criteria for assessing the animals' conditions represent a refinement strategy. 

QUESTIONS:
1.	True/False.  A humane endpoint is defined as the point at which an animal's pain and /or distress is terminated, minimized or reduced by such action as killing the animal humanely, terminating a painful procedure or giving treatment to relieve pain/distress. 
2.	True/False.  The establishment of humane endpoints is a replacement strategy. 
3.	The following are examples of objective parameters for establishing human endpoints:
a.	Body temperature
b.	Vocalizations
c.	Tremors
d.	Serum creatinine determinations
e.	Responsiveness

ANSWERS:
1.	True
2.	False
3.	a, d

Hess et al.  Measurements of blood pressure and electrocardiogram in conscious freely moving guinea pigs: a model for screening QT interval prolongation effects, pp. 470-480

Task 9 - Collaborate on the Selection and Development of Animal Models
Secondary species: Guinea Pig (Cavia porcellus)

SUMMARY: The guinea pig has three advantages over the dog and the primate.
    * It has specific ion channels similar to man;
    * A smaller amount of test article is required for the investigation and,
    * Its housing is less expensive.

Four reference compounds were used in this study: three positive (sotalol, terfenadine and dofetilide) and one negative reference (enalapril). Electrophysiological in vitro assays are commonly used in the pharmaceutical industry to examine drug effects on the human ether-a-go-go-related gene (hERG) channel. However, the predictive value of these studies is limited, verapamil being a notable example of a false-positive. Verapamil blocks the hERG Kþ channel, but is reported to have no potential to generate torsades de pointes (torsades de pointes refers to a specific variety of ventricular tachycardia and is associated with Long QT syndrome). Because anaesthetics are known to alter the sensitivity to drugs, it is highly desirable to study ECGs in conscious unrestrained animals. The authors show in this study that it is possible to monitor ECG intervals and arterial blood pressure (ABP) in conscious freely moving guinea pigs by using an intraperitoneal approach for the implantation of the telemetry system. 

Guinea pigs lack an enzyme that catalyses the conversion of glucose to vitamin C. They cannot produce their own vitamin C, so that it must be supplied in food or drinking water. The pre- and post-treatment with ascorbic acid is essential to achieve good postoperative care. However, as in the rat, a recovery period of 2–3 weeks is necessary. The authors show in this study that guinea pigs can also be used as an alternative to dog and primate models for the assessment of cardiovascular safety. QT is known to inversely correlate with heart rate. In this study, the authors used Bazett’s formula to correct QT for heart rate (The formula is as follows:

QTc = \frac{QT}{\sqrt {RR} },

where QTc is the QT interval corrected for rate, and RR is the interval from the onset of one QRS complex to the onset of the next QRS complex, measured in seconds). Single oral administration of all three positive references dose-dependently induced bradycardia and QT corrected (QTc) prolongation. In contrast, neither enalapril nor its vehicle prolonged the QTc. The guinea pig model is suitable for detecting QT prolongations after oral administration of reference compounds.

QUESTIONS:
1. 	What are the advantages that the guinea pig offers over the dog and the primate?
2. 	What causes verapamil on the human ether-a-go-go-related gene (hERG) channel?
3. 	True or False: QT is known to directly correlate with heart rate.
4. 	True or False: The guinea pig model is suitable for detecting QT prolongations after oral administration of reference compounds.

ANSWERS:
1. 	It has specific ion channels similar to man; a smaller amount of test article is required for the investigation and, its housing is less expensive.
2. 	A blockade.
3. 	False.
4. 	True.

Boot and Reubsaet.  Identification of bacterial strains by laboratories participating in the Deutshes Krebsforschungszentrum quality assurance programme, pp. 481-491.

SUMMARY: The quality assurance programme (QAP) was developed to study the capabilities of laboratory animal laboratories to correctly identify bacterial species. From 1980 to 2000, the bacterial strains sent to the laboratories were from culture colelctions. From then on, bacterial strains were selected from these obtained from clinical samples during routine monitoring. The information supplied with the bacteria is limited to host species (and sometimes isolation site) or environmental origin. Genus and species identification reported from laboratories were compared with results from a reference laboratory. Samples submitted were mostly of animal origin (different animal species), but also environmental strains, from water and watering systems, cages or bedding and unspecified origin. Identifications were assigned a score. Only  the 60% of species identification agreed. 23% of the identification were within the genus but differed in s pecies designations, and 17% were outside the genus. The capabilities of laboratories in the laboratory animal discipline to correctly identify bacterial species are well below what are considered acceptable limits for human diagnostic laboratories. Misidentification of bacteria may lead to:
1	Unrecognized spread of a pathogen
2	Unnecessary eradication measures
3	Unnecessary or inappropriate therapy
 
No questions were submitted with this summary.

Schutze et al.  A surgical technique for catheterization of the sagittal sinus in pigs, pp. 492-495

Task 1: prevent, Diagnose, Control and Treat Disease
Primary Species
 
Purpose: To describe an efficient, reliable and inexpensive surgical method for cerebral venous blood gas sampling in acutely instrumented pigs in a research setting. 
 
Animals: female domestic pigs; 4 months, weight 34-36kg
 
Anesthetic protocol: pre-medicated with flunitrazepam and ketamine; induced via auricular vein with fentanyl and flunitrazepqam; maintained with IV infusion of propofol, fentanyl, and rocuronium
 
Surgical technique: 
1	Placed in prone position
2	3cm skin incision made midway between the eyes
3	5mm burr hole placed in midline and widened with bone biopsy punch
4	Frontal sinus was placed directly under the burr hole
5	Back wall of frontal sinus was opened and dura mater prepared
6	Pig was placed with head pointed downward 30 degrees
7	Used the Seldinger technique for insertion of the catheter
8	For cannulation of the sagittal sinus, a pediatric jugular catheterization set was used
9	Dura was grasped and a 1mm incision was made
10	Spring-wire guide 0.025” in diameter was placed in the sinus
11	20 Ga x 5” catheter was inserted via the wire about 2cm into the sinus
12	Burr hole was closed with an absorbable gelatin sponge and the catheter was fixed to the skin, which was sutured regularly.
13	It was locked with heparinized saline
 
QUESTIONS:
1.	Why is the catheterization of the sagittal sinus in pigs an easier way?
2.	What technique was used to insert the catheter? 
3.	What are the anatomical landmarks for orientation of the optimal trepanation area?
 
ANSWERS:
1.	Radiograph is not required to check placement
2.	Seldinger
3.	The midline and the frontal orbital line