Experimentación animal

General information regarding the Behavioral Rooms:

1. All the rooms must be booked through the INTRANET before their use. Rooms can be booked in blocks (8:00 to 14:00 and 14:00 to 20:00). The reservations can be up to 4 weeks in some of the rooms: for instance room E4 can be reserved for 1 whole month due to extended length of Morris Water maze testing. This scheme will be revised depending on users’ demands. For specific questions or reservations that do not adjust to the duration/requirements indicated, please contact directly with the scientific responsible otano@umh.es

2. To work in the service you must be registered in the following list: in.zona-experimental.rmg@listas.umh.es. For any questions, room release or help in the experimental area write to the e-mail : in.zona-experimental.rmg@listas.umh.es

3. NOISE perturbs other people´s experiments. Please do not speak in the corridors, and conduct all procedures with the doors closed.

4. The phone in the store room should NOT be used (x9358), except for UNAVOIDABLE communication between the people working in the experimental area and the exterior, but not vice versa

5. Cell phones are NOT PERMITTED

6. Every use and small incidence should be recorded in the Incidence sheet located in each room or to this email in.zona-experimental.rmg@listas.umh.es. Major incidences, such as damage or malfunction of the equipment or software, noise issues and temperature problems should be discussed directly with scientific responsible: otano@umh.es.

7. The users should make sure that the room and equipment is in order and clean after their use.

8. If common materials (gloves, paper towel, cleaning liquids, etc) are close to be finished, inform the personnel in the facility in charge of their reposition.

9. Some equipment MUST STAY IN specific rooms. Other is kept in the storeroom and moved to rooms 1, 3, 4 and 7 for their use. After use, equipment should be thoughtfully cleaned and moved back to the storeroom except when if has to be used the day after. It this forbidden to change the location of the equipment associated to a specific room without authorization of the scientific responsible.

COMPUTERS

All the computers are connected to the UMHNET domain. To log you have to select UMHNET in the box “Connect to”, and use the “user name” and “password” that we use to connect to our e-mail and other computers of the institute.
The files created should be transferred electronically to external computers (by email, google drive. dropbox etc..). Because we have limited space on the computer’s hard drive, please delete all files once they have been sent and can be read from other computers.

EQUIPMENT INVENTORY:

Housing area
2 parks for enriched environment (please ask Angel Barco, abarco@umh.es if you need to use them)

Room Comp E2. Sensory and motor testing
1. Hot/Cold Plate
2. Plantar Test (Hargreaves Apparatus)
3. Von Frey
4. RotaRod
5. Activity wheel
6. Treadmill (2 lanes)

Room Comp E3 and Room 7:
Video Tracking Smart license (Panlab) (one in each room) with computer and camera associated.

Room Comp E4:
Morris water-maze (2 m diameter) SMART Video tracking (Panlab) for the Water maze.

Room Comp E5:
1. Startle and fear conditioning boxes (X2) with computer and software Panlab associated.
2. Skinner Operant boxes. (x4)

Room Comp E6:
Phenotyper (Ethovision Noldus)

Testing apparatus in Store Room
1. Radial arm maze
2. T-maze
3. Y-maze
4. Open field boxes (50x50cm)
5. Elevate plus maze
6. Wireless running Wheels (x8)
7. Intelligent cages
8. Light-dark boxes
9. Passive avoidance
10. Tail Suspension
12. Forced swiming test
13. Conditioned Place Preference/Aversion boxes (x2)
14. Catwalk

SHIRPA kit:
1. Viewing jar (10 x 20 cm clear perspex glass cilinder)
2. Clear perspex glass sheet (the base for the viewing jar)
3. Metal grid (for measuring visual placing, negative geotaxis and grip strength)
4. Vertical pole wrapped with yellow tape (for vertical pole test)
5. Rod (for measuring provoked biting)
6. Forceps (for measuring toe pinch)
7. Von-frey hair attached to a wooden stick (for measuring corneal and pinna reflexes)
8. Clear persplex glass 5×30 cm cilinder (for measuring contact righting reflex)
9. White perspex glass box with attached wire (for measuring wire manoeuvre)
10. Electronic balance (for weight measurement)
11. Small dynamometer to measure strength
12. Stop watch
13. One or two wheeled tables.
14. Moving staircase.

Cryopreservation offers the opportunity of archiving valuable laboratory mice strains such as inbred or genetically modified mice, slowing down the onset of genetic drift and insuring them against any natural or sanitary disaster, mismanagement, reproductive problem, or transgene silencing expression.

The use of cryopreservation in this area also represents a good opportunity to apply the 3Rs policies, since on the one hand is an indirect method of reduction by avoiding the loss of these lines and therefore avoiding a new investment of animals in its creation, but on the other hand offers the possibility of directly reducing the number of animals used in research by archiving and eliminating strains that are kept alive but nevertheless are not being used. In this sense, cryopreservation is also a mechanism that makes possible the good management of the animal facilities since the elimination of obsolete lines is cost-effective (Table 1) and not only reduces the total maintenance costs of animals, but releases space that is being wasted and could be used by other developing research lines.

In short, cryopreservation prevents us from losing our model, ensures its quality, is an opportunity to apply the 3Rs and reduce the number of animals used in research, and is also advisable from an economic and management point of view.

Table 1. Ratio of Laboratory mice strains maintenance costs depending on whether they are kept alive or cryopreserved.

The Transgenic Service of the Institute of Neurosciences CSIC-UMH provides support to researchers for the generation and establishment of genetically modified mouse models. Our team offers technical advice on the best strategy to achieve the desired model through additive transgenesis or targeted mutagenesis.

The laboratory is located inside an SPF barrier and the work is performed under aseptic conditions, ensuring the best health status for the new strains generated.

Technical responsible: Gonzalo Moreno Del Val
email: g.moreno@umh.es »

Technical: Patricia Muñoz Robledano
email: pmunoz@umh.es »

Transgenic and Cryopreservation
Instituto de Neurociencias
Universidad Miguel Hernández – CSIC
Campus de San Juan
Sant Joan d’Alacant
Alicante | España

Tel. + 34 965 23 37 00
(ext. service 9371 / 9408)
Fax + 34 965 91 95 61

• It is mandatory for new users to receive the corresponding training by the Facility’s personnel before using any of these equipments.
  
  
• Working area should be always kept clean, particularly after finalizing the session.

This facility is located on the basement floor of the IN and organized as follows:

• Operating room 1
• Operating room 2

Electroporator CUY21 SC. Nepa Gene

The CUY21 SC can measure the electric current from the low ampere (1mA) up to 1.60A. The CUY21 SC was developed for the electroporation of genetic constructs that are delivered into the sensitive target region such as mouse’s brain, tissue slices and embryos (chick, mouse). The CUY21 SC can be used with different NEPA GENE’s CUY series electrodes to perform a wide range of electroporation experiments. In our animal house there are the CUY650P3 and CUY650P5 Tweezers w/3mm or 5mm diameter platinum disk electrode (Nepa Gene Co., Ltd.)

Estereotaxic Stoelting 51730

Carry a basic stereotaxic frame, together with all the usual accessories. Precision alignment ensures accurate placement of electrodes, micropipettes, and other devices. Is ideal for researchers in need of a versatile, reliable instrument for stereotaxic procedures with mice and other small rodents.

Nano injector. NANOLITER 2010-WPI

The Nanoliter Injector offers significant options and controls ideal for ultra-delicate work with oocytes, embryos, and tissues. Injection volumes range between 2.3 nanoliters and 69.0 nanoliters in 16 selectable increments.

Vetario TS INTENSIVE CARE UNITS

Designed for helping the recovery of mice after an experimental procedure. It avoids the hypothermia, shock and other complications derived from the experimental procedure performed.

This facility is located in room 105 (First floor).

Common surgery equipment:

• Isofluorane and oxygen anesthesia system
• Anesthesia induction chamber
• Stereomicroscope and light source
• Heating blancket
• Minifugue for eppendorf tubes
• Waterbath
• Heating plate
• Fluorescence Estereomicroscope Leica MZ16FA (GFP and DsRed filters)

Electroporator in vivo Cuy21Edit

CUY21EDIT(Electroporator) can introduce DNAs, siRNAs, drugs, into tissues including brain or embryos.
• General characteristics: -Voltage 1~500V -Pulse time 0.1~999.9ms -Pulse Interval 0.1~999.9ms -Pulse number 1~99 time(s) -Impedance measurement ~18kO -Effective voltage 1~500V -Effective current 0.01~4.00
• Applications: In vivo, in utero, ex utero, in ovo, ex ovo, ex vivo electroporation.

Vevo 3100 micro Ultrasound Imaging System

Ultrasonic imaging system adapted for use in small animals including mouse and zebrafish. This system makes possible a high-resolution anatomical visualization to perform cardiovascular, renal, reproductive system, oncological or embryonic development studies in real time.
• General characteristics. Equipment includes:
-Microinjector
-Isofluorane anesthesia system
-MX201 transducer: 10-22 MHz, Centre Transmit: 15 MHz, Axial Resolution: 100 µm
-MX550D transducer: Centre Transmit: 40 MHz, Axial Resolution: 40 µm
• Applications:
Developmental biology:
-Gestation monitorization and determination of embryonic stage
-Analysis of placental structure and function
-Monitoring of organ development in the embryo
-Injection of viruses, cells or drugs into intra-uterine embryos: in the brain, eye, spinal cord or any other developing organ
Oncology:
-Tumor detection and tumour burden quantification
-Visualization and quantification of tumour angiogenesis, vascularization and perfusion
-Therapy responses (non invasive measure of tumors over time)
-Guided implantation of tumor cells
-Precise injection of drugs into tumors
-Ultrasound guided biopsy
Abdominal analysis:
-Function of kidneys, liver and reproductive system
Cardiovascular studies:
-Cardiac function in 2D, 3D or 4D
-Hemodynamics

IVIS Lumina XR

Real-Time in vivo bioluminescence, fluorescence & X-Ray Imaging System for small animals.
• General characteristics:
– 10 filter sets to image reporters that emit from green to near-infrared (capable of imaging all common fluorescent and bioluminescent reporters).
– Heated stage to keep animals from becoming hypothermic.
– Input and output connection ports for the anesthesia system.
– X-ray. Precise optical and X-ray overlay allows you to bring your optical signal into.
• Applications:
The IVIS Lumina XR can be used in oncology research to follow tumor progression, monitor response to therapeutic treatments, metastasis, cell tracking, and tumor burden monitoring.
Other uses include: infectious disease, inflammation studies, metabolic disease, neurology, gene therapy, stem cell biology, cardiovascular disease, immunology, transplantation biology, and drug metabolism studies.

• Working in silence is strictly required in this area.
• It is forbidden to talk on the phone inside this area, especially in the housing rooms.
• Animals are housed in reversed cycle, thus extreme attention should be paid with the use of white lights in this area, in order not to interfere with the rat’s normal cycles.
• It is mandatory for new users to receive an introduction to the code of behavior and organization rules of this experimental area by the Facility’s personnel.
• Once you have received this training, your secured access (card) will be activated, and you will be invited to the main communication channel for users (Slack) and google calendar.
• In order to receive this training, please write an e-mail to cmarquez@umh.es

• Organization of spaces is performed through Slack and users’ google calendar. Access will be provided after introductory training (see rules).

Scientific Director: Cristina Márquez Vega
email: cmarquez@umh.es »

El servicio de cirugía ofrece los siguientes servicios:

• Sala equipada con mesa quirúrgica, estereoscopio y cámara de recuperación de animales con temperatura regulable
• Marco estereotáxico adaptable para ratones y ratas (Kopf) y accesorios
• Vaporizador de isoflurano
• Concentrador de oxígeno

La sala y el equipo se pueden reservar de forma independiente, ya que la mayoría de los equipos pueden ser retirados para realizar procedimientos en laboratorios individuales.

La reserva de la sala y el equipo debe realizarse a través de la intranet en: acceso usuarios.
Hay que tener en cuenta que deben de hacerse reservas independientes para la sala (Cirugía) y el equipo requerido. Las opciones se enumeran a continuación:

• Cirugía
• Cirugía-concentrador de oxígeno
• Cirugía-Estereotáxico
• Cirugía-Isoflurano

Los nuevos usuarios deben ponerse en contacto con la Dra. Sandra Jurado: sjurado@umh.es para recibir una breve formación y familiarizarse con las pautas de uso. Este primer contacto es obligatorio para acceder al quirófano.

Para cualquier consulta o incidencia con la instalación, contactar con la Dra. Sandra Jurado: sjurado@umh.es.