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Scandinavian Journal of Laboratory Animal Science

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Original scientific article


A modified technique for thymectomy in adult mouse with no ventilation support

by Chenchen Zhao1, Hui Zeng2, Zhengya Yu3


1Department of General Surgery, Beijing Tongren Hospital, Capital Medical University, Beijing 100730, China
2Institute of Infectious Diseases, Beijing Ditan Hospital, Capital Medical University, Beijing 100015, China
3Department of General Surgery, Beijing Tongren Hospital, Capital Medical University, Beijing 100730, China

 

PDFCorrespondence: Zhengya Yu, Department of General Surgery
Beijing Tongren Hospital, Capital Medical University
No.1 Dong Jiao Min Xiang Street, Dongcheng District, Beijing 100730, China
Tel/Fax          +86-10-58268504
Email            zhengya_yu@ccmu.edu.cn


Summary

Thymectomy in adult mice, a method to eliminate newly produced naïve T cells, has proved valuable in various immunological studies. However, pneumothorax is the primary cause of high mortality while preforming the traditional thymectomy technique. Although modified techniques utilizing mechanical ventilation support can reduce the occurrence of pneumothorax, it may increase operational complexity and cause ventilation-induced lung injury, which could interfere with subsequent investigations by causing inflammation and a series of immune responses. To solve this problem, we developed a novel technique using pleural ligations to replace ventilation support before thymus removal. Our method not only reduced the incidence of pneumothorax but also caused less disturbance to the immune system. No thymic residue was found by postoperative autopsy and a distinct decrease of T cells in peripheral blood was detected by flow cytometry analysis. The mortality rate was 3.3%, which is comparable to the ventilation-support technique, yet the new procedure is simpler and faster. This technique provides both reliability and simplicity for thymectomy in adult mice.

Introduction

Thymectomy has been widely used as an effective method for studying the function of the thymus and T cells in various fields, such as infection, transplantation and tumor immunity etc. As distinct from thymectomy in neonatal mice and genetically engineered mice such as SCID and nude mice, in which T cells are depleted at a very young age, thymectomy in adult mice can create a unique immune status at a selected time at which no newborn T cells are released to the circulation (Miller, 1965). In mice, naïve T cells are almost exclusively sustained by thymus output ( den Braber et al., 2012 ). Thus, this method may not only help researchers to manipulate the T cell pool according to their interests, but also be an effective tool to study the role of the thymus under different pathological conditions.


The  original  technique  of  thymectomy  in  adult mice  was  described  in  1963,  which  mainly  used  suction to remove thymus lobes (Sjodin et al., 1963). However, there were two shortcomings that rendered this method unsatisfactory. Firstly, suction may not assure complete removal of the thymus, because suction through  the  constrictive  tube  could  tear  the  thymus into   pieces. Secondly,  the  mortality rate was high, up  to  5-30%  (Castro  JE,  1974;  DeMatteo  et  al.,  1995; Vrisekoop  et  al.,  2008).  Pneumothorax  was  the  primary cause of death followed by hemorrhage of the heart or  mediastinal  vessels  (DeMatteo  et  al.,  1995).  Our experience  is  that  about  20-30%  of  mice  succumb  to this procedure (unpublished data). To overcome this, researchers developed a method using intubation and mechanical  ventilation  to  prevent  respiratory  failure (DeMatteo   et   al.,   1995).   Despite   the   mortality   rate decreasing to 3-6%, this modified method not only prolonged   the   operating   time,   increased   complexity and added experimental cost, but also resulted in unexpected negative consequences such as ventilation lung injury. The aim of the present study was to develop  a  new  method,  which  provides  complete  removal of  thymus  tissue,  as  well  as  improving  animal  survival with  minimal  side  effects.

Materials and methods

Mice. Thirty   C57BL/6J   male  mice  (6-8  weeks  old, weighing  19-21g)  were  purchased  from  the  Institute of  Experimental  Animals,  Chinese  Academy  of  Medical Science (Beijing, China). To avoid fighting, mice from the same litter were housed in groups of five in polypropylene  cages  (290×178×160mm)  with  sufficient specific pathogen free (SPF) soft wood shavings (GB14924.3-2010, HFK bioscience, Beijing, China). Cotton  wool  was  added  to  the  shavings  to  help  the mice nesting, and disposable cardboard boxes were provided for hiding and occupation, which might also prevent  aggressive  behavior.  Mice  were  given  ad  libitum access to double-distilled water and commercial SPF pelleted food (GB14924.3-2010, HFK bioscience, Beijing, China). All animals were kept at a room temperature   of   24±2 o C   and   50±10%   relative   humidity on a 12h light/dark photoperiod. All procedures performed on animals were approved by the Animal Care Research  Ethics  Committee  of  the  Capital  Medical University  of  China.

 

Equipment.  Stereomicroscope  ( Zeiss  Stemi  2000-C, Oberkochen, Germany ), 6-0 silk braided non-absorbable suture (Ethicon, Johnson-Johnson,  NJ,  USA),  and 7-0  polypropylene  suture  (Ethicon,  Johnson-Johnson, NJ,  USA).

Procedure of thymectomy  in adult mouse.  All  operations were performed in a special disinfected operating room. Surgical instruments were sterilized and disposable sterile gloves were used during the operation. Mice were anesthetized by intraperitoneal injection of narcotic  fluid  (ketamine  100mg/kg,  10mg/kg  xylazine
0.1ml/10g), and placed on a dissecting board in supine position.  The  feet  of  the  mice were fixed with tape to keep a stable position during the operation. The skin of neck  and  upper  chest  was  clipped  and  disinfected  with
70%  alcohol.  Under  a  stereomicroscope,  a  15-20mm skin incision was made along the anterior median line from  the suprasternal  notch  to  the  level  of  the  third ri b. Then, the suprasternal fossa was exposed by dividing the superficial fascia and moving the submaxillary glands aside. A central sternal incision was made to the level  of  the  second  rib  using  microdissection  scissors. In  order  to  prevent  pneumothorax  and  hemorrhage, this  incision  was  made  strictly  along  the  midline  with the   scissor   tips   tilting   towards   the   dorsal   aspect   of the  sternum  (Figure1,  step  1).  The  bisected  sternum was retracted laterally with 6-0 silk braided sutures implanted on the bilateral edges in order to expose the mediastinum.  Suturing  was  performed  from  interior to exterior to avoid punctures inside the chest cavity. Such  punctures  might  easily  have  led  to  pneumothorax,   even   hemorrhage   and   organ   damage   (Figure 1, step  2).  Two  white  thymus  lobes  were  exposed.  Each lobe was enveloped separately in a sack-like thin membrane connected to the pleura, which was hard to discern.  Careful  dissection  was  performed  to  open  the sack and separate the lobe from its membrane without damaging  the  pleura.  The  right  lobe  was  gently  held and  drawn  by  iris forceps  and  the  inferior  pole  of  the lobe  was  exposed  (Figure1,  step  3).  To  perform  pleura ligation at the bottom of the thymus lobe, a 7-0 polypropylene  suture  loose  knot  was  made  in  advance  to avoid  making  risky  movements inside  the  chest  cavitywhile tying knots. Then the circle was set at the caudal edge of the lobe (Figure1, step 4). After the pleura and the  nourishing  blood  vessels  of  the  lobe  were  ligated, the entire lobe was cut along the edge and removed carefully  under  clear  vision  (Figure1,  step  5).  The  left lobe  of  the  thymus  was  removed  in  a  similar  manner. The  chest  cavity  was  checked  for  thymic  remnants and  accessory  injuries  before  being  sealed  with  6-0 silk suture lines, which were formerly used for sternal retraction (Figure1, step 6). After the skin had been approximated  with  single  silk  sutures  or  wound  clips, the  incision  was  cleaned  with  0.9%  physiological saline. Following thymectomy, animals were given fentanyl citrate (2.5 μg /kg, sc.) and placed in clean cages in groups of five with warming lights until they recovered from anesthesia. Mice were kept for 14 days until euthanized  for  examination.

Post-operation assessment. To test the success of the thymectomy, autopsy and flow cytometry analysis were performed  14  days  after  the  operation.  With  the  help of  narcotic  fluid,  mice  were  deeply  anesthetized  and blood  samples  were  collected  from  the  orbital  sinus into  heparinized  tubes.  Then,  mice  were  euthanized by cervical dislocation. After the diaphragm had been dissected, the thoracic cavity was exposed under a stereomicroscope  in  order  to  search  for  thymic  residues. In case of small thymic residues invisible to the naked eye, we harvested all soft tissue from the original position  of  the  thymus  and  stained  the  cells  with  fluorescein    isothiocyanate    (FITC)-conjugated    anti-mouse CD45, PE-conjugated CD8 and APC-conjugated CD4 antibodies (BD   Bioscience,   San   Diego,   CA , USA). We next detected the influence of thymectomy on  peripheral  blood.  After  erythrocyte  lysis  with  BD Pharm   Lyse   solution   (BD   Bioscience,   Sparks,   MD, USA),   blood   cells   were   stained   with   phycoerythrin (PE)-conjugated   anti-mouse   CD8   and   allophycocyanin  (APC)-conjugated anti-mouse  CD4  antibodies (BD Bioscience, San Diego, CA , USA). All cells were examined  tested  by  a  BD  FACSCalibur  flow  cytometer  (BD,  CA ,  USA).  Flow  cytometry  data were analyzed using Flowjo (7.6.5, Tree- Star Inc., OR, USA) and Prism (6.0c, GraphPad Software Inc., CA , USA). Data were  analyzed  by  using  the  Student  t  test. P<0.05  was  defined  as  significant.


Figure 1

Figure 1. Procedure of pleura ligation and thymectomy in adult mice (10×). (1) The thymus was exposed after sternotomy. (2) Sternum was suspended and two thymus lobes were separated by dissecting their membrane. (3) The right lobe was gently pulled out, and the bottom edge and nourishing blood vessels were clearly visible. (4) A loose knot was set at the edge ready to ligate the pleura. (5) After the ligation, the complete right lobe was cut and removed. (6) Both thymus lobes were removed.

Click image to enlarge
Figure 2

Figure 2. Results of postoperative autopsy (10×). Compared with wild type mouse (left), thymectomized mouse (right) showed no residue of thymus in the mediastinum.
In addition, two blue knots were still visible (white arrow).

Click image to enlarge
Figure 3

Figure 3. ACompared to wild type mouse (left), thymectomized mouse showed no group of CD4+ and CD8+ double positive T cells (right).
B. Fourteen days after thymectomy, significant decreases
in CD4+ T cells and CD8+ T cells were observed in peripheral blood (P<0.01).

Click image to enlarge

Results

By  using  legation  thymectomy, the mortality  rate was 3.3% (1 of 30). Only one animal succumbed to severe pneumothorax during sternotomy. All other mice had successful  operations  and  lived  until  the  endpoint  of the experiment with no postoperative complications (such as tachypnea, wound infection etc.). Autopsy confirmed that no residue or regenerated tissue of thymus was observable in the mediastinum (Figure 2, blue  polypropylene  knots  were  still  visible).  No  signs of pulmonary or cardiovascular injury were observed. Results from flow cytometry showed that no group of CD4/CD8 double positive cells was found in tissue harvested from the former  thymus  location (Figure 3A),  and a  significant  decrease  of  CD4 +  T  and  CD8 + T cells in peripheral blood was found (P <0.01) (Figure 3B). Results from both autopsy and flow cytometry indicated  a  complete  removal  of  thymus.  Moreover, the average time of the operation for each mouse was around ten minutes in our laboratory.

 

Discussion

The  primary  challenge  of  thymectomy  in  adult  mice is  manipulation  of  the  delicate  pleura.  Animals  can suffer  from  instant  death  due  to  anoxia  and  mediastinal flutter triggered by the change in intrapleural pressure. In such cases, researchers use mechanical ventilation  to  support  pulmonary  function  and  prevent  progression  of  small pneumothorax  ( DeMatteo et al., 1995 ). Although this method seems to be a successful  solution,  the  extra  apparatus and  procedural steps  needed  may  render  the  whole  method  too  complicated. Moreover, ventilation support could lead to ventilation-induced lung injury (VILI) including collapse  of  alveolar  units,  edema  and  inflammation  etc. In particular, VILI has been associated with increased bronchoalveolar lavage fluid (BAL) levels of cytokines such  as   TNF-α  and   IL-6,   and  chemokines  such  as macrophage inflammatory protein (MIP)-2 (Chiumello et al., 1999; Cheng et al., 2002). Both impaired pulmonary function and inflammation might adversely affect subsequent immunological experiments. To eliminate these  problems,  it  is  necessary  to  develop  an  innocuous  technique  free  from  ventilation support and with high reliability as well.

We noted that the inferior pole of the thymus lobe was intimately attached to the pleura and cardiac pericardium with several nourishing blood vessels embedded. Because the pleura was more soft and fragile than blood vessels, it was easier to tear while blood vessels were still attached to thymus lobes. A small pleural leakage could quickly be enlarged by chest movement and become fatal. We also observed that in most failed cases, pneumothorax often occurred when the inferior pole of the thymus was pulled or bluntly dissected. In fact, strong or sudden traction of thymus lobes could even lead to aorta and atrium dislocation and rupture. Hence,  thymectomy  with  strong  direct  traction,  such as  suction  described  in  the  conventional  technique, may not be appropriate. Furthermore,  the  sucking force is difficult  to  control  and  the  cannula  tip  needs fine adjustment. Suitable modification of the cannula tip is extremely difficult because the pleura is likely to be involved during suction using a large diameter cannula, yet thymic residue is hard to avoid since a small diameter tip could easily tear tissue into pieces instead of  removing  the  complete  thymus.
To maintain the integrity of the pleura, we abandoned suction thymectomy and introduced ligation thymectomy by ligating the pleura at the bottom of thymus lobes. Firstly, ligation could prevent small pleural  leakage  by  tying  up  the  nourishing  blood  vessels with surrounding pleura, which could greatly minimize the risk of pneumothorax. Secondly, thymus excision is more reliable and safer with ligation, because the edge of the thymus lobe is clearly visible. In this way, complete  removal  of  the  thymus  is  ensured.  Post-operation examination also confirmed that no residue of thymus remained.  For  better  operational  sight  and  handling, we strongly  suggest  this  procedure be performed with the help of a stereomicroscope and microinstruments.

This method of thymectomy in adult mice is as efficient  as  procedures  with  ventilation  support  yet  it is simpler, easier and cheaper. More importantly, there are no ventilation-induced injuries to the respiratory system and subsequent immunological disturbance is eliminated.  Our  method  conforms  to  the  concept  of the 3Rs (replacement, reduction, refinement) and provides a stable animal model for future immunological investigation.


Acknowledgements

This work was supported by the Innovative Research Program of Capital Medical University, Beijing, China (No. xsky2012089).
Authors  declare  no  competing  interests.


References