Published: Vol 9, Iss 15, Aug 5, 2019 DOI: 10.21769/BioProtoc.3317 Views: 5468
Reviewed by: Meenal SinhaLuis Alberto Sánchez VargasAnonymous reviewer(s)
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Abstract
Splenectomy in an animal model requires a standardized technique utilizing best practice to avoid variability which can result in adverse impact to the animal resulting in flawed physiologic responses simply due to technique rather than to the studied variables. In the case of the spleen, often investigators are analyzing the animal immune or inflammatory responses. Surgical splenectomy involves many variables from the training and expertise of the surgeon, which directly correlates to surgical technique to the length of operation and ease of the procedure. This operation, in turn, impacts blood loss and insensible fluid losses, sterile technique, unintended trauma to the spleen and surrounding organs, the length of the incision and the duration of the operation with more prolonged exposure to anesthetic agents. All these variables ultimately play a significant role in the experiment since they directly affect the response of the model in terms of inflammation, immune activation, or even suppression. Undesired variables such as these go unnoticed and lead to inaccurate and misleading data.
Keywords: Blunt traumaBackground
The spleen produces protective humoral antibodies via plasma cells, harbors transient and stable populations of B and T cells, filters particulate antigens, and is a reservoir for red and white blood cells. There are specific helper T cells that migrate to and from the spleen serving a role in tumor surveillance and antigen recognition. It has also long been recognized as a domicile for specific B cell populations affecting the individual’s ability to respond to specific gram-negative encapsulated bacteria such as Meningococcus, Streptococcus, and Haemophilus. Splenectomy is sometimes necessary in cases of hemolytic anemias with splenomegaly from hereditary spherocytosis or sickle cell disease or in cases of thrombocytopenia, trauma, and even in some instances of malaria. In many instances, pediatric surgeons try to preserve some splenic function to preserve some immune function and avert long term risks of pulmonary hypertension by performing a partial splenectomy saving about 20-30% of the total splenic mass (Tastaldi et al., 2019). Sometimes due to the inadequate vascular supply of the upper pole of the spleen or in cases of hilar involvement of lesions or massive trauma, total splenectomy may be the only option. Asplenia may result in increased risks of post-splenectomy sepsis, pulmonary hypertension and even some speculation of increased risk of Type II diabetes, childhood obesity (Szendroi et al., 1997) and even inadequate cellular repair responses after cardiac ischemia (Costi et al., 2018).
Recent reports show that therapeutic modality for efficient antigen delivery to spleen shows promising for conquering tumor growth and progression (Shimizu et al., 2018). Current immunotherapy elevated the splenic level of IL-1β, IL-4, IL-10, IL-17, IFN-γ, and TNF-α for immunomodulatory effects of cancer progression (Merheb et al., 2019). Pediatric surgeons have advanced laparoscopic partial splenectomy to preserve hematological functions of the organ while trying to blunt or ameliorate hemolytic dysfunction (Serra et al., 2018). All these studies require the optimal performance of splenectomy to gain immune benefits of spleen function.
Surgery for splenectomy can involve many variables from training and expertise of the surgeon, surgical technique, length of operation, ease of the procedure, blood and fluid loss, sterile technique, trauma to the spleen or other tissues and traumatic manipulation, length of incision and length of exposure to anesthetic agents.
We provide some guidelines for the standardization of the technique for splenectomy and some thoughts in order to create a procedural best practice protocol such that resulting data can have less contamination by factors that can truly impact the right metrics being evaluated such as the degree of inflammation or immunity.
Materials and Reagents
Equipment
Procedure
Data analysis
Representative results:
Surgical splenectomy involves many variables from the training and expertise of the surgeon, which directly correlates to surgical technique to the length of operation and ease of the procedure. The actual surgery time varied from the skilled surgeon to two trainees, as shown in Figure 1. The actual surgery time and related complications impacted the recovery time as shown from 5.4 min (running around after the surgery, refer to the end of Video 1), to 2-3 h. Full recovery usually required about a week. Specifically, the skilled surgeon took an average of 7.98 min in the range of 5.32-10.54 min, with ten mice (total 10 mice) to survive up to 42-63 days (sacrificed). The Trainee 1 took the average 11.49 min in the range of 11.49-18.12 min, with six mice (total 6) survived 2-14 days; the trainee 2, 20.43 min, in the range of 18.54-22.36 min, with three mice (total 3) survived 1-5 days (Figure 1).
Figure 1. Relationship of survival with the duration of the surgical procedure
Discussion:
We describe a step by step technique for splenectomy with emphasis on performing a quick and easy surgery by limiting the length of the operation and being familiar with mouse anatomy and some techniques to limit the trauma and therefore the risk of blood loss. This technique minimizes fluid and blood loss by limiting the time and focusing on avoiding traumatic manipulation. Also, the length of the surgical incision limits the pain experienced and the shorter duration of surgery means that there is less exposure to anesthetic agents, which can also affect the physiologic responses. Many variations exist in the procedure performed, and therefore, the responses measured can be highly variable and inaccurate. When the procedure is performed swiftly, and without trauma, it limits the adverse effects of surgery and anesthesia such that the stress responses from the body are less significant in their effects on inflammation and infection. All these can explain why the relationship of survival with the duration of the surgical procedure (Figure 1). We have worked on two strains (BalbC and C57BL/6), and both strains worked. We did not work on male mice, and we found ideally, the ages of the mice are eight weeks. The tips and tricks and critical steps include: (1) the incision location is above the spleen, (2) precision cut (minimum incision opening in size sufficient to pull out the spleen); (3) to avoid hitting blood vessels, (4) seal the blood vessel as soon as possible to avoid loss of the blood, (5) seal the incision as soon as possible to avoid body fluidic loss. Cited literature below supports our observation.
The spleen is one of the largest lymphoid organs in the body. It harbors immune cells, including antigen presenting cells, B and T lymphocytes. Many studies have demonstrated the importance of the spleen in mounting a cellular and humoral immune response. Splenectomized Balb/C mice have reduced expression of IL-4 and IL-10, making them more susceptible to infection (Maioli et al., 2007). Humans have been recognized to be susceptible to certain gram-negative organisms when splenectomized. They are at risk of overwhelming post-splenectomy infection (OPSI) since King and Schumacker landmark reported in 1952. Loss of the spleen leads to reduced abilities to mount an adequate humoral response to certain bacteria and antigens due to loss of specific subsets of B lymphocytes (including plasma B cells, memory B cells, and follicular B cells) but also potential reduction in cell-mediated immunity given there are specific Helper T cells (TH1 and TH2) that shuttle around serving in surveillance and possess splenic homing receptors. T cells include cytotoxic T cells, memory T cells, helper T cells, natural killer T cells, suppressor T cells, and gamma delta T cells, natural killer cells, and progenitor lymphopoietic stem cells. Also, the actual physical structure of the spleen allowing large volumes of blood to enter within sinusoids that expose the resident cells to monitor and respond to potential antigens.
Splenocytes and specific fractions may have utility for immunotherapy (Li and Kabeer, 2013) in cancer and its responses within the tumor microenvironment (Li and Kabeer, 2018). Autologous splenic lymphocytes may be harvested for reperfusion to preserve specific memory T cell responses against cancer, especially when combined with cancer genome diagnostics (Li et al., 2014). The long-term outcomes of optimal laparoscopic splenectomy for immune thrombocytopenia reached curative in 68% of a cohort study of 109 patients as it is related to relapse-free rates, and predictors of long-term response (Tastaldi et al., 2019). Minimally invasive surgery improves splenic vessel preservation for reducing surgical complications, which was documented in fifteen relevant cohorts with 769 patients (Nakata et al., 2018). Pathologic enlargement (splenomegaly) results in an expansion of cellular proliferation outside of the bone marrow compartment, i.e., extramedullary hematopoiesis (EMH) (Mo et al., 2009), which is not yet fully understood. T- and B-cell recruitment into the restoration of the central (thymus) and peripheral (spleen and lymph node) is required. As T cell compartments are necessary for optimal adaptive immune responses to pathogens and tumor antigens, a comprehensive assessment of immune responses to splenectomy needs to be elucidated in achieving global immunization against cancer (Li et al., 2018) and infection. In particular, a recent study shows that “a novel antigen delivery platform for cancer vaccines that delivers an encapsulated Ag to splenic marginal zone B (MZ-B) cells via the aid of a PEGylated liposome (PL) system shows promise as a therapeutic modality for conquering tumor growth and/or progression” (Shimizu et al., 2018). Such antigen delivery platform for cancer vaccines relies on the quality of control surgery on the spleen. Furthermore, autologous splenic lymphocyte isolation for preserving an individual’s humoral responses to gram-negative bacteria and potentially have a much more robust response to vaccination in patients who have been splenectomized may be of significant benefit to patients.
Taken together, we wanted to emphasize that a precision procedure for splenectomy is essential in spleen related studies. We noted that a previously published procedure (Reeves et al., 2001) differs from our procedure. Specifically, (1) They made skin incision, ∼2.5 cm long, midway between the last rib and the hip joint, while we made only 0.5 cm incision in vertical midline abdomen just below the xyphoid process with fine scissors. (2) They did not seal the blood vessels, which bleeding along with the procedure for up to 60 min of recovery. Note that we used the cautery pen to seal the blood vessel and connective tissue to avoid the ever-lasting wound–natural healing takes much longer time, which needs only 5 min for mice to recovery. (3) Their two stitches for 2.5 cm incision (i.e., subject to infection) were compared with our five stitches for 0.5 cm incision. (4) Their survival rate of 95% following adult splenectomy was in contrast, to our surgeon gained a 100% survival rate.
Conclusions:
This precision technique for splenectomy limits mouse stress responses for accurate and realistic measurements for investigating inflammation and immunity.
Acknowledgments
Author Contributions: SCL and MHK conceived of the study and drafted the manuscript, and all other authors participated in its experimentation of the manuscript. All authors read and approved the final manuscript.
We thank Brent A. Dethlefs and Maria Minon, MD, for their enthusiasm and support.
DISCLOSURES: Funding sources: This work was supported in part by the National Institutes of Health (NCI, R01CA164509; NIEHS, R01ES021801-04S1), by CHOC Children’s Foundation, CHOC-UCI Joint Research Awards (2014, 2015, 2016), and by CHOC Children’s Physician’s Research Pilot grant.
Competing interests
The authors declare that they have no conflicts of competing interest.
Ethics
All protocols were approved by the specific ethics committee (IACUC) that approved the described experiment. IACUC #160501was the approval ID of the animal experiment in this protocol and the validity period was from June 01, 2016 to March 25, 2019.
References
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© 2019 The Authors; exclusive licensee Bio-protocol LLC.
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Category
Immunology > Animal model > Mouse
Microbiology > in vivo model > Bacterium
Cell Biology > Tissue analysis > Injury model
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