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Table of Contents
ORIGINAL ARTICLE
Year : 2019  |  Volume : 3  |  Issue : 3  |  Page : 174-177

The effect of ketamine on the immature granulocyte and C-reactive protein concentration in rat models of sepsis


Department of Anesthesiology and Intensive Care, Faculty of Medicine, Brawijaya University, Dr. Saiful Anwar General Hospital, Malang, Indonesia

Date of Submission21-Nov-2019
Date of Acceptance21-Nov-2019
Date of Web Publication23-Jan-2020

Correspondence Address:
Dr. Aswoco Andyk Asmoro
Department of Anesthesiology and Intensive Care, Faculty of Medicine, Brawijaya University, Dr. Saiful Anwar General Hospital, Jl. Mayjend Moch. Wiyono C-13, Kesatrian, Blimbing, Malang
Indonesia
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/BJOA.BJOA_5_19

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  Abstract 


Background: Sepsis directly affects the immune system, leads tissue damage, and forms system organ dysfunction. The immunological biomarker of sepsis has a possibility to become an immunotherapy target of sepsis. This study was conducted to determine the effect of ketamine on the number of immature granulocyte and C-reactive protein (CRP) concentration in rat models of sepsis. Materials and Methods: This study used thirty white mice (Rattus norvegicus) divided into six treatment groups. The negative control group received nothing, and the positive control (sepsis) group was fecal-induced peritonitis (FIP) by fecal administration (i. p). The treatment groups (A, B, C, and D) treat with ketamine 5 mg/kg body weight (i. p.) right after FIP, 3 h after FIP, 5 h after FIP, and intermittent every 2 h. The peripheral mononuclear blood cell was isolated 6 h after FIP. The immature granulocytes counted using a hematology analyzer while CRP concentration analyzed using kit enzyme-linked immunosorbent assay. Data were analyzed statistically using the one-way ANOVA test using SPSS version 20 software (P < 0.05). Results: Sepsis induction with FIP increases the number of immature granulocytes in animal models from 0.48% to 9.12% (P < 0.05) but did not affect CRP concentration (P > 0.05). The ketamine administration significantly decreases the immature granulocytes in Groups C (1.04%) and D (1.58%). Ketamine administration did not have a significant effect on CRP concentration. Conclusion: The ketamine administration at 5 h after FIP and intermittently every 2 h can be an alternative to be sepsis immunotherapy with immature granulocyte as the potential target.

Keywords: C-Reactive protein, immature granulocyte, ketamine, sepsis


How to cite this article:
Asmoro AA, Isngadi I, Laksono RM, Firdiansayah I, Supriyanto A. The effect of ketamine on the immature granulocyte and C-reactive protein concentration in rat models of sepsis. Bali J Anaesthesiol 2019;3:174-7

How to cite this URL:
Asmoro AA, Isngadi I, Laksono RM, Firdiansayah I, Supriyanto A. The effect of ketamine on the immature granulocyte and C-reactive protein concentration in rat models of sepsis. Bali J Anaesthesiol [serial online] 2019 [cited 2020 May 25];3:174-7. Available from: http://www.bjoaonline.com/text.asp?2019/3/3/174/276625




  Introduction Top


Sepsis is one of the most common health problems in the medical field. Sepsis case reaches 13 million each year and killed >250.000 people.[1],[2] The study by Sodik et al.[1] showed that the mortality rate of sepsis is about 70.2% in Indonesia. The study by Asmoro et al.[3] said that the mortality rate of sepsis is about 76.8% in Malang city. Sepsis occurs due to over-inflammation which leads to tissue damage. Tissue damage then develops system organ dysfunction and increases the sequential organ failure assessment score by 2 points or more.[4]

The body's response to sepsis begins with hyperinflammation characterized by the increase of pro-inflammatory cytokine production. Sepsis then continues toward the hypoinflammation or immunosuppression phase to balance the immune system. The hypoinflammation phase is characterized by a decrease in the amount of pro-inflammatory cytokines.[5] Immune cells play an important role in the inflammatory process. Immature granulocyte is an immune cell involved in pathological sepsis. The study conducted by Nierhaus et al.[6] explained the increases in the number of immature granulocytes in sepsis conditions. The number of immature granulocytes is then used as a sepsis biomarker. C-reactive protein (CRP) is a molecule produced by the liver and plays a role in the body's defense against infection. CRP can bind to antigens through a calcium mechanism. The CRP increases the phagocytic activity of the phagocyte cells. The CRP synthesis is stimulated by interleukin-6 (IL-6).[7],[8] The overproduction of CRP in sepsis is dangerous because it can activate nuclear factor-kappa B (NF-κB). The NF-κB activation is associated with high production of pro-inflammatory cytokines.[9],[10]

Ketamine is an anesthetic agent that has immunomodulatory activity. Ketamine prevents exacerbation and modulates progressed inflammation.[11] Research conducted by Gurfinkel et al.[12] states that ketamine in subanesthetic doses increases the survival rate of a sepsis model animal by decreasing the production of tumor necrosis factor-α and IL-6. The anesthetic doses of ketamine reduce lipopolysaccharide induced-liver inflammation. Ketamine decreases the cyclooxygenase-2, protein nitric oxide kinase, and NF-κB.[13] Based on several studies, ketamine has the potential to be the sepsis immunotherapy. Sepsis directly affects the immune system homeostasis such as influencing the number of immature granulocyte and CRP concentration. Both immune components have the potential to be a therapeutic target of ketamine. This study was conducted to determine the effect of ketamine on the number of immature granulocyte and CRP concentration in rat models of sepsis.


  Materials and Methods Top


This study was a true experimental study with a randomized posttest-only controlled group design. The research was conducted at the Clinical Pathology Laboratory of the Faculty of Medicine, Brawijaya University, Malang, in December 2016–January 2017. The research method has been approved by the Health Research Ethics Committee of Faculty of Medicine, Brawijaya University. This study used 30 white mice (Rattus norvegicus) with inclusion criteria including male rats, age 5 months, weight 200–250 g, active moves, and hair did not fall out. The samples were divided into six treatment groups. The negative control is not treated. Positive controls are fecal-induced peritonitis (FIP) or sepsis model. Feces from mice dissolved with normal saline until the concentration becomes 200 mg/ml. Feces 1 mg/g were administrated to induced peritonitis (i. p). Group A received ketamine 5 mg/kg body weight (i. p.) right after FIP. Group B received ketamine 5 mg/kg body weight (i. p.) at 3 h after FIP. Group C received ketamine 5 mg/kg body weight (i. p.) at 5 h after FIP. Group D received ketamine 5 mg/kg body weight (i. p.) intermittent every 2 h (0, 2, and 4 h after FIP). Peripheral blood mononuclear cell blood is isolated at 6 h after FIP (cardiac puncture). The number of immature granulocytes is calculated using a hematology analyzer. CRP concentration analyzes using kit enzyme-linked immunosorbent assay. Data were analyzed statistically using the one-way ANOVA test using SPSS software version 20 IBM Corp. Released 2013. IBM SPSS Statistics for Windows, Version 22.0. Armonk, NY: IBM Corp. (P < 0.05).


  Results Top


The rats were injected intraperitoneally with feces (FIP) to make a sepsis model. The sepsis model was determined by the murine sepsis score.[14] In the study, the rats that were injected with feces showed piloerection, closed eyes, periocular discharge, lethargy, decreased appetite, and decreased respiration rate. All animal models experienced sepsis (P = 0.094).

The immature granulocyte percentage in each group showed a varied result. The immature granulocytes in the negative control group were 0.48%, while in the positive control group, it was 9.12%. Sepsis induction with FIP increases the number of immature granulocytes in animal models. The administration of ketamine caused a decrease in the number of immature granulocytes in Groups A (1.78%), B (1.78%), C (1.04%), and D (1.58%) [Figure 1].
Figure 1: The immature granulocyte in the control group and in the ketamine group

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The CRP concentration in all treatment groups did not have a significant difference. The sepsis induction did not affect CRP concentration. The negative control group (normal) had the same CRP concentration as the positive control group (sepsis) (0.01 mg/dl). The CRP concentration after ketamine administration in Groups A and B was 0.012 mg/dl, 0.01 mg/dl in Group C, and 0.016 mg/dl in Group D [Figure 2].
Figure 2: The CRP concentration in the control groups and in the treatment groups

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  Discussion Top


Fecal administration in the control group showed a significant increase in the number of immature granulocytes but did not affect the CRP concentration. The immature granulocyte elevation is one of the innate immune responses. The immature granulocyte elevation is caused by the increase of granulocyte production in the bone marrow.[6] The study is similar to Nierhaus et al.[6] study about the immature granulocyte elevation in sepsis conditions. The presence of immature granulocytes can distinguish between infected patients and uninfected patients, especially at critical hours after the initial warning of systemic inflammatory response syndrome. The study was also similar to Ha et al.[15] who showed that immature granulocytes are more accurate to be sepsis biomarkers than CRP. The CRP concentrations after ketamine administration did not show a significant difference likely due to differences in sample surgical time and CRP production time after antigen exposure. The CRP concentration in plasma would increase between 4 and 6 h after the antigen exposure and reach the peak after 24–48 h and synthesized in the acute phase.[16],[17],[18] In this study, the PMBC isolation was carried out 6 h after FIP induction.

Ketamine is an anesthetic agent that has immunomodulatory properties and potential to become sepsis immunotherapy. The ketamine administration in the 5th h after FIP and intermittent every 2 h significantly reduced the number of immature granulocytes in the sepsis group. However, ketamine did not affect CRP concentration (P = 0.536) in the sepsis group. Immature granulocytes and CRP help the immune system to increase immune activity against infection. Nevertheless, granulocytes and CRP overproduction in sepsis conditions are dangerous because they can trigger acute inflammation. Ketamine administration in the sepsis group significantly reduces the immature granulocyte but not affects the CRP concentration.

Ketamine administration in Group C (5 h after FIP) and D (intermittent every 2 h) significantly reduces the immature granulocyte. Granulocyte activity regulates by the granulocyte-macrophage colony-stimulating factor (GM-CSF). GM-CSF activates NF-κB through IKK2-GMRβ interaction and leading to cell proliferation and survival. Ketamine reduces the phosphorylation of various kinases involved in cellular signaling toll-like receptor-2/4 (TLR-2/4). The stimulation of lipoteichoic acid (TLR-2 antagonist) on macrophages exposed to ketamine can reduce NF-κB production. Ketamine also increases NF-κB kinase inhibitors (IκB kinase) activation. The IκB kinase activation decreases the NF-κB translocation in the nucleus.[19],[20] The ketamine administration in the sepsis group did not affect the CRP concentration. However, Groups A and B experienced an increase in CRP concentration to 0.012 mg/dl. Intermittent administration of ketamine in the sepsis group had the highest CRP concentration (0.16 mg/dl). The study by Ibrahim et al.[21] showed a significant increase in CRP concentration after the administration of ketamine to the patients. The weakness of this study is the lack of ketamine dose variations, and the sepsis induction duration is too short. Therefore, further research is needed using various ketamine doses and longer sepsis induction duration.


  Conclusion Top


Sepsis significantly increases the number of immature granulocytes but does not affect CRP concentration. Ketamine administration at the 5th h after FIP and intermittently every 2 h can be an alternative to sepsis immunotherapy. The effect of ketamine modulation on CRP concentrations has not been determined.

Acknowledgment

The authors report no conflict of interest.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Sodik DC, Pradipta IS, Lestari K. Manajemen Terapi Sepsis. Stud J 2012;1:38. Availabe from: http://jurnal.unpad.ac.id/ejournal/article/view/1117. [Last accessed on 2016 December 23].  Back to cited text no. 1
    
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Hotchkiss RS, Monneret G, Payen D. Sepsis-induced immunosuppression: From cellular dysfunctions to immunotherapy. Nat Rev Immunol 2013;13:862-74.  Back to cited text no. 2
    
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Asmoro AA, Rakhmatullah R, Puspitasari S, Tarimah K, Saleh SC, Widodo MA, et al. The effect of ketamine on the lipopolysaccharide-induced inflammation in in vitro culture of HUVEC. Asian Pacific Journal of Tropical Disease. 2015;5:894-6.  Back to cited text no. 3
    
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Singer M, Deutschman CS, Seymour CW, Shankar-Hari M, Annane D, Bauer M, et al. The third international consensus definitions for sepsis and septic shock (Sepsis-3). JAMA 2016;315:801-10.  Back to cited text no. 4
    
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Boomer JS, Green JM, Hotchkiss RS. The changing immune system in sepsis: Is individualized immuno-modulatory therapy the answer? Virulence 2014;5:45-56.  Back to cited text no. 5
    
6.
Nierhaus A, Klatte S, Linssen J, Eismann NM, Wichmann D, Hedke J, et al. Revisiting the white blood cell count: Immature granulocytes count as a diagnostic marker to discriminate between SIRS and sepsis – A prospective, observational study. BMC Immunol 2013;14:8.  Back to cited text no. 6
    
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Del Giudice M, Gangestad SW. Rethinking IL-6 and CRP: Why they are more than inflammatory biomarkers, and why it matters. Brain Behav Immun 2018;70:61-75.  Back to cited text no. 7
    
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Fraj M, Salem N. C-Reactive Protein. Department of Neurosurgery, Hospital of Neuroscience. Available from: http://www.interchopen.com. [Last accessed on 2019 Jun 03].  Back to cited text no. 8
    
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Liu T, Zhang L, Joo D, Sun SC. NF-κB signaling in inflammation. Signal Transduct Target Ther. 2017;2:17023.  Back to cited text no. 9
    
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Verma S, Badiwala MV, Weisel RD, Li SH, Wang CH, Fedak PW, et al. C-reactive protein activates the nuclear factor-kappaB signal transduction pathway in saphenous vein endothelial cells: Implications for atherosclerosis and restenosis. J Thorac Cardiovasc Surg 2003;126:1886-91.  Back to cited text no. 10
    
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Loix S, De Kock M, Henin P. The anti-inflammatory effects of ketamine: State of the art. Acta Anaesthesiol Belg 2011;62:47-58.  Back to cited text no. 11
    
12.
Gurfinkel R, Czeiger D, Douvdevani A, Shapira Y, Artru AA, Sufaro Y, et al. Ketamine improves survival in burn injury followed by sepsis in rats. Anesth Analg 2006;103:396-402.  Back to cited text no. 12
    
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Hirota K, Lambert DG. Ketamine: New uses for an old drug? Br J Anaesth 2011;107:123-6.  Back to cited text no. 13
    
14.
Shrum B, Anantha RV, Xu SX, Donnelly M, Haeryfar SM, McCormick JK, et al. A robust scoring system to evaluate sepsis severity in an animal model. BMC Res Notes 2014;7:233.  Back to cited text no. 14
    
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Ha SO, Park SH, Park SH, Park JS, Huh JW, Lim CM, et al. Fraction of immature granulocytes reflects severity but not mortality in sepsis. Scand J Clin Lab Invest 2015;75:36-43.  Back to cited text no. 15
    
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Douraiswami B, Dilip PK, Harish BN, Jagdish M. C-reactive protein and interleukin-6 levels in the early detection of infection after open fractures. J Orthop Surg (Hong Kong) 2012;20:381-5.  Back to cited text no. 16
    
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Husain TM, Kim DH. C-reactive protein and erythrocyte sedimentation rate in orthopaedics. Univ Pa Orthop J 2002;15:13-6.  Back to cited text no. 17
    
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Ingle PV, Patel DM. C-reactive protein in various disease condition – An overview. Asian J Pharm Clin Res 2011;4:9-13.  Back to cited text no. 18
    
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Peters K, Unger RE, Brunner J, Kirkpatrick CJ. Molecular basis of endothelial dysfunction in sepsis. Cardiovasc Res 2003;60:49-57.  Back to cited text no. 19
    
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Bhattacharya P, Budnick I, Singh M, Thiruppathi M, Alharshawi K, Elshabrawy H, et al. Dual Role of GM-CSF as a Pro-Inflammatory and a Regulatory Cytokine: Implications for Immune Therapy. J Interferon Cytokine Res 2015;35:585-99.  Back to cited text no. 20
    
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Ibrahim TH, Abdelrahman H, Alharbi M, Zabani I, Ismail F, Kary H. Original Article Effect of ketamine on pro- and antiinflammatory cytokine response in paediatric cardiac surgery: A prospective randomised controlled study. Indian J Anesth 2017;61:549-55.  Back to cited text no. 21
    


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