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Original Research Article

DTT 2022; 1(1): 45-50

Published online July 31, 2022 https://doi.org/10.58502/DTT.22.008

Copyright © The Pharmaceutical Society of Korea.

Effect of Anesthesia/Euthanasia Method Used for Brain Collection on GSK3β Activation in Male Sprague-Dawley Rats

Suryun Jung , Sooyeun Lee

College of Pharmacy, Keimyung University, Daegu, Korea

Correspondence to:Sooyeun Lee, sylee21@kmu.ac.kr

Received: May 2, 2022; Revised: June 8, 2022; Accepted: June 16, 2022

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ((http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

GSK3β is strongly associated with pathophysiological conditions and neuropsychiatric diseases by regulating synaptic plasticity and the activity of structural proteins and signaling molecules in the brain. Therefore, the change in GSK3β activity is an important factor in interpreting the results of the study. We analyzed the effect of the tissue extraction method on the expression of GSK3β in rat brain. Male Sprague–Dawley rats were randomly assigned to two groups [Group A: decapitate after anesthesia with pentobarbital (n = 8) and Group D: decapitate without anesthesia (n = 6)]. The phosphorylation of GSK3β in Group D was significantly higher than that in Group A in the four regions of the brain, and ERK1/2 activity was significantly increased in Group D compared to that in Group A in the prefrontal cortex. Therefore, we confirmed that the method of anesthesia/euthanasia can affect GSK3β activity, which in turn can contaminate the results of experimental studies on novel chemo-therapeutic approaches involving GSK3β. We recommend that the effect of anesthesia/euthanasia method must be considered to obtain accurate results regarding the activity and expression of GSK3β.

KeywordsGSK3β, anesthesia, ERK1/2, neuropsychiatric disorder

Glycogen synthase kinase 3 (GSK3) is a serine/threonine kinase that is known for its ability to phosphorylate and inactivate glycogen synthase. However, it was later found to be a member of the mitogen-activated protein kinase (MAPK) family, involved in energy metabolism, neuronal development, cell proliferation, and survival regulation (Grimes and Jope 2001; Doble and Woodgett 2003; Luo 2009). Mammals possess two homologues encoded by two separate genes: GSK3α (51 kDa) and GSK3β (47 kDa). GSK3β was originally isolated from skeletal muscle (Embi et al. 1980; Rylatt et al. 1980), but is widely expressed in all tissues, especially in the brain (Woodgett 1990). GSK3β phosphorylates transcription factors, structural proteins, and signaling molecules that regulate synaptic plasticity (Camiletti-Moirón et al. 2013; Speck et al. 2014). GSK3β is strongly associated with pathophysiological conditions such as neurodegenerative diseases, inflammation, diabetes, cancer, and neuropsychiatric diseases such as drug addiction, anxiety, and depressive-like behaviors (Wang et al. 2014; Firth et al. 2015; Park et al. 2016;). Most studies investigating the function of GSK3β in relation to neuropsychiatric diseases or drug efficacy are performed by immunohistochemistry analysis or immunoblotting of extracted brains. However, the effect of tissue collection on intracellular signaling pathways is not well understood. Ko et al. (2019b) investigated the effects of different methods of anesthesia or euthanasia (ketamine/xylazine, isoflurane anesthesia, carbon dioxide asphyxiation, and decapitation) on the activity of mitogen-activated protein kinase (MAPK) in the brain tissue of C57BL/6 mice. Since MAPKs regulate neural functions, such as neurodifferentiation, proliferation, and apoptosis, their signaling pathways are crucial to neuropsychiatric studies (Huang and Lin 2006; Duman et al. 2007; Wefers et al. 2012). Ko et al. (2019b) showed that there was no sex-associated difference in ERK1/2 activity; however, in comparison with carbon dioxide asphyxiation, the use of isoflurane followed by decapitation significantly increased ERK1/2 activity in the regions of male and female brains. In addition, euthanasia by carbon dioxide asphyxiation induces hypoxia and affects MAPK signaling activity (Risbud et al. 2005), and rapid decapitation without anesthesia induces a stress response and may modulate MAPK signaling in the prefrontal cortex and hippocampus (Meller et al. 2003). Xylazine and ketamine have been shown to affect MAPK signaling (Réus et al. 2015), and isoflurane, which is commonly used in brain enucleation, can induce neuroinflammation and increase JNK phosphorylation (Altay et al. 2014). Therefore, the process of brain tissue collection can seriously contaminate the experimental results of neurochemical studies by directly affecting the MAPK signaling pathway. GSK3β is a member of the MAPK family whose activity is known to be affected by stress. Therefore, the possibility that the tissue extraction method affects GSK3β expression cannot be excluded. However, very little is known about the effect of tissue extraction methods on GSK3β expression. In this study, we analyzed the effect of the tissue extraction method on the expression of GSK3β in rat brain.

Animals

A pair of male Sprague–Dawley rats weighing approximately 200 g each were housed in a cage. The rats were acclimated to a 12-h light/dark cycle with food (#2014, Harlan Telkad, Madison, WI, USA) and tap water available ad libitum. The rats were randomly assigned to two groups – Group A (n = 8) was decapitated after anesthesia with pentobarbital and Group D (n = 6) was decapitated without anesthesia. All animal studies were approved by the Institutional Animal Care and Use Committee of Keimyung University (KM2020-008).

Anesthesia/euthanasia methods

The rat brains were collected in a separate suite at the same time of the day during their active cycle using two different anesthesia and euthanasia methods: (1) pentobarbital:100 mg/kg sodium pentobarbital (Entobar inj., Hanlim Pharmaceuticals Co., Ltd., Seoul, Korea) was injected intraperitoneally. If there was no plantar reflex, the rats were decapitated. (2) Decapitation: The rats were gently restrained and decapitated in a new cage to minimize exposure to blood from conspecific rats (Fig. 1).

Figure 1.Overview of experimental methods.

Tissue collections and sample preparation

Brains were swiftly removed, and the nucleus accumbens, striatum, prefrontal cortex, and hippocampal regions were placed on an ice-cold glass plate. Tissues were immediately frozen in liquid nitrogen and stored at −80℃ for downstream processing.

Western blot

The extracted brain tissue was mixed with RIPA buffer (w/protease inhibitor (#1861280, Fisher Scientific, USA) and phosphatase inhibitor (P2850, Sigma-Aldrich, USA) and homogenized with a Wheaton® tissue grinder (#357535 and 357537, DWK Life Sciences, USA). The homogenized samples were subjected to three freeze/thaw cycles and centrifuged (1000 × g, 15 min, 4℃). The supernatant of the samples was quantified using a BSA assay (#5000006, Bio-Rad, Hercules, CA, USA) in preparation for western blotting. Samples were prepared using Laemmli buffer (#1610747, Bio-Rad) and boiled at 100℃ for 10 min prior to loading. The prepared samples were loaded onto a sodium dodecyl sulfate-polyacrylamide gel, electrophoresed, and transferred onto a nitrocellulose membrane. The membrane was blocked for 60 min using 5% non-fat dry milk and Tris-buffered saline + 0.1% Tween 10 (TBST; pH 7.5), washed with TBST, and incubated overnight at 4℃ with the following primary antibodies: GSK3β (Santa Cruz Bio, sc-81462), phospho-GSK3β (Santa Cruz Bio, sc-373800), phospho (Thr202/Tyr204)-extracellular signal-regulated kinases 1/2 (Erk1/2, 1:1000, Cell Signaling, #9101), and Erk1/2 (1:3000, Cell signaling, #9102). After another wash with TBST, the samples were treated with secondary antibody (anti-mouse or anti-rabbit, Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 60 min. The bands were visualized using ECL (Genekhan Scientific, St. Louis, MO, USA), and the relative intensities of the bands were assessed using SigmaGel (Jandel Scientific Corp., Erkrath, Germany).

Statistical analysis

Statistical analyses were performed using GraphPad Prism 7.0 (GraphPad Software Inc., La Jolla, CA, USA). The results are presented as mean ± standard error (SE). Significant differences between groups were evaluated using an unpaired t-test where p-value < 0.05 was considered statistically significant. The analysis was performed by a statistician blinded to the experimental groups.

Fig. 2 shows the expression pattern of GSK3β in the regions of the brain. The phosphorylation of GSK3β in rats that were decapitated without anesthesia (Group D) was significantly higher than in those that were decapitated after anesthesia with pentobarbital (Group A). This was observed in the prefrontal cortex, hippocampus, striatum, and nucleus accumbens of the brain.

Figure 2.Expression pattern of GSK3β in the regions of the brain. (A) Expression of phospho-GSK3β. (B) Expression of total GSK3α, GSK3β. (C) p/t GSK3β ratio in hippocampus (Group A, n = 8; Group D, n = 6). (D) p/t GSK3β ratio in prefrontal cortex (Group A, n = 8; Group D, n = 5). (E) p/t GSK3β ratio in nucleus accumbens (Group A, n = 8; Group D, n = 5). (F) p/t GSK3β ratio in striatum (Group A, n = 8; Group D, n = 5). Values are means ± SE, ***p < 0.001, **p < 0.01, *p < 0.05. AU, arbitrary unit; GSK3β, glycogen synthase kinase 3β.

The expression pattern of ERK1/ERK2 in the prefrontal cortex was also observed. The ERK1/2 activity, an upstream signaling factor regulating GSK3β phosphorylation, was significantly higher in Group D than that in in Group A, as shown in Fig. 3.

Figure 3.Expression pattern of ERK1/ERK2 in the prefrontal cortex. (A) Expression level of ERK1/ERK2. (B) p/t ERK1 ratio (Group A, n = 7; Group D, n = 5). (C) p/t ERK2 ratio (Group A, n = 7; Group D, n = 5). Values are means ± SE, ***p < 0.001. AU, arbitrary unit; ERK1/ERK2, extracellular signal-regulated kinases 1 and 2.

GSK3β is widely expressed in all tissues, and is particularly abundant in the central nervous system (Woodgett 1990). GSK3β, unlike most protein kinases, is constitutively active in resting cells, and is rapidly and transiently inhibited in response to multiple external signals (Grimes and Jope 2001; Doble and Woodgett 2003). GSK3β activity is regulated by site-specific phosphorylation, and the complete activity of GSK3β generally requires phosphorylation at tyrosine 216 (Tyr216), whereas phosphorylation at serine 9 (Ser9) inhibits GSK3β activity (Lin et al. 1997; Shaw et al. 1998; Bijur and Jope 2000). The phosphorylation of Ser9 is the most important mechanism regulating GSK3β activity and is mediated by several kinases, including p70S6 kinase, extracellular signaling kinase (ERK), p90Rsk (MAPKAP kinase-1), protein kinase B, and cyclic AMP-dependent protein kinase C (Grimes and Jope 2001; Kaytor and Orr 2002).

GSK-3β plays a significant role in physiological and pathological conditions in many neurological and psychiatric disorders (Noori et al. 2020) such as diabetes, Alzheimer's disease, bipolar disorder, inflammatory disease, and cancer (Benedetti et al. 2004; Jope and Roh 2006; Cole 2013). Since GSK3β plays a key role in the pathogenesis of these neurodegenerative and psychiatric disorders, novel therapeutic approaches based on GSK3β inhibition are being explored. However, GSK3β activity is directly affected by the stress generated during tissue harvesting, a process integral to the experimental studies on therapeutic strategies involving GSK3β (Ko et al. 2019a). This can seriously contaminate experimental results. Additionally, appropriate assessment of GSK3β activity or its inhibition is important to study the neurochemical basis of these disorders and how drug treatment modifies or alters cellular signaling. In this study, we analyzed the effect of commonly used experimental protocols such as decapitating after anesthesia vs. decapitating without anesthesia, on the activity of GSK3β in various regions of the brain. Pentobarbital is the most commonly used barbiturate for euthanasia (Laferriere and Pang 2020). Therefore, in our study, Male SD rats were decapitated with pentobarbital or without anesthesia. We selected four brain regions, prefrontal cortex, hippocampus, striatum, and nucleus accumbens, that are closely related to neuropsychiatric disorders (Alviña et al. 2021; Saigusa et al. 2021). The hippocampus is closely associated with anxiety, memory, attention deficit, hyperactivity, and autism spectrum disorders (Bannerman et al. 2004; Onnink et al. 2014; Tottenham et al. 2014). The striatum and nucleus accumbens are strongly associated with substance use disorders, Parkinson's disease, and epilepsy (Deransart et al. 2000; Robbins and Everitt 2002; Surmeier et al. 2014). Abnormalities in the prefrontal cortex have been associated with attention-deficit/hyperactivity disorder, autism spectrum disorder, and various mood disorders (Drevets et al. 1997; Halperin and Schulz 2006; Morgan et al. 2010; Courchesne et al. 2011).

The present study demonstrated that the anesthesia/euthanasia method can affect GSK3β activity, which can seriously contaminate the results of investigative studies on the therapeutic role of GSK3β inhibition in neurodegenerative and psychiatric disorders. However, there are some limitations in the study. The regulation of the signaling pathway of GSK3β was not investigated and hence the causative factors responsible for the change in GSK3β levels were not be identified. In addition, the effects of more various anesthetics need to be investigated. Nonetheless, the results in the present study could be referenced in the experimental field by comparing and analyzing the effect of decapitation with or without anesthesia on GSK3β.

No potential conflict of interest relevant to this article was reported.

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Article

Original Research Article

DTT 2022; 1(1): 45-50

Published online July 31, 2022 https://doi.org/10.58502/DTT.22.008

Copyright © The Pharmaceutical Society of Korea.

Effect of Anesthesia/Euthanasia Method Used for Brain Collection on GSK3β Activation in Male Sprague-Dawley Rats

Suryun Jung , Sooyeun Lee

College of Pharmacy, Keimyung University, Daegu, Korea

Correspondence to:Sooyeun Lee, sylee21@kmu.ac.kr

Received: May 2, 2022; Revised: June 8, 2022; Accepted: June 16, 2022

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ((http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

GSK3β is strongly associated with pathophysiological conditions and neuropsychiatric diseases by regulating synaptic plasticity and the activity of structural proteins and signaling molecules in the brain. Therefore, the change in GSK3β activity is an important factor in interpreting the results of the study. We analyzed the effect of the tissue extraction method on the expression of GSK3β in rat brain. Male Sprague–Dawley rats were randomly assigned to two groups [Group A: decapitate after anesthesia with pentobarbital (n = 8) and Group D: decapitate without anesthesia (n = 6)]. The phosphorylation of GSK3β in Group D was significantly higher than that in Group A in the four regions of the brain, and ERK1/2 activity was significantly increased in Group D compared to that in Group A in the prefrontal cortex. Therefore, we confirmed that the method of anesthesia/euthanasia can affect GSK3β activity, which in turn can contaminate the results of experimental studies on novel chemo-therapeutic approaches involving GSK3β. We recommend that the effect of anesthesia/euthanasia method must be considered to obtain accurate results regarding the activity and expression of GSK3β.

Keywords: GSK3&beta,, anesthesia, ERK1/2, neuropsychiatric disorder

Introduction

Glycogen synthase kinase 3 (GSK3) is a serine/threonine kinase that is known for its ability to phosphorylate and inactivate glycogen synthase. However, it was later found to be a member of the mitogen-activated protein kinase (MAPK) family, involved in energy metabolism, neuronal development, cell proliferation, and survival regulation (Grimes and Jope 2001; Doble and Woodgett 2003; Luo 2009). Mammals possess two homologues encoded by two separate genes: GSK3α (51 kDa) and GSK3β (47 kDa). GSK3β was originally isolated from skeletal muscle (Embi et al. 1980; Rylatt et al. 1980), but is widely expressed in all tissues, especially in the brain (Woodgett 1990). GSK3β phosphorylates transcription factors, structural proteins, and signaling molecules that regulate synaptic plasticity (Camiletti-Moirón et al. 2013; Speck et al. 2014). GSK3β is strongly associated with pathophysiological conditions such as neurodegenerative diseases, inflammation, diabetes, cancer, and neuropsychiatric diseases such as drug addiction, anxiety, and depressive-like behaviors (Wang et al. 2014; Firth et al. 2015; Park et al. 2016;). Most studies investigating the function of GSK3β in relation to neuropsychiatric diseases or drug efficacy are performed by immunohistochemistry analysis or immunoblotting of extracted brains. However, the effect of tissue collection on intracellular signaling pathways is not well understood. Ko et al. (2019b) investigated the effects of different methods of anesthesia or euthanasia (ketamine/xylazine, isoflurane anesthesia, carbon dioxide asphyxiation, and decapitation) on the activity of mitogen-activated protein kinase (MAPK) in the brain tissue of C57BL/6 mice. Since MAPKs regulate neural functions, such as neurodifferentiation, proliferation, and apoptosis, their signaling pathways are crucial to neuropsychiatric studies (Huang and Lin 2006; Duman et al. 2007; Wefers et al. 2012). Ko et al. (2019b) showed that there was no sex-associated difference in ERK1/2 activity; however, in comparison with carbon dioxide asphyxiation, the use of isoflurane followed by decapitation significantly increased ERK1/2 activity in the regions of male and female brains. In addition, euthanasia by carbon dioxide asphyxiation induces hypoxia and affects MAPK signaling activity (Risbud et al. 2005), and rapid decapitation without anesthesia induces a stress response and may modulate MAPK signaling in the prefrontal cortex and hippocampus (Meller et al. 2003). Xylazine and ketamine have been shown to affect MAPK signaling (Réus et al. 2015), and isoflurane, which is commonly used in brain enucleation, can induce neuroinflammation and increase JNK phosphorylation (Altay et al. 2014). Therefore, the process of brain tissue collection can seriously contaminate the experimental results of neurochemical studies by directly affecting the MAPK signaling pathway. GSK3β is a member of the MAPK family whose activity is known to be affected by stress. Therefore, the possibility that the tissue extraction method affects GSK3β expression cannot be excluded. However, very little is known about the effect of tissue extraction methods on GSK3β expression. In this study, we analyzed the effect of the tissue extraction method on the expression of GSK3β in rat brain.

Materials and Methods

Animals

A pair of male Sprague–Dawley rats weighing approximately 200 g each were housed in a cage. The rats were acclimated to a 12-h light/dark cycle with food (#2014, Harlan Telkad, Madison, WI, USA) and tap water available ad libitum. The rats were randomly assigned to two groups – Group A (n = 8) was decapitated after anesthesia with pentobarbital and Group D (n = 6) was decapitated without anesthesia. All animal studies were approved by the Institutional Animal Care and Use Committee of Keimyung University (KM2020-008).

Anesthesia/euthanasia methods

The rat brains were collected in a separate suite at the same time of the day during their active cycle using two different anesthesia and euthanasia methods: (1) pentobarbital:100 mg/kg sodium pentobarbital (Entobar inj., Hanlim Pharmaceuticals Co., Ltd., Seoul, Korea) was injected intraperitoneally. If there was no plantar reflex, the rats were decapitated. (2) Decapitation: The rats were gently restrained and decapitated in a new cage to minimize exposure to blood from conspecific rats (Fig. 1).

Figure 1. Overview of experimental methods.

Tissue collections and sample preparation

Brains were swiftly removed, and the nucleus accumbens, striatum, prefrontal cortex, and hippocampal regions were placed on an ice-cold glass plate. Tissues were immediately frozen in liquid nitrogen and stored at −80℃ for downstream processing.

Western blot

The extracted brain tissue was mixed with RIPA buffer (w/protease inhibitor (#1861280, Fisher Scientific, USA) and phosphatase inhibitor (P2850, Sigma-Aldrich, USA) and homogenized with a Wheaton® tissue grinder (#357535 and 357537, DWK Life Sciences, USA). The homogenized samples were subjected to three freeze/thaw cycles and centrifuged (1000 × g, 15 min, 4℃). The supernatant of the samples was quantified using a BSA assay (#5000006, Bio-Rad, Hercules, CA, USA) in preparation for western blotting. Samples were prepared using Laemmli buffer (#1610747, Bio-Rad) and boiled at 100℃ for 10 min prior to loading. The prepared samples were loaded onto a sodium dodecyl sulfate-polyacrylamide gel, electrophoresed, and transferred onto a nitrocellulose membrane. The membrane was blocked for 60 min using 5% non-fat dry milk and Tris-buffered saline + 0.1% Tween 10 (TBST; pH 7.5), washed with TBST, and incubated overnight at 4℃ with the following primary antibodies: GSK3β (Santa Cruz Bio, sc-81462), phospho-GSK3β (Santa Cruz Bio, sc-373800), phospho (Thr202/Tyr204)-extracellular signal-regulated kinases 1/2 (Erk1/2, 1:1000, Cell Signaling, #9101), and Erk1/2 (1:3000, Cell signaling, #9102). After another wash with TBST, the samples were treated with secondary antibody (anti-mouse or anti-rabbit, Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 60 min. The bands were visualized using ECL (Genekhan Scientific, St. Louis, MO, USA), and the relative intensities of the bands were assessed using SigmaGel (Jandel Scientific Corp., Erkrath, Germany).

Statistical analysis

Statistical analyses were performed using GraphPad Prism 7.0 (GraphPad Software Inc., La Jolla, CA, USA). The results are presented as mean ± standard error (SE). Significant differences between groups were evaluated using an unpaired t-test where p-value < 0.05 was considered statistically significant. The analysis was performed by a statistician blinded to the experimental groups.

Results

Fig. 2 shows the expression pattern of GSK3β in the regions of the brain. The phosphorylation of GSK3β in rats that were decapitated without anesthesia (Group D) was significantly higher than in those that were decapitated after anesthesia with pentobarbital (Group A). This was observed in the prefrontal cortex, hippocampus, striatum, and nucleus accumbens of the brain.

Figure 2. Expression pattern of GSK3β in the regions of the brain. (A) Expression of phospho-GSK3β. (B) Expression of total GSK3α, GSK3β. (C) p/t GSK3β ratio in hippocampus (Group A, n = 8; Group D, n = 6). (D) p/t GSK3β ratio in prefrontal cortex (Group A, n = 8; Group D, n = 5). (E) p/t GSK3β ratio in nucleus accumbens (Group A, n = 8; Group D, n = 5). (F) p/t GSK3β ratio in striatum (Group A, n = 8; Group D, n = 5). Values are means ± SE, ***p < 0.001, **p < 0.01, *p < 0.05. AU, arbitrary unit; GSK3β, glycogen synthase kinase 3β.

The expression pattern of ERK1/ERK2 in the prefrontal cortex was also observed. The ERK1/2 activity, an upstream signaling factor regulating GSK3β phosphorylation, was significantly higher in Group D than that in in Group A, as shown in Fig. 3.

Figure 3. Expression pattern of ERK1/ERK2 in the prefrontal cortex. (A) Expression level of ERK1/ERK2. (B) p/t ERK1 ratio (Group A, n = 7; Group D, n = 5). (C) p/t ERK2 ratio (Group A, n = 7; Group D, n = 5). Values are means ± SE, ***p < 0.001. AU, arbitrary unit; ERK1/ERK2, extracellular signal-regulated kinases 1 and 2.

Discussion

GSK3β is widely expressed in all tissues, and is particularly abundant in the central nervous system (Woodgett 1990). GSK3β, unlike most protein kinases, is constitutively active in resting cells, and is rapidly and transiently inhibited in response to multiple external signals (Grimes and Jope 2001; Doble and Woodgett 2003). GSK3β activity is regulated by site-specific phosphorylation, and the complete activity of GSK3β generally requires phosphorylation at tyrosine 216 (Tyr216), whereas phosphorylation at serine 9 (Ser9) inhibits GSK3β activity (Lin et al. 1997; Shaw et al. 1998; Bijur and Jope 2000). The phosphorylation of Ser9 is the most important mechanism regulating GSK3β activity and is mediated by several kinases, including p70S6 kinase, extracellular signaling kinase (ERK), p90Rsk (MAPKAP kinase-1), protein kinase B, and cyclic AMP-dependent protein kinase C (Grimes and Jope 2001; Kaytor and Orr 2002).

GSK-3β plays a significant role in physiological and pathological conditions in many neurological and psychiatric disorders (Noori et al. 2020) such as diabetes, Alzheimer's disease, bipolar disorder, inflammatory disease, and cancer (Benedetti et al. 2004; Jope and Roh 2006; Cole 2013). Since GSK3β plays a key role in the pathogenesis of these neurodegenerative and psychiatric disorders, novel therapeutic approaches based on GSK3β inhibition are being explored. However, GSK3β activity is directly affected by the stress generated during tissue harvesting, a process integral to the experimental studies on therapeutic strategies involving GSK3β (Ko et al. 2019a). This can seriously contaminate experimental results. Additionally, appropriate assessment of GSK3β activity or its inhibition is important to study the neurochemical basis of these disorders and how drug treatment modifies or alters cellular signaling. In this study, we analyzed the effect of commonly used experimental protocols such as decapitating after anesthesia vs. decapitating without anesthesia, on the activity of GSK3β in various regions of the brain. Pentobarbital is the most commonly used barbiturate for euthanasia (Laferriere and Pang 2020). Therefore, in our study, Male SD rats were decapitated with pentobarbital or without anesthesia. We selected four brain regions, prefrontal cortex, hippocampus, striatum, and nucleus accumbens, that are closely related to neuropsychiatric disorders (Alviña et al. 2021; Saigusa et al. 2021). The hippocampus is closely associated with anxiety, memory, attention deficit, hyperactivity, and autism spectrum disorders (Bannerman et al. 2004; Onnink et al. 2014; Tottenham et al. 2014). The striatum and nucleus accumbens are strongly associated with substance use disorders, Parkinson's disease, and epilepsy (Deransart et al. 2000; Robbins and Everitt 2002; Surmeier et al. 2014). Abnormalities in the prefrontal cortex have been associated with attention-deficit/hyperactivity disorder, autism spectrum disorder, and various mood disorders (Drevets et al. 1997; Halperin and Schulz 2006; Morgan et al. 2010; Courchesne et al. 2011).

The present study demonstrated that the anesthesia/euthanasia method can affect GSK3β activity, which can seriously contaminate the results of investigative studies on the therapeutic role of GSK3β inhibition in neurodegenerative and psychiatric disorders. However, there are some limitations in the study. The regulation of the signaling pathway of GSK3β was not investigated and hence the causative factors responsible for the change in GSK3β levels were not be identified. In addition, the effects of more various anesthetics need to be investigated. Nonetheless, the results in the present study could be referenced in the experimental field by comparing and analyzing the effect of decapitation with or without anesthesia on GSK3β.

Conflict of Interest

No potential conflict of interest relevant to this article was reported.

Fig 1.

Figure 1.Overview of experimental methods.
Drug Targets and Therapeutics 2022; 1: 45-50https://doi.org/10.58502/DTT.22.008

Fig 2.

Figure 2.Expression pattern of GSK3β in the regions of the brain. (A) Expression of phospho-GSK3β. (B) Expression of total GSK3α, GSK3β. (C) p/t GSK3β ratio in hippocampus (Group A, n = 8; Group D, n = 6). (D) p/t GSK3β ratio in prefrontal cortex (Group A, n = 8; Group D, n = 5). (E) p/t GSK3β ratio in nucleus accumbens (Group A, n = 8; Group D, n = 5). (F) p/t GSK3β ratio in striatum (Group A, n = 8; Group D, n = 5). Values are means ± SE, ***p < 0.001, **p < 0.01, *p < 0.05. AU, arbitrary unit; GSK3β, glycogen synthase kinase 3β.
Drug Targets and Therapeutics 2022; 1: 45-50https://doi.org/10.58502/DTT.22.008

Fig 3.

Figure 3.Expression pattern of ERK1/ERK2 in the prefrontal cortex. (A) Expression level of ERK1/ERK2. (B) p/t ERK1 ratio (Group A, n = 7; Group D, n = 5). (C) p/t ERK2 ratio (Group A, n = 7; Group D, n = 5). Values are means ± SE, ***p < 0.001. AU, arbitrary unit; ERK1/ERK2, extracellular signal-regulated kinases 1 and 2.
Drug Targets and Therapeutics 2022; 1: 45-50https://doi.org/10.58502/DTT.22.008

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