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Sepsis-induced encephalopathy impairs descending nociceptive pathways in rats
Journal of Neuroimmunology 342 (2020) 577198
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Sepsis-induced encephalopathy impairs descending nociceptive pathways in rats
T
Rafael Alves Cazuzaa,1, Nilton Nascimento Santos-Júniorb,1, Luís Henrique Angenendt da Costab,c, Carlos Henrique Rocha Catalãob,c, Joyce Mendes-Gomesa,d, Maria José Alves da Rochab, ⁎ Christie Ramos Andrade Leite-Panissia, a
Department of Psychology, School of Philosophy, Science and Literature of Ribeirão Preto, University of São Paulo, Ribeirão Preto, 14040-901, SP, Brazil Department of Basic and Oral Biology, Ribeirão Preto Dentistry Faculty, University of São Paulo, Ribeirão Preto 14040-904, SP, Brazil c Department of Neurosciences and Behavioral Sciences, Ribeirao Preto Medical School, University of Sao Paulo, Ribeirao Preto 14049-900, SP, Brazil d UNIFADRA-FUNDEC Medical School, Dracena 17900-000, SP, Brazil b
A R T I C LE I N FO
A B S T R A C T
Keywords: SAE Neuroinflammation Brain damage Glial cells Pain perception
Sepsis-associated encephalopathy (SAE) is a significant problem in patients with sepsis, and it is associated with a decrease in cognitive and sensitivity capability induced by systemic inflammation. SAE is implicated in reversible brain damage of several regions related to cognition, emotion, and sensation; however, it is not well established if it could affect brain regions associated with nociceptive modulation. Here were evaluated the nociceptive thresholds in rats with systemic inflammation induced by cecal ligation puncture (CLP). After 24 h of CLP, it was observed an increase in nociceptive threshold in all tests. Periaqueductal gray, rostroventral medulla, critical regions for descending nociceptive modulation, were evaluated and showed enhanced pro-inflammatory cytokines as well as glial activation. These results suggest that systemic inflammation could compromise descending facilitatory pathways, impairing nociceptive sensory functioning.
1. Introduction Sepsis is defined as a potentially fatal organic dysfunction caused by a dysregulated immune response to an infection (Singer et al., 2016). Despite all clinical efforts and intense investigation, it remains a significant issue in public health, even in developed countries, concerning mortality rates and high costs (Stoller et al., 2015; Williams et al., 2004). Clinical and experimental studies have elucidated that hemodynamic and immunological alterations result from a complex interaction between the organism and the infectious agent leading to the impairment of several organs, being the brain one of the first to be affected (Remick, 2007; Sonneville et al., 2013; Stearns-Kurosawa et al., 2011). Sepsis-associated encephalopathy (SAE) has been well described in humans and experimental models (Catalão et al., 2017; Gofton and Young, 2012; Michels et al., 2015a) and represents a risk factor for mortality (Eidelman et al., 1996; Ziaja, 2013). A combination of inflammatory mediators produced both peripheral and locally contributes to brain damage. Circulating cytokines, reactive oxygen species, and
other mediators can directly reach the brain since the blood-brain barrier became dysfunctional. Additionally, these inflammatory mediators can also be synthesized by brain parenchymal cells, like astrocytes and microglia (Flierl et al., 2010; Michels et al., 2015b; Sonneville et al., 2013). This process potentiates the neuroinflammation and leads to metabolic impairment, synaptic alterations, cell death, endothelial activation, and other deleterious consequences (Mazeraud et al., 2016). Acute and long-term alterations in the central nervous system have been found after systemic inflammation, resulting in cognitive (Comim et al., 2009; Mina et al., 2014), autonomic (Pancoto et al., 2008; Pinto et al., 2017), neuroendocrine (Oliveira-Pelegrin et al., 2009; SantosJunior et al., 2017; Stare et al., 2015) and psychological (Comim et al., 2010) impairment following sepsis. However, the sensory component of brain function during sepsis is poorly investigated. Nociception is defined as the encoding and processing of noxious stimuli in the nervous system, which is projected to the brain where it is interpreted as pain. This perception has an essential protective role alerting us to potential tissue damage that can compromise bodily integrity (Bell, 2018). The noxious stimulus is carried out from the
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Corresponding author at: Department of Psychology, School of Philosophy, Science and Literature of Ribeirão Preto of the University of São Paulo, 14040-901 Ribeirão Preto, SP, Brazil. E-mail address: [email protected] (C.R.A. Leite-Panissi). 1 the authors contributed equally to the work https://doi.org/10.1016/j.jneuroim.2020.577198 Received 15 September 2019; Received in revised form 19 February 2020; Accepted 24 February 2020 0165-5728/ © 2020 Elsevier B.V. All rights reserved.
Journal of Neuroimmunology 342 (2020) 577198
R.A. Cazuza, et al.
2.3. Cecal ligation and puncture (CLP)
nociceptors through dorsal horns of the spinal cord and reaches structures of the brainstem that send both descending facilitatory and inhibitory pathways to control the stimulus intensity and program suitable responses to escape or to avoid the stimulus (De Felice and Ossipov, 2016; Zhuo, 2017). Among the essential structures that modulate the nociceptive input are the periaqueductal gray (Chu et al., 2012), nucleus raphe magnus (NRM) and the alfa part of the gigantocellular reticular nucleus (GIa), that is considered as part of the rostroventral medulla (RVM) (Burgess et al., 2002; Huang et al., 2014). Previous studies regarding pain and systemic inflammation showed that endotoxemia by lipopolysaccharide (LPS) injection could play some role in sensory pain modulation, possible due to its peripheral impairment of sensory neurons (Calil et al., 2014; Hsieh et al., 2018; Meseguer et al., 2014; Ruiz-Miyazawa et al., 2015; Sun et al., 2015). Moreover, in the sciatic nerve was found that the CLP (cecal ligation and puncture) model-induced excitability impairment may enhance mechanical antinociception in the von Frey test (Diniz et al., 2018); although the authors did not state the role of central descending pain modulation in the sepsis model. Leading the same way, Imamura et al. (2016) had also shown thermal antinociception on hot plate test in animals with systemic inflammation induced by LPS. Furthermore, they found an association with increased microglial activation and inflammatory cytokines in the cerebral cortex. However, no study has so far investigated central brain structures and their relationship with pain perception in the CLP model. Therefore, our objective in the present study was to investigate the sensory status in a clinically relevant model of sepsis (CLP) and the alterations in supraspinal structures of the brainstem that are part of descending pathways of nociceptive modulation. In particular, we evaluated whether the CLP protocol promotes alterations in the thermal nociception using the tail-flick and acetone tests, as the mechanical nociception through the von Frey test. Additionally, we analyzed the expression of proinflammatory cytokines (IL-1β, IL-6, and TNF-α), as well as the expression of the anti-apoptotic protein (Bcl-2) and the expression of the astrocytes and microglia markers (GFAP and Iba-1, respectively) in the PAG and RVM brainstem regions.
Experimental sepsis was induced by surgical cecal ligation and puncture (CLP) method. This model has been widely used in previous studies to evaluate neuroimmunoendocrine alterations during sepsis (Catalão et al., 2017; Mishra and Choudhury, 2018; Oliveira-Pelegrin et al., 2009; Oliveira-Pelegrin et al., 2014; Santos-Junior et al., 2017). Briefly, the rats were anesthetized with tribromoethanol (25 mg/0.1 kg, intraperitoneally), and after that, their abdomen was incised, followed by exposure and partial obstruction of the cecum, at the distal ileocecal valve level. Subsequently, the cecum was perforated once with a 14G needle. After checking the stool extravasation, the cecum was re-inserted into the abdominal cavity, and it was sutured. Sterile saline solution was applied subcutaneously as a resuscitation fluid (20 mL/kg). Non-manipulated animals (Naïve) were used as control (Catalão et al., 2017; da Costa et al., 2017; Santos-Junior et al., 2018; Wahab et al., 2016). Baseline measures for nociceptive tests were obtained one day before CLP and then tested again 24 h after the surgery. The Humane endpoint in shock research (Nemzek et al., 2004) was used as the criterion to euthanize rats in high suffering. 2.4. Mechanical sensitivity An electronic von Frey test analyzed the mechanical sensitivity. In this way, the mechanical stimulation threshold was evaluated with the von Frey electronic device (Insight Ltda, Ribeirão Preto, SP, Brazil), which consists of a cone-shaped plastic tip (tip area = 0.7 mm2) connected to a hand-held probe. The rat was placed in an acrylic cage (12 cm × 10 cm × 17 cm) with a wire floor for 30 min to allow adaptation to the environment. After the habituation period, an increase in upward pressure was applied with the plastic tip against the plantar hind paw, and force applied in grams (g) was recorded continuously by the main unit connected to the probe. The removal of the paw determined the withdrawal threshold. At this point, the probe movement was interrupted, and the threshold pressure automatically determined. The withdrawal threshold of each rat was calculated as the mean ± SEM based on three values obtained in each session (baseline and 24 h after CLP surgery or not – Naïve group). The mechanical threshold was then transformed into a percentage of maximal possible effect (MPE%), calculated as the percentage difference between the measured response and the baseline response, divided by the difference between the maximum response and the baseline response (Chaplan et al., 1995).
2. Material and methods 2.1. Animals Experiments were performed on Wistar adult male rats (350–400 g, n = 6–8 per group) obtained from the Animal Facility of the University of São Paulo, Ribeirão Preto, SP, Brazil. Animals were housed in a temperature-controlled room (24 ± 1 °C) on a 12-h light/dark cycle (lights on at 06:00 h) with food and water ad libitum. All procedures were carried out in compliance with the recommendations of the Conselho Nacional de Controle de Experimentação Animal - Ministério da Ciência e Tecnologia, Brazil and with the approval of the Animal Care and Use Committee of the University of São Paulo, Brazil at the Ribeirão Preto (Protocol number # 2017.1.888.58.1).
2.5. Thermal sensitivity to cold The acetone plantar instillation test was used to evaluate the effect of the sepsis on cold sensitivity. For this test, the rats were placed in acrylic boxes with a 5 mm2 floor corresponding to a mesh net made of non-malleable 1 mm wire. After the habituation period (10 min), a 100 μl acetone jet was instilled in the left or right hind paw of the animal with an insulin syringe at a distance of approximately 5 mm, through the mesh of the observation box. After applying the stimulus, the behavior was evaluated for 40 s. This test was performed before (baseline) and 24 h after the CLP surgery or not (Naïve group). The behavioral responses were evaluated according to the classification described by (Flatters and Bennett, 2004). Briefly, this method used a scale of 4 points thus presented: score “0” for the absence of any painful behavior; “1” score for a rapid withdrawal of the paw and/or with tremor or beat of the paw on the ground (the characteristic tremor, or flicking, was critical in determining whether paw withdrawal was due to pain or fear of the animal); score “2” for a prolonged withdrawal of the paw with or without repeated flicking of it, and score “3” for the situation where the animal licks the ventral face of the paw after repeated flicking. The analysis was made by the gross scores added to the number of times the animal has emitted such behaviors.
2.2. Experimental protocol Animals submitted to CLP surgery were left to recover for 24 hs. Following the behavioral tests (nociceptive and locomotor tests), they were divided into two groups. In the first one, the animals were decapitated for blood and brain tissue collection for analyses of plasma cytokines by ELISA and astroglial (GFAP), microglia (Iba-1) activation, and anti-apoptotic marker (Bcl-2) by Western Blot technique. In the second group, the animals were anesthetized, perfused, and the brains were collected, fixed, and frozen for immunohistochemistry assay for GFAP and Iba-1 analysis in the PAG and RVM areas. Naïve animals (without any surgery) were used as a control in each group. 2
Journal of Neuroimmunology 342 (2020) 577198
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2.6. Thermal sensitivity to heat
(Synergy™ H1, Biotek Instruments, Inc.).
The tail-flick test evaluated the thermal hyperalgesia to heat before (baseline) and 24 h after the CLP surgery or not (Naïve group). In this test, the rats were wrapped gently on a soft cotton cloth and held so that the tail extends through a chromed steel spiral at room temperature (23 ± 1 °C). The experimenter manipulated the apparatus, and the latency of tail withdrawal occurs between 2.5 and 3.5 s after the elevation of the spiral temperature by the passage of electric current. The heat was applied to the portion of the ventral surface of the tail between 4 and 6 cm from the tip. If tail removal did not occur, a cut-off time of 6 s was used to eliminate the possibility of skin damage. Latencies were measured at 5-min intervals until a stable baseline is achieved over three consecutive trials. At 24 h after CLP or not (Naïve group), the latencies were measured in 5-min intervals until the maximum time of 30 min. Thermal hyperalgesia is expressed as Analgesic Index (AI) calculated using the following formula: AI = 100 × [(latency of tail withdrawal − baseline value)/(6 − baseline value)] and the resulting in an increase or decrease in thermal hyperalgesia is plotted on a negative or positive axis, respectively.
2.10. Immunohistochemistry for GFAP and Iba-1 analyses Briefly, after the behavioral procedures, the animals were deeply anesthetized with 4% xylazine (30 mg/kg) and 10% ketamine (225 mg/ kg) and submitted to transcardial perfusion with 0.01 phosphate-buffered-saline (PBS) followed by 4% paraformaldehyde (4% PFA). The brains were removed and post-fixed in 4% PFA during 4 h, followed by a 30% sucrose solution until tissue saturation. Fixed brains were then frozen in isopentane at −20 °C and cut in coronal sections of the brainstem through RVM and PAG (bregma −12.00 mm to 10.32 mm and − 8.40 mm to −6.00 mm, respectively) in a Leica CM1850 cryostat (Leica Microsystems Nussloch GmbH, Nussloch, Germany). Freefloating sections (40 μm) were incubated in normal 10% goat serum in 0.01 M PBS + 0.1% Triton Triton +0.04% NaN3 for 1 h. After being washed 3 times with 0.01 M PBS for 5 min, the tissues were incubated overnight at 4 °C with primary antibodies - GFAP (Sigma-Aldrich, 1:1000) or Iba-1 (Wacko, 1:1000) in 0.01 M PBS + 0.1% Triton +0.04% NaN3. After new rinses, they were incubated with secondary antibody (goat anti-rabbit IgG H&L AlexaFluor 488, Abcam, 1:750) for 3 h. The sections were mounted on gelatin-coated slides, covered with antifading media (Vectashield), and stored in the refrigerator before analysis. The anatomical description of brain regions was identified and delimited according to the rat brain atlas of Paxinos and Watson (Paxinos and Watson, 2007).
2.7. Locomotor activity After completion of behavioral experiments, each experimental group was evaluated in an open field test to assess motor behavior for 5 min. The apparatus consists of a circular arena with raised acrylic walls measuring 50 cm (height) × 60 cm (diameter), divided into 12 quadrants, four central and eight lateral. The rats were placed individually in the center of the arena, and locomotor activity was recorded during a 5 min period for posterior analysis in TotalMedia 5.0 software. The number of total crosses of the animal and rearing movements was quantified.
2.11. Statistical analysis For von Frey and open field tests, analyses were performed by an unpaired t-test. Two-way ANOVA (with time and group as factors) was used for tail-flick and acetone tests followed by the Tukey test. Western Blot and ELISA were statistically analyzed by an unpaired Student's ttest or Kolmogorov-Smirnov test. Data were considered statistically significant when P < .05. Results are shown as mean ± S.E.M.
2.8. Western blot for GFAP, Iba-1 and Bcl-2 quantification The periaqueductal gray (PAG) and rostral ventromedial medulla (RVM) were dissected from brain samples and immersed in RIPA buffer containing a 10% protease inhibitor cocktail and 0.5% of phenylmethylsulfonyl fluoride (PMSF). Following homogenization and centrifugation, the supernatant was collected. Proteins (30 μg/sample) were separated electrophoretically (125 V, 90 min) in 12% SDS-polyacrylamide gels. After electrophoresis, proteins were blotted to a nitrocellulose membrane (0.45 μm pore size; Millipore) in a tank blotting system (125 V, 90 min). The membranes were blocked (5% BSA in PBS, with 0.2% Tween 20) for 1 h and then incubated overnight at 4 °C with specific primary antibodies for B-cell lymphoma 2 (Bcl-2) (Santa Cruz, 1:1000), Glial Fibrillary Acidic Protein (GFAP) (Sigma-Aldrich, 1:10000) and ionized calcium-binding adapter molecule 1 (Iba-1) (Wacko, 1:1000), and then, incubated for 2 h at 4 °C with secondary HRP-conjugated antibodies (Abcam, 1:10000 dilution). A chemiluminescence reaction kit (GE Healthcare) was used for detection, and immunolabeled protein bands were visualized in a ChemiDoc MP System (BioRad) and analyzed by ImageLab 5.2.1 software. A β-actin-specific antibody was used for the normalization of the samples.
3. Results 3.1. Mechanical and thermal sensitivity CLP-induced sepsis was able to produce an alteration in mechanical and thermal sensitivity (Figs. 1A - C). Mechanical sensitivity measured by the electronic von Frey apparatus showed that CLP animals presented an increase in the percentage of maximal possible effect (%MPE) when compared with the control group [T(14) = 2.623; P = .02, unpaired Student's-t-test, Fig. 1A]. In the tail-flick test, the statistical analyses (Two-way ANOVA) revealed a difference in time [F(1, 7) = 61.92; P = .0001], group [F(1, 7) = 99.45; P < .0001] and interaction time and group [F(1, 7) = 99.45; P < .000]. Tukey's post-test showed an increase in the analgesic index 24 h after the CLP induction when compared to respective baseline, and to the Naïve group in the same period (P < .05, Fig. 1B); however, in the control group (Naïve) any difference in the analgesic index was observed. The same was verified when scores of cold sensitivity in the acetone test were analyzed. Two-way ANOVA evidenced a time-group interaction [F(1, 7) = 13.44; P = .008], and Tukey post-test showed a significant decrease at the score in the CLP group when compared 24 h after the surgery to its respective baseline (P < .05, Fig. 1C).
2.9. ELISA for cytokines quantification The collected blood was centrifugated at 4 °C, 3000 rpm for 10 min, and sonicated tissue extracted at 4 °C, 13000 rpm for 20 min. The supernatant of processed tissues and plasma was used for cytokine quantification. The IL1-β, IL-6, and TNF-α cytokines levels were determined using appropriate ELISA kits (R&D Systems, Minneapolis, Minn., USA) according to the manufacturer's instructions. The detection limits for TNF-α, IL1-β, and IL-6 were 10 pg/mL. The total protein concentration in the tissue was measured using the BCA protein assay kit (Pierce). The samples were analyzed in a microplate reader
3.2. Locomotor activity Compared to Naïve group, animals submitted to CLP surgery did not show compromising locomotor behavior, measured by a total of crossings between squares in open field [T(14) = 1.036; P = .31, unpaired Student's-t-test] and total rearing movements [T(14) = 0.0314; P = .97, unpaired Student's-t-test, Fig. 1D). 3
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Fig. 1. Sensitivity and locomotor evaluation in rats 24 h after the surgery to induce sepsis (CLP) or not (control group, Naïve). A: Mechanical sensitivity evaluated by the percentage of maximal possible effect (MPE %); B: Thermal sensitivity to heat evaluated in the tail-flick test; C: Thermal sensitivity to cold evaluated in the acetone test; D: Locomotor activity during the 5 min-exposure to the open field test, evaluated by the horizontal (number of squares crossing) and vertical (number of rearing movement) activity. a P < .05, unpaired Student's-ttest when compared to Naïve group. b P < .05 Tukey test when compared CLP group 24 h after surgery with respective baseline. c P < .05 Tukey test when compared with Naïve group at the same time. The data represent means ± SEM. N = 6–8 per group.
compared to its respective Naïve group. Besides, in the RVM tissue were observed an increase in the IL 1-β [T(7) = 2.513; P = .0402; IL-6 [T(7) = 3.18; P = .0191] and TNF-α [T(6) = 2.454; P = .0495] compared to Naïve group (Fig. 4).
3.3. Astrocytes/microglia activation, and Bcl-2 expression A non-parametric statistical analyses (Kolmogorov-Smirnov test) showed an increased in the GFAP expression in PAG (P = .0095, Fig. 2A) and RVM (P = .0159, Fig. 2B) and also for Iba-1 in PAG (P = .0286, Fig. 2C) when compared to the respective Naïve group. A reduction in anti-apoptotic protein Bcl-2 expression in PAG (P = .0159, Fig. 2E) and RVM (P = .0159, Fig. 2F) was also seen, when compared to Naïve group. Representative photomicrography showing astrocytes and microglial expression in PAG and RVM of Naïve and CLP animals can be seen in Fig. 3.
4. Discussion Our results showed that sepsis induces an increase in overall nociceptive threshold in three different nociceptive tests, presenting cold, heat, and mechanical antinociception without alterations in general locomotor activity. It was accompanied by an increase in glial activation and production of proinflammatory mediators in RVM and PAG, two nuclei involved in descending control of pain. Previous studies regarding pain and an immune challenge have different results concerning nociception alterations (de Goeij et al., 2013; Diniz et al., 2018; Reeve et al., 2000; Seo et al., 2008). We believe that these diversified data result from the distinct models used (CLP, LPS injection, or cytokine administration), the dose of LPS, the route of administration (intraperitoneally, intravenous, intracerebroventricular) and the time points analyzed (minutes, hours or days after the
3.4. Cytokines levels The plasma citokynes levels analyses (unpaired Student's-t-test) showed that in the CLP group there was a significant increase in IL 1-β pro-inflammatory citokyne [T(9) = 6.593; P = .0001) when compared with Naïve group. In the PAG tissues, the statistical analyses evidenced a difference in IL 1-β [T(7) = 3.005; P = .0198], but not in IL-6 [T(7) = 1.796; P = .1156] or in TNF-α [T(8) = 0.0258; P = .980] 4
Journal of Neuroimmunology 342 (2020) 577198
R.A. Cazuza, et al.
Fig. 2. Expression of the GFAP, Iba-1, and Bcl-2 proteins in the periaqueductal gray (PAG) and rostroventral medulla (RVM). Relative quantification of the expression of the GFAP (A, B), Iba-1 (C, D), and Bcl-2 (E, F) proteins by western blotting assay 24 h after sepsis induction (CLP) or not (Naïve group). * P < .05, KolmogorovSmirnov test when compared to Naïve group. The data represent means ± SEM. N = 6–8 per group.
In the midbrain, PAG receives inputs from higher brain nuclei like the cerebral cortex and amygdala and has a major opioid-mediated inhibitory control of descending nociceptive pathways (Loyd and Murphy, 2009; Staud, 2013). RVM has bidirectional connections with PAG and other brain centers and is considered a final relay of descending inhibitory pathways (Ossipov et al., 2014; Staud, 2013). We believe that in septic animals, the overproduction of inflammatory agents could disturb the physiological functions of these nuclei. The increased Iba-1 protein expression and immunoreactivity indicates microglial activation, which is a potential source of cytokines. When activated, these cells may assume an M1 or proinflammatory phenotype
challenge). In the present study, we used the cecal ligation and puncture, which is considered a golden standard and clinically relevant model of sepsis, since it mimics in a better manner the human sepsis, with polymicrobial infection, presence of infectious focus, hyper- and hypoinflammatory phase and similar hemodynamic progression (Dejager et al., 2011; Ruiz et al., 2016). The impairment herein observed in discriminative sensory pathways indicates a potential consequence of SAE. In fact, in patients, the reduction of cortical and subcortical low latency sensory evoked potential, mainly in the sensory cortex and brainstem nuclei, indicates generalized dysfunction of the clinical condition (Zauner et al., 2002). 5
Journal of Neuroimmunology 342 (2020) 577198
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Fig. 3. Representative photomicrographs were taken in the regions of the lateral periaqueductal gray (lPAG) and rostroventral medulla (RVM) showing GFAP (green fluorescence) and Iba-1 (green fluorescence) immunostaining in 40 μm coronal sections of Naïve rats and rats 24 h after CLP surgery. Plates adapted from Paxinos and Watson (2007). Photomicrographs at 10× (smaller box) and 20×. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
changes in other brain structures, which are intimately involved with nociceptive modulation (Kato et al., 2018; Llorca-Torralba et al., 2018). In this way, there is intense microglial activation and neuronal apoptosis in the amygdala and locus coeruleus (Carlson et al., 2007; Sharshar et al., 2003), as well as morphological anomalies in the sensorimotor cortex (Polyanin and Bardakhch'yan, 1984) during sepsis. Thus, these changes can also contribute to the reduction of nociception that is observed in the present study. Even though peripheral mechanisms were not the focus of our investigation, we cannot exclude that it may be involved in the reduced nociception in septic animals. A recent study has also shown a reduction in mechanical sensitivity in CLP animals on early sepsis (24 h after surgery) accompanied by a reduction in sciatic nerve excitability (Diniz et al., 2018). In our study, the reduced nociception was observed both in paw (mechanical) and in the tail (thermal), suggesting an involvement of other possible mechanisms beyond the sciatic nerve in sepsisinduced antinociception. The endogenous opioids system also has potential effects in regulating nociception during sepsis since its circulating levels are increased in septic patients (Glattard et al., 2010). Yirmiya et al. (Yirmiya et al., 1994) have demonstrated that the reduced pain sensitivity observed four hours after LPS injection was reversed by the administration of naltrexone, an opiate antagonist. In conclusion, our results indicate that CLP-induced sepsis leads to a reduction in nociception, and it is can be associated, at least in part, with glial cell activation and an inflammatory microenvironment in brain areas related to pain regulation. We believe that the investigation of cellular mechanisms and neurotransmission is essential to a better understanding of the sensory alterations in septic pathophysiology.
and secrete inflammatory cytokines, like IL-1β, IL-6, TNF-α, and IFN-γ. (Eggen et al., 2013; Morrison and Filosa, 2013). Astrocyte activation also leads to cytokine expression, and more importantly, it alters the regulation of local neuro- and glia transmitters (Gorina et al., 2011; Pascual et al., 2012). Also, even though TNF-α may induce an enhanced the expression of BCL-2, an essential mitochondrial protein that serves as an inflammatory and apoptotic inhibitor (Kim, 2005), it seems to be downregulated in severe sepsis patients (Bilbault et al., 2004), animals with SAE in CLP (Deng et al., 2017) and LPS models of sepsis (Ning et al., 2017) what means a possible vulnerability to cell death in brain tissue. Moreover, it influences the synthesis, uptake, and release of several neurotransmitters, like GABA and glutamate, in both excitatory and inhibitory synapses in different brain regions (Camacho-Arroyo et al., 2009; Miller et al., 2013). Although we did not investigate the precise modulation of activation of neurotransmitters in each of these areas, we believe that systemic inflammation profoundly alters the local synaptic circuitry resulting in reduced nociception. Sepsis and microglia-derived cytokines could deeply deregulate the brain content of GABA (Serantes et al., 2006), acetylcholine (Pavlov et al., 2006) and amines (serotonin, dopamine, norepinephrine) (Dal-Pizzol et al., 2014) levels in the brain. Besides, astrocyte plays a vital role in glutamatergic regulation in the brain (Araque et al., 1999; Malarkey and Parpura, 2008). The presence of TNF-α and IL-β induces the release of this neurotransmitter in both human and rat cortex and hippocampal primary neurons, as well as in astrocyte cultures (Sama et al., 2008; Ye et al., 2013). We hypothesize that in septic animals there is an excessive cytokine-mediate glutamate release in PAG and RVM that could result in the reduced nociception since it has been already shown that glutamate in these areas exerts an antinociceptive effect in pain modulation (Morgan et al., 2009; Samineni et al., 2017). All these transmitters play an important role in nociception control in the PAG-RVM system, remarkably by regulating “on” and “off” cells in the RVM being “off” cells, a subpopulation that when tonically activated inhibit nociception, while “on” cells facilitate pain behavior. We presume that during sepsis, the inflammatory milieu in the brain favors the inhibitory component of RVM. Another possibility is that SAE can promote morphofunctional
Authors contributions R.A.C., N.N.S.-J., L.H.A.C., C.H.R.C, and J.M.G. performed the experiments and analyzed the data. R.A.C., N.N.S.-J., M.J.A.R., and C.R.A.L.P designed the study and wrote the manuscript. All authors read and approved the final manuscript.
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Journal of Neuroimmunology 342 (2020) 577198
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Fig. 4. Up-regulation of inflammatory mediators (IL-1 β, IL-6, and TNF-α) in the plasma, periaqueductal gray (PAG), and rostroventral medulla (RVM) at 24 h after sepsis induction (CLP) or not (Naïve group). * P < .05 unpaired Student's-t-test when compared to Naïve. The data represent means ± SEM. N = 6–8 per group.
Compliance with ethical standards
unacknowledged partner. Trends Neurosci. 22, 208–215. Bell, A., 2018. The neurobiology of acute pain. Vet. J. 237, 55–62. Bilbault, P., Lavaux, T., Lahlou, A., Uring-Lambert, B., Gaub, M.P., Ratomponirina, C., Meyer, N., Oudet, P., Schneider, F., 2004. Transient Bcl-2 gene down-expression in circulating mononuclear cells of severe sepsis patients who died despite appropriate intensive care. Intensive Care Med. 30, 408–415. Burgess, S.E., Gardell, L.R., Ossipov, M.H., Malan Jr., T.P., Vanderah, T.W., Lai, J., Porreca, F., 2002. Time-dependent descending facilitation from the rostral ventromedial medulla maintains, but does not initiate, neuropathic pain. J. Neurosci. 22, 5129–5136. Calil, I.L., Zarpelon, A.C., Guerrero, A.T., Alves-Filho, J.C., Ferreira, S.H., Cunha, F.Q., Cunha, T.M., Verri Jr., W.A., 2014. Lipopolysaccharide induces inflammatory hyperalgesia triggering a TLR4/MyD88-dependent cytokine cascade in the mice paw. PLoS One 9, e90013. Camacho-Arroyo, I., Lopez-Griego, L., Morales-Montor, J., 2009. The role of cytokines in the regulation of neurotransmission. Neuroimmunomodulation 16, 1–12. Carlson, D.E., Chiu, W.C., Fiedler, S.M., Hoffman, G.E., 2007. Central neural distribution of immunoreactive Fos and CRH in relation to plasma ACTH and corticosterone during sepsis in the rat. Exp. Neurol. 205, 485–500. Catalão, C.H.R., Santos-Júnior, N.N., da Costa, L.H.A., Souza, A.O., Alberici, L.C., Rocha, M.J.A., 2017. Brain oxidative stress during experimental sepsis is attenuated by simvastatin administration. Mol. Neurobiol. 54, 7008–7018. Chaplan, S.R., Bach, F.W., Shafer, S.L., Yaksh, T.L., 1995. Prolonged alleviation of tactile allodynia by intravenous lidocaine in neuropathic rats. Anesthesiology 83, 775–785. Chu, H., Sun, J., Xu, H., Niu, Z., Xu, M., 2012. Effect of periaqueductal gray melanocortin 4 receptor in pain facilitation and glial activation in rat model of chronic constriction injury. Neurol. Res. 34, 871–888. Comim, C.M., Constantino, L.C., Barichello, T., Streck, E.L., Quevedo, J., Dal-Pizzol, F., 2009. Cognitive impairment in the septic brain. Curr. Neurovasc. Res. 6, 194–203. Comim, C.M., Cassol-Jr, O.J., Constantino, L.C., Petronilho, F., Constantino, L.S., Stertz, L., Kapczinski, F., Barichello, T., Quevedo, J., Dal-Pizzol, F., 2010. Depressive-like parameters in sepsis survivor rats. Neurotox. Res. 17, 279–286. da Costa, L.H.A., Júnior, N.N.D.S., Catalão, C.H.R., Sharshar, T., Chrétien, F., da Rocha,
All animal experiments in this study were carried out according to a protocol approved by the Animal Care and Use Committee of the University of São Paulo, Brazil at the Ribeirão Preto (protocol number # 2017.1.888.58.1). Declaration of Competing Interest The authors declare that no conflict of interest could be perceived as prejudicing the impartiality of the reported research. Acknowledgments The authors thank Nadir M. Fernandes, Bruna Barissa and Patrícia Adriana Basile for technical assistance. Financial support from Fundação de Amparo à Pesquisa do Estado de São Paulo (grant number # 2017/11213-6), Conselho Nacional de Pesquisa – Brazil (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brazil (Capes Finance Code 001). C.R.A.L.P. was granted a research fellowship from CNPq (grant number # 304214/2016-3). References Araque, A., Parpura, V., Sanzgiri, R.P., Haydon, P.G., 1999. Tripartite synapses: glia, the
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Sepsis-induced encephalopathy impairs descending nociceptive pathways in rats
T
Rafael Alves Cazuzaa,1, Nilton Nascimento Santos-Júniorb,1, Luís Henrique Angenendt da Costab,c, Carlos Henrique Rocha Catalãob,c, Joyce Mendes-Gomesa,d, Maria José Alves da Rochab, ⁎ Christie Ramos Andrade Leite-Panissia, a
Department of Psychology, School of Philosophy, Science and Literature of Ribeirão Preto, University of São Paulo, Ribeirão Preto, 14040-901, SP, Brazil Department of Basic and Oral Biology, Ribeirão Preto Dentistry Faculty, University of São Paulo, Ribeirão Preto 14040-904, SP, Brazil c Department of Neurosciences and Behavioral Sciences, Ribeirao Preto Medical School, University of Sao Paulo, Ribeirao Preto 14049-900, SP, Brazil d UNIFADRA-FUNDEC Medical School, Dracena 17900-000, SP, Brazil b
A R T I C LE I N FO
A B S T R A C T
Keywords: SAE Neuroinflammation Brain damage Glial cells Pain perception
Sepsis-associated encephalopathy (SAE) is a significant problem in patients with sepsis, and it is associated with a decrease in cognitive and sensitivity capability induced by systemic inflammation. SAE is implicated in reversible brain damage of several regions related to cognition, emotion, and sensation; however, it is not well established if it could affect brain regions associated with nociceptive modulation. Here were evaluated the nociceptive thresholds in rats with systemic inflammation induced by cecal ligation puncture (CLP). After 24 h of CLP, it was observed an increase in nociceptive threshold in all tests. Periaqueductal gray, rostroventral medulla, critical regions for descending nociceptive modulation, were evaluated and showed enhanced pro-inflammatory cytokines as well as glial activation. These results suggest that systemic inflammation could compromise descending facilitatory pathways, impairing nociceptive sensory functioning.
1. Introduction Sepsis is defined as a potentially fatal organic dysfunction caused by a dysregulated immune response to an infection (Singer et al., 2016). Despite all clinical efforts and intense investigation, it remains a significant issue in public health, even in developed countries, concerning mortality rates and high costs (Stoller et al., 2015; Williams et al., 2004). Clinical and experimental studies have elucidated that hemodynamic and immunological alterations result from a complex interaction between the organism and the infectious agent leading to the impairment of several organs, being the brain one of the first to be affected (Remick, 2007; Sonneville et al., 2013; Stearns-Kurosawa et al., 2011). Sepsis-associated encephalopathy (SAE) has been well described in humans and experimental models (Catalão et al., 2017; Gofton and Young, 2012; Michels et al., 2015a) and represents a risk factor for mortality (Eidelman et al., 1996; Ziaja, 2013). A combination of inflammatory mediators produced both peripheral and locally contributes to brain damage. Circulating cytokines, reactive oxygen species, and
other mediators can directly reach the brain since the blood-brain barrier became dysfunctional. Additionally, these inflammatory mediators can also be synthesized by brain parenchymal cells, like astrocytes and microglia (Flierl et al., 2010; Michels et al., 2015b; Sonneville et al., 2013). This process potentiates the neuroinflammation and leads to metabolic impairment, synaptic alterations, cell death, endothelial activation, and other deleterious consequences (Mazeraud et al., 2016). Acute and long-term alterations in the central nervous system have been found after systemic inflammation, resulting in cognitive (Comim et al., 2009; Mina et al., 2014), autonomic (Pancoto et al., 2008; Pinto et al., 2017), neuroendocrine (Oliveira-Pelegrin et al., 2009; SantosJunior et al., 2017; Stare et al., 2015) and psychological (Comim et al., 2010) impairment following sepsis. However, the sensory component of brain function during sepsis is poorly investigated. Nociception is defined as the encoding and processing of noxious stimuli in the nervous system, which is projected to the brain where it is interpreted as pain. This perception has an essential protective role alerting us to potential tissue damage that can compromise bodily integrity (Bell, 2018). The noxious stimulus is carried out from the
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Corresponding author at: Department of Psychology, School of Philosophy, Science and Literature of Ribeirão Preto of the University of São Paulo, 14040-901 Ribeirão Preto, SP, Brazil. E-mail address: [email protected] (C.R.A. Leite-Panissi). 1 the authors contributed equally to the work https://doi.org/10.1016/j.jneuroim.2020.577198 Received 15 September 2019; Received in revised form 19 February 2020; Accepted 24 February 2020 0165-5728/ © 2020 Elsevier B.V. All rights reserved.
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2.3. Cecal ligation and puncture (CLP)
nociceptors through dorsal horns of the spinal cord and reaches structures of the brainstem that send both descending facilitatory and inhibitory pathways to control the stimulus intensity and program suitable responses to escape or to avoid the stimulus (De Felice and Ossipov, 2016; Zhuo, 2017). Among the essential structures that modulate the nociceptive input are the periaqueductal gray (Chu et al., 2012), nucleus raphe magnus (NRM) and the alfa part of the gigantocellular reticular nucleus (GIa), that is considered as part of the rostroventral medulla (RVM) (Burgess et al., 2002; Huang et al., 2014). Previous studies regarding pain and systemic inflammation showed that endotoxemia by lipopolysaccharide (LPS) injection could play some role in sensory pain modulation, possible due to its peripheral impairment of sensory neurons (Calil et al., 2014; Hsieh et al., 2018; Meseguer et al., 2014; Ruiz-Miyazawa et al., 2015; Sun et al., 2015). Moreover, in the sciatic nerve was found that the CLP (cecal ligation and puncture) model-induced excitability impairment may enhance mechanical antinociception in the von Frey test (Diniz et al., 2018); although the authors did not state the role of central descending pain modulation in the sepsis model. Leading the same way, Imamura et al. (2016) had also shown thermal antinociception on hot plate test in animals with systemic inflammation induced by LPS. Furthermore, they found an association with increased microglial activation and inflammatory cytokines in the cerebral cortex. However, no study has so far investigated central brain structures and their relationship with pain perception in the CLP model. Therefore, our objective in the present study was to investigate the sensory status in a clinically relevant model of sepsis (CLP) and the alterations in supraspinal structures of the brainstem that are part of descending pathways of nociceptive modulation. In particular, we evaluated whether the CLP protocol promotes alterations in the thermal nociception using the tail-flick and acetone tests, as the mechanical nociception through the von Frey test. Additionally, we analyzed the expression of proinflammatory cytokines (IL-1β, IL-6, and TNF-α), as well as the expression of the anti-apoptotic protein (Bcl-2) and the expression of the astrocytes and microglia markers (GFAP and Iba-1, respectively) in the PAG and RVM brainstem regions.
Experimental sepsis was induced by surgical cecal ligation and puncture (CLP) method. This model has been widely used in previous studies to evaluate neuroimmunoendocrine alterations during sepsis (Catalão et al., 2017; Mishra and Choudhury, 2018; Oliveira-Pelegrin et al., 2009; Oliveira-Pelegrin et al., 2014; Santos-Junior et al., 2017). Briefly, the rats were anesthetized with tribromoethanol (25 mg/0.1 kg, intraperitoneally), and after that, their abdomen was incised, followed by exposure and partial obstruction of the cecum, at the distal ileocecal valve level. Subsequently, the cecum was perforated once with a 14G needle. After checking the stool extravasation, the cecum was re-inserted into the abdominal cavity, and it was sutured. Sterile saline solution was applied subcutaneously as a resuscitation fluid (20 mL/kg). Non-manipulated animals (Naïve) were used as control (Catalão et al., 2017; da Costa et al., 2017; Santos-Junior et al., 2018; Wahab et al., 2016). Baseline measures for nociceptive tests were obtained one day before CLP and then tested again 24 h after the surgery. The Humane endpoint in shock research (Nemzek et al., 2004) was used as the criterion to euthanize rats in high suffering. 2.4. Mechanical sensitivity An electronic von Frey test analyzed the mechanical sensitivity. In this way, the mechanical stimulation threshold was evaluated with the von Frey electronic device (Insight Ltda, Ribeirão Preto, SP, Brazil), which consists of a cone-shaped plastic tip (tip area = 0.7 mm2) connected to a hand-held probe. The rat was placed in an acrylic cage (12 cm × 10 cm × 17 cm) with a wire floor for 30 min to allow adaptation to the environment. After the habituation period, an increase in upward pressure was applied with the plastic tip against the plantar hind paw, and force applied in grams (g) was recorded continuously by the main unit connected to the probe. The removal of the paw determined the withdrawal threshold. At this point, the probe movement was interrupted, and the threshold pressure automatically determined. The withdrawal threshold of each rat was calculated as the mean ± SEM based on three values obtained in each session (baseline and 24 h after CLP surgery or not – Naïve group). The mechanical threshold was then transformed into a percentage of maximal possible effect (MPE%), calculated as the percentage difference between the measured response and the baseline response, divided by the difference between the maximum response and the baseline response (Chaplan et al., 1995).
2. Material and methods 2.1. Animals Experiments were performed on Wistar adult male rats (350–400 g, n = 6–8 per group) obtained from the Animal Facility of the University of São Paulo, Ribeirão Preto, SP, Brazil. Animals were housed in a temperature-controlled room (24 ± 1 °C) on a 12-h light/dark cycle (lights on at 06:00 h) with food and water ad libitum. All procedures were carried out in compliance with the recommendations of the Conselho Nacional de Controle de Experimentação Animal - Ministério da Ciência e Tecnologia, Brazil and with the approval of the Animal Care and Use Committee of the University of São Paulo, Brazil at the Ribeirão Preto (Protocol number # 2017.1.888.58.1).
2.5. Thermal sensitivity to cold The acetone plantar instillation test was used to evaluate the effect of the sepsis on cold sensitivity. For this test, the rats were placed in acrylic boxes with a 5 mm2 floor corresponding to a mesh net made of non-malleable 1 mm wire. After the habituation period (10 min), a 100 μl acetone jet was instilled in the left or right hind paw of the animal with an insulin syringe at a distance of approximately 5 mm, through the mesh of the observation box. After applying the stimulus, the behavior was evaluated for 40 s. This test was performed before (baseline) and 24 h after the CLP surgery or not (Naïve group). The behavioral responses were evaluated according to the classification described by (Flatters and Bennett, 2004). Briefly, this method used a scale of 4 points thus presented: score “0” for the absence of any painful behavior; “1” score for a rapid withdrawal of the paw and/or with tremor or beat of the paw on the ground (the characteristic tremor, or flicking, was critical in determining whether paw withdrawal was due to pain or fear of the animal); score “2” for a prolonged withdrawal of the paw with or without repeated flicking of it, and score “3” for the situation where the animal licks the ventral face of the paw after repeated flicking. The analysis was made by the gross scores added to the number of times the animal has emitted such behaviors.
2.2. Experimental protocol Animals submitted to CLP surgery were left to recover for 24 hs. Following the behavioral tests (nociceptive and locomotor tests), they were divided into two groups. In the first one, the animals were decapitated for blood and brain tissue collection for analyses of plasma cytokines by ELISA and astroglial (GFAP), microglia (Iba-1) activation, and anti-apoptotic marker (Bcl-2) by Western Blot technique. In the second group, the animals were anesthetized, perfused, and the brains were collected, fixed, and frozen for immunohistochemistry assay for GFAP and Iba-1 analysis in the PAG and RVM areas. Naïve animals (without any surgery) were used as a control in each group. 2
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2.6. Thermal sensitivity to heat
(Synergy™ H1, Biotek Instruments, Inc.).
The tail-flick test evaluated the thermal hyperalgesia to heat before (baseline) and 24 h after the CLP surgery or not (Naïve group). In this test, the rats were wrapped gently on a soft cotton cloth and held so that the tail extends through a chromed steel spiral at room temperature (23 ± 1 °C). The experimenter manipulated the apparatus, and the latency of tail withdrawal occurs between 2.5 and 3.5 s after the elevation of the spiral temperature by the passage of electric current. The heat was applied to the portion of the ventral surface of the tail between 4 and 6 cm from the tip. If tail removal did not occur, a cut-off time of 6 s was used to eliminate the possibility of skin damage. Latencies were measured at 5-min intervals until a stable baseline is achieved over three consecutive trials. At 24 h after CLP or not (Naïve group), the latencies were measured in 5-min intervals until the maximum time of 30 min. Thermal hyperalgesia is expressed as Analgesic Index (AI) calculated using the following formula: AI = 100 × [(latency of tail withdrawal − baseline value)/(6 − baseline value)] and the resulting in an increase or decrease in thermal hyperalgesia is plotted on a negative or positive axis, respectively.
2.10. Immunohistochemistry for GFAP and Iba-1 analyses Briefly, after the behavioral procedures, the animals were deeply anesthetized with 4% xylazine (30 mg/kg) and 10% ketamine (225 mg/ kg) and submitted to transcardial perfusion with 0.01 phosphate-buffered-saline (PBS) followed by 4% paraformaldehyde (4% PFA). The brains were removed and post-fixed in 4% PFA during 4 h, followed by a 30% sucrose solution until tissue saturation. Fixed brains were then frozen in isopentane at −20 °C and cut in coronal sections of the brainstem through RVM and PAG (bregma −12.00 mm to 10.32 mm and − 8.40 mm to −6.00 mm, respectively) in a Leica CM1850 cryostat (Leica Microsystems Nussloch GmbH, Nussloch, Germany). Freefloating sections (40 μm) were incubated in normal 10% goat serum in 0.01 M PBS + 0.1% Triton Triton +0.04% NaN3 for 1 h. After being washed 3 times with 0.01 M PBS for 5 min, the tissues were incubated overnight at 4 °C with primary antibodies - GFAP (Sigma-Aldrich, 1:1000) or Iba-1 (Wacko, 1:1000) in 0.01 M PBS + 0.1% Triton +0.04% NaN3. After new rinses, they were incubated with secondary antibody (goat anti-rabbit IgG H&L AlexaFluor 488, Abcam, 1:750) for 3 h. The sections were mounted on gelatin-coated slides, covered with antifading media (Vectashield), and stored in the refrigerator before analysis. The anatomical description of brain regions was identified and delimited according to the rat brain atlas of Paxinos and Watson (Paxinos and Watson, 2007).
2.7. Locomotor activity After completion of behavioral experiments, each experimental group was evaluated in an open field test to assess motor behavior for 5 min. The apparatus consists of a circular arena with raised acrylic walls measuring 50 cm (height) × 60 cm (diameter), divided into 12 quadrants, four central and eight lateral. The rats were placed individually in the center of the arena, and locomotor activity was recorded during a 5 min period for posterior analysis in TotalMedia 5.0 software. The number of total crosses of the animal and rearing movements was quantified.
2.11. Statistical analysis For von Frey and open field tests, analyses were performed by an unpaired t-test. Two-way ANOVA (with time and group as factors) was used for tail-flick and acetone tests followed by the Tukey test. Western Blot and ELISA were statistically analyzed by an unpaired Student's ttest or Kolmogorov-Smirnov test. Data were considered statistically significant when P < .05. Results are shown as mean ± S.E.M.
2.8. Western blot for GFAP, Iba-1 and Bcl-2 quantification The periaqueductal gray (PAG) and rostral ventromedial medulla (RVM) were dissected from brain samples and immersed in RIPA buffer containing a 10% protease inhibitor cocktail and 0.5% of phenylmethylsulfonyl fluoride (PMSF). Following homogenization and centrifugation, the supernatant was collected. Proteins (30 μg/sample) were separated electrophoretically (125 V, 90 min) in 12% SDS-polyacrylamide gels. After electrophoresis, proteins were blotted to a nitrocellulose membrane (0.45 μm pore size; Millipore) in a tank blotting system (125 V, 90 min). The membranes were blocked (5% BSA in PBS, with 0.2% Tween 20) for 1 h and then incubated overnight at 4 °C with specific primary antibodies for B-cell lymphoma 2 (Bcl-2) (Santa Cruz, 1:1000), Glial Fibrillary Acidic Protein (GFAP) (Sigma-Aldrich, 1:10000) and ionized calcium-binding adapter molecule 1 (Iba-1) (Wacko, 1:1000), and then, incubated for 2 h at 4 °C with secondary HRP-conjugated antibodies (Abcam, 1:10000 dilution). A chemiluminescence reaction kit (GE Healthcare) was used for detection, and immunolabeled protein bands were visualized in a ChemiDoc MP System (BioRad) and analyzed by ImageLab 5.2.1 software. A β-actin-specific antibody was used for the normalization of the samples.
3. Results 3.1. Mechanical and thermal sensitivity CLP-induced sepsis was able to produce an alteration in mechanical and thermal sensitivity (Figs. 1A - C). Mechanical sensitivity measured by the electronic von Frey apparatus showed that CLP animals presented an increase in the percentage of maximal possible effect (%MPE) when compared with the control group [T(14) = 2.623; P = .02, unpaired Student's-t-test, Fig. 1A]. In the tail-flick test, the statistical analyses (Two-way ANOVA) revealed a difference in time [F(1, 7) = 61.92; P = .0001], group [F(1, 7) = 99.45; P < .0001] and interaction time and group [F(1, 7) = 99.45; P < .000]. Tukey's post-test showed an increase in the analgesic index 24 h after the CLP induction when compared to respective baseline, and to the Naïve group in the same period (P < .05, Fig. 1B); however, in the control group (Naïve) any difference in the analgesic index was observed. The same was verified when scores of cold sensitivity in the acetone test were analyzed. Two-way ANOVA evidenced a time-group interaction [F(1, 7) = 13.44; P = .008], and Tukey post-test showed a significant decrease at the score in the CLP group when compared 24 h after the surgery to its respective baseline (P < .05, Fig. 1C).
2.9. ELISA for cytokines quantification The collected blood was centrifugated at 4 °C, 3000 rpm for 10 min, and sonicated tissue extracted at 4 °C, 13000 rpm for 20 min. The supernatant of processed tissues and plasma was used for cytokine quantification. The IL1-β, IL-6, and TNF-α cytokines levels were determined using appropriate ELISA kits (R&D Systems, Minneapolis, Minn., USA) according to the manufacturer's instructions. The detection limits for TNF-α, IL1-β, and IL-6 were 10 pg/mL. The total protein concentration in the tissue was measured using the BCA protein assay kit (Pierce). The samples were analyzed in a microplate reader
3.2. Locomotor activity Compared to Naïve group, animals submitted to CLP surgery did not show compromising locomotor behavior, measured by a total of crossings between squares in open field [T(14) = 1.036; P = .31, unpaired Student's-t-test] and total rearing movements [T(14) = 0.0314; P = .97, unpaired Student's-t-test, Fig. 1D). 3
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Fig. 1. Sensitivity and locomotor evaluation in rats 24 h after the surgery to induce sepsis (CLP) or not (control group, Naïve). A: Mechanical sensitivity evaluated by the percentage of maximal possible effect (MPE %); B: Thermal sensitivity to heat evaluated in the tail-flick test; C: Thermal sensitivity to cold evaluated in the acetone test; D: Locomotor activity during the 5 min-exposure to the open field test, evaluated by the horizontal (number of squares crossing) and vertical (number of rearing movement) activity. a P < .05, unpaired Student's-ttest when compared to Naïve group. b P < .05 Tukey test when compared CLP group 24 h after surgery with respective baseline. c P < .05 Tukey test when compared with Naïve group at the same time. The data represent means ± SEM. N = 6–8 per group.
compared to its respective Naïve group. Besides, in the RVM tissue were observed an increase in the IL 1-β [T(7) = 2.513; P = .0402; IL-6 [T(7) = 3.18; P = .0191] and TNF-α [T(6) = 2.454; P = .0495] compared to Naïve group (Fig. 4).
3.3. Astrocytes/microglia activation, and Bcl-2 expression A non-parametric statistical analyses (Kolmogorov-Smirnov test) showed an increased in the GFAP expression in PAG (P = .0095, Fig. 2A) and RVM (P = .0159, Fig. 2B) and also for Iba-1 in PAG (P = .0286, Fig. 2C) when compared to the respective Naïve group. A reduction in anti-apoptotic protein Bcl-2 expression in PAG (P = .0159, Fig. 2E) and RVM (P = .0159, Fig. 2F) was also seen, when compared to Naïve group. Representative photomicrography showing astrocytes and microglial expression in PAG and RVM of Naïve and CLP animals can be seen in Fig. 3.
4. Discussion Our results showed that sepsis induces an increase in overall nociceptive threshold in three different nociceptive tests, presenting cold, heat, and mechanical antinociception without alterations in general locomotor activity. It was accompanied by an increase in glial activation and production of proinflammatory mediators in RVM and PAG, two nuclei involved in descending control of pain. Previous studies regarding pain and an immune challenge have different results concerning nociception alterations (de Goeij et al., 2013; Diniz et al., 2018; Reeve et al., 2000; Seo et al., 2008). We believe that these diversified data result from the distinct models used (CLP, LPS injection, or cytokine administration), the dose of LPS, the route of administration (intraperitoneally, intravenous, intracerebroventricular) and the time points analyzed (minutes, hours or days after the
3.4. Cytokines levels The plasma citokynes levels analyses (unpaired Student's-t-test) showed that in the CLP group there was a significant increase in IL 1-β pro-inflammatory citokyne [T(9) = 6.593; P = .0001) when compared with Naïve group. In the PAG tissues, the statistical analyses evidenced a difference in IL 1-β [T(7) = 3.005; P = .0198], but not in IL-6 [T(7) = 1.796; P = .1156] or in TNF-α [T(8) = 0.0258; P = .980] 4
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Fig. 2. Expression of the GFAP, Iba-1, and Bcl-2 proteins in the periaqueductal gray (PAG) and rostroventral medulla (RVM). Relative quantification of the expression of the GFAP (A, B), Iba-1 (C, D), and Bcl-2 (E, F) proteins by western blotting assay 24 h after sepsis induction (CLP) or not (Naïve group). * P < .05, KolmogorovSmirnov test when compared to Naïve group. The data represent means ± SEM. N = 6–8 per group.
In the midbrain, PAG receives inputs from higher brain nuclei like the cerebral cortex and amygdala and has a major opioid-mediated inhibitory control of descending nociceptive pathways (Loyd and Murphy, 2009; Staud, 2013). RVM has bidirectional connections with PAG and other brain centers and is considered a final relay of descending inhibitory pathways (Ossipov et al., 2014; Staud, 2013). We believe that in septic animals, the overproduction of inflammatory agents could disturb the physiological functions of these nuclei. The increased Iba-1 protein expression and immunoreactivity indicates microglial activation, which is a potential source of cytokines. When activated, these cells may assume an M1 or proinflammatory phenotype
challenge). In the present study, we used the cecal ligation and puncture, which is considered a golden standard and clinically relevant model of sepsis, since it mimics in a better manner the human sepsis, with polymicrobial infection, presence of infectious focus, hyper- and hypoinflammatory phase and similar hemodynamic progression (Dejager et al., 2011; Ruiz et al., 2016). The impairment herein observed in discriminative sensory pathways indicates a potential consequence of SAE. In fact, in patients, the reduction of cortical and subcortical low latency sensory evoked potential, mainly in the sensory cortex and brainstem nuclei, indicates generalized dysfunction of the clinical condition (Zauner et al., 2002). 5
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Fig. 3. Representative photomicrographs were taken in the regions of the lateral periaqueductal gray (lPAG) and rostroventral medulla (RVM) showing GFAP (green fluorescence) and Iba-1 (green fluorescence) immunostaining in 40 μm coronal sections of Naïve rats and rats 24 h after CLP surgery. Plates adapted from Paxinos and Watson (2007). Photomicrographs at 10× (smaller box) and 20×. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
changes in other brain structures, which are intimately involved with nociceptive modulation (Kato et al., 2018; Llorca-Torralba et al., 2018). In this way, there is intense microglial activation and neuronal apoptosis in the amygdala and locus coeruleus (Carlson et al., 2007; Sharshar et al., 2003), as well as morphological anomalies in the sensorimotor cortex (Polyanin and Bardakhch'yan, 1984) during sepsis. Thus, these changes can also contribute to the reduction of nociception that is observed in the present study. Even though peripheral mechanisms were not the focus of our investigation, we cannot exclude that it may be involved in the reduced nociception in septic animals. A recent study has also shown a reduction in mechanical sensitivity in CLP animals on early sepsis (24 h after surgery) accompanied by a reduction in sciatic nerve excitability (Diniz et al., 2018). In our study, the reduced nociception was observed both in paw (mechanical) and in the tail (thermal), suggesting an involvement of other possible mechanisms beyond the sciatic nerve in sepsisinduced antinociception. The endogenous opioids system also has potential effects in regulating nociception during sepsis since its circulating levels are increased in septic patients (Glattard et al., 2010). Yirmiya et al. (Yirmiya et al., 1994) have demonstrated that the reduced pain sensitivity observed four hours after LPS injection was reversed by the administration of naltrexone, an opiate antagonist. In conclusion, our results indicate that CLP-induced sepsis leads to a reduction in nociception, and it is can be associated, at least in part, with glial cell activation and an inflammatory microenvironment in brain areas related to pain regulation. We believe that the investigation of cellular mechanisms and neurotransmission is essential to a better understanding of the sensory alterations in septic pathophysiology.
and secrete inflammatory cytokines, like IL-1β, IL-6, TNF-α, and IFN-γ. (Eggen et al., 2013; Morrison and Filosa, 2013). Astrocyte activation also leads to cytokine expression, and more importantly, it alters the regulation of local neuro- and glia transmitters (Gorina et al., 2011; Pascual et al., 2012). Also, even though TNF-α may induce an enhanced the expression of BCL-2, an essential mitochondrial protein that serves as an inflammatory and apoptotic inhibitor (Kim, 2005), it seems to be downregulated in severe sepsis patients (Bilbault et al., 2004), animals with SAE in CLP (Deng et al., 2017) and LPS models of sepsis (Ning et al., 2017) what means a possible vulnerability to cell death in brain tissue. Moreover, it influences the synthesis, uptake, and release of several neurotransmitters, like GABA and glutamate, in both excitatory and inhibitory synapses in different brain regions (Camacho-Arroyo et al., 2009; Miller et al., 2013). Although we did not investigate the precise modulation of activation of neurotransmitters in each of these areas, we believe that systemic inflammation profoundly alters the local synaptic circuitry resulting in reduced nociception. Sepsis and microglia-derived cytokines could deeply deregulate the brain content of GABA (Serantes et al., 2006), acetylcholine (Pavlov et al., 2006) and amines (serotonin, dopamine, norepinephrine) (Dal-Pizzol et al., 2014) levels in the brain. Besides, astrocyte plays a vital role in glutamatergic regulation in the brain (Araque et al., 1999; Malarkey and Parpura, 2008). The presence of TNF-α and IL-β induces the release of this neurotransmitter in both human and rat cortex and hippocampal primary neurons, as well as in astrocyte cultures (Sama et al., 2008; Ye et al., 2013). We hypothesize that in septic animals there is an excessive cytokine-mediate glutamate release in PAG and RVM that could result in the reduced nociception since it has been already shown that glutamate in these areas exerts an antinociceptive effect in pain modulation (Morgan et al., 2009; Samineni et al., 2017). All these transmitters play an important role in nociception control in the PAG-RVM system, remarkably by regulating “on” and “off” cells in the RVM being “off” cells, a subpopulation that when tonically activated inhibit nociception, while “on” cells facilitate pain behavior. We presume that during sepsis, the inflammatory milieu in the brain favors the inhibitory component of RVM. Another possibility is that SAE can promote morphofunctional
Authors contributions R.A.C., N.N.S.-J., L.H.A.C., C.H.R.C, and J.M.G. performed the experiments and analyzed the data. R.A.C., N.N.S.-J., M.J.A.R., and C.R.A.L.P designed the study and wrote the manuscript. All authors read and approved the final manuscript.
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Fig. 4. Up-regulation of inflammatory mediators (IL-1 β, IL-6, and TNF-α) in the plasma, periaqueductal gray (PAG), and rostroventral medulla (RVM) at 24 h after sepsis induction (CLP) or not (Naïve group). * P < .05 unpaired Student's-t-test when compared to Naïve. The data represent means ± SEM. N = 6–8 per group.
Compliance with ethical standards
unacknowledged partner. Trends Neurosci. 22, 208–215. Bell, A., 2018. The neurobiology of acute pain. Vet. J. 237, 55–62. Bilbault, P., Lavaux, T., Lahlou, A., Uring-Lambert, B., Gaub, M.P., Ratomponirina, C., Meyer, N., Oudet, P., Schneider, F., 2004. Transient Bcl-2 gene down-expression in circulating mononuclear cells of severe sepsis patients who died despite appropriate intensive care. Intensive Care Med. 30, 408–415. Burgess, S.E., Gardell, L.R., Ossipov, M.H., Malan Jr., T.P., Vanderah, T.W., Lai, J., Porreca, F., 2002. Time-dependent descending facilitation from the rostral ventromedial medulla maintains, but does not initiate, neuropathic pain. J. Neurosci. 22, 5129–5136. Calil, I.L., Zarpelon, A.C., Guerrero, A.T., Alves-Filho, J.C., Ferreira, S.H., Cunha, F.Q., Cunha, T.M., Verri Jr., W.A., 2014. Lipopolysaccharide induces inflammatory hyperalgesia triggering a TLR4/MyD88-dependent cytokine cascade in the mice paw. PLoS One 9, e90013. Camacho-Arroyo, I., Lopez-Griego, L., Morales-Montor, J., 2009. The role of cytokines in the regulation of neurotransmission. Neuroimmunomodulation 16, 1–12. Carlson, D.E., Chiu, W.C., Fiedler, S.M., Hoffman, G.E., 2007. Central neural distribution of immunoreactive Fos and CRH in relation to plasma ACTH and corticosterone during sepsis in the rat. Exp. Neurol. 205, 485–500. Catalão, C.H.R., Santos-Júnior, N.N., da Costa, L.H.A., Souza, A.O., Alberici, L.C., Rocha, M.J.A., 2017. Brain oxidative stress during experimental sepsis is attenuated by simvastatin administration. Mol. Neurobiol. 54, 7008–7018. Chaplan, S.R., Bach, F.W., Shafer, S.L., Yaksh, T.L., 1995. Prolonged alleviation of tactile allodynia by intravenous lidocaine in neuropathic rats. Anesthesiology 83, 775–785. Chu, H., Sun, J., Xu, H., Niu, Z., Xu, M., 2012. Effect of periaqueductal gray melanocortin 4 receptor in pain facilitation and glial activation in rat model of chronic constriction injury. Neurol. Res. 34, 871–888. Comim, C.M., Constantino, L.C., Barichello, T., Streck, E.L., Quevedo, J., Dal-Pizzol, F., 2009. Cognitive impairment in the septic brain. Curr. Neurovasc. Res. 6, 194–203. Comim, C.M., Cassol-Jr, O.J., Constantino, L.C., Petronilho, F., Constantino, L.S., Stertz, L., Kapczinski, F., Barichello, T., Quevedo, J., Dal-Pizzol, F., 2010. Depressive-like parameters in sepsis survivor rats. Neurotox. Res. 17, 279–286. da Costa, L.H.A., Júnior, N.N.D.S., Catalão, C.H.R., Sharshar, T., Chrétien, F., da Rocha,
All animal experiments in this study were carried out according to a protocol approved by the Animal Care and Use Committee of the University of São Paulo, Brazil at the Ribeirão Preto (protocol number # 2017.1.888.58.1). Declaration of Competing Interest The authors declare that no conflict of interest could be perceived as prejudicing the impartiality of the reported research. Acknowledgments The authors thank Nadir M. Fernandes, Bruna Barissa and Patrícia Adriana Basile for technical assistance. Financial support from Fundação de Amparo à Pesquisa do Estado de São Paulo (grant number # 2017/11213-6), Conselho Nacional de Pesquisa – Brazil (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brazil (Capes Finance Code 001). C.R.A.L.P. was granted a research fellowship from CNPq (grant number # 304214/2016-3). References Araque, A., Parpura, V., Sanzgiri, R.P., Haydon, P.G., 1999. Tripartite synapses: glia, the
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