In our study, we aimed to investigate the effects of inflammation on the brain tissue of rats with LPS-induced sepsis, and how these effects could be changed using an ionotropic GABAA receptor antagonist, also acetylcholinesterase inhibitor-bicuculline. The mechanism of neurologic complications in sepsis-induced encephalopathy has not been fully elucidated. Researchers in many studies reported that the brain GABA receptor density and serum GABA levels were increased in experimental septic encephalopathy models, bacteria were reported to possibly produce large amounts of GABA, and bacterial sepsis was a cause of encephalopathy. Furthermore, recent studies reported that a GABA agonist, benzodiazepine, exacerbated septic encephalopathy (Jones et al. 1984; Minuk et al. 1985; Sallam et al. 2006; Ramirez- Jarquin et al. 2017; Shulyatnikova et al., 2020).
Acute brain dysfunction was reported in LPS-induced sepsis, especially with the increase in GABA levels in the hippocampus, and with a decrease in the level of consciousness and neuronal activity (Kadoi et al. 1996; Hellstrom et al. 2005; Serantes et al. 2006; Jacob et al. 2011; Malaguarnera et al. 2019). Rossi et al. showed that the released proinflammatory molecules increased the central GABA activity during autoimmune encephalomyelitis (Rossi et al. 2011). Sugiura et al. illustrated that macrophage GABA receptors increased the susceptibility of mice to endotoxemia through regulation by increasing the NF κß, and MAPKERK (Sugiura et al. 2011). For blocking GABAergic activity using the GABAA receptor blocker bicuculline, and GABAB receptor blocker baclofen, Salam et al. reported that administration of bicuculline improved cardiac, hemodynamic, and inflammatory findings compared with a baclofen-treated group, and ionotropic GABAA receptors were more effective in the inflammatory process (Sallam et al. 2006).
Variable levels of neurotransmitters in sepsis led to alterations in cognitive processes, in addition to the damage of neuronal mechanisms and brain tissue (Ramirez- Jarquin et al. 2017; Malaguarnera et al. 2019). Inflammatory mediators were reported to cause increased receptor activity by binding to GABAA receptors by modulating the expression of GABAA receptor subunits with infectious factors, hypoxia, or stress (Wang et al. 2012). In our study, we observed that GABA levels increased in total brain tissue; however, the increase was not statistically significant (p > 0.05). In the bicuculline and LPS + bicuculline group, we found similar GABA levels to those in the control group.
In their experimental study, Winder et al. observed that GABA levels were elevated in the brain tissue of rats with sepsis. They reported that GABA levels increased, especially in the sub-brain region; however, in a total brain investigation, this increase was not as significant as in our study (Winder et al. 1988). Our findings of GABA levels in total brain tissue were similar to the levels in other studies in the literature.
Recent studies showed that acetylcholine and bicuculline methiodide could activate nicotinic acetylcholine receptors (Hsu et al. 2004; Thomsen et al. 2012; Ramírez-Jarquín et al. 2017). Wang et al. demonstrated that activation of nicotinic acetylcholine receptors inhibited TNF-α synthesis and decreased inflammatory response by increasing IL-10 release (Wang et al. 2003). In addition, activation of nicotinic acetylcholine receptors was reported to be associated with a decrease in the production of NO and reactive oxygen species (Li et al. 2000; Hoover et al. 2017). Bicuculline methiodide was suggested to have a protective effect after cecal ligation and perforation in a study examining the change of cytokines with bicuculline methiodide. TNF-α is known to trigger the production of free radicals in neonatal rat hepatocytes and reduce oxidative metabolism (New et al. 2001). This activity is positively controlled by various up-regulatory stimuli, including TNF-α and IL-1ß, and is negatively controlled by pro-inflammatory cytokines such as IL-10 (Szabo et al. 1993; Thiemermann et al. 1993).
In our study, we investigated the effects of bicuculline methiodide on IL-10 levels from TNF-α and anti-inflammatory cytokines from proinflammatory cytokines in brain tissue in the sepsis group. The TNF-α levels of the bicuculline and LPS + bicuculline groups were close to those in the control group. We observed that TNF-α levels increased in total brain tissue in the sepsis group (P < 0.05). TNF-α is known to reach peak values at minute 90 and its half-life is shorter. In addition, studies showed that TNF-α concentrations were decreased by stimulating several pathways such as anti-inflammatory pathways in long-term sepsis models (Thiemermann et al. 1993). We found that IL-10 levels significantly decreased in the sepsis group compared with the other experimental groups (P < 0.05). Our other experimental groups were similar to the control group.
A decrease in IL-10 levels is reported as an indicator of the inflammatory pathway induced by TNF-α in the literature (Munford et al. 2005). Although the NF-κß pathway plays an important role in the regulation of cytokine production, it has been supported in studies that bicuculline methiodide might decrease proinflammatory cytokine production by inducing activation of nicotinic acetylcholine receptors instead of using the NF-κß pathway (Loop et al. 2003). However, researchers reported that nicotinic acetylcholine receptors might be associated with the anti-inflammatory effect of bicuculline methiodide in sepsis (Wang et al. 2003). In another study, bicuculline methiodide was suggested to inhibit the pro-inflammatory cytokine release through nicotinic α-7 receptors (Sallam et al. 2006; Wang et al. 2003). We observed that TNF and IL-10 cytokine values decreased in the sepsis group in the LPS + bicuculline group. This may be explained by the fact that bicuculline blocks GABAA ionotropic receptors and inhibits acetylcholinesterase and blocks the inflammatory process.
The alpha-7 nicotinic Ach receptor agonist GTS-21 was reported to decrease cytokine levels in studies conducted with volunteers who were given endotoxin treatment. Some studies reported that cholinergic neurons were particularly sensitive to systemic inflammation (Dal-Pizzol et al. 2014). These findings are in line with our results. Bicuculline methiodide, which is known to have acetylcholinesterase inhibition, was suggested to possibly be a good anti-inflammatory agent as a result of stimulating cholinergic pathways and was supported by other studies (Sallam et al. 2006; Hsu et al. 2004; Thomsen et al. 2012; Malaguarnera et al. 2019 ).
Increased ROS production in sepsis causes neuropathy and myopathy leading to the destruction of the brain and tissue. In studies with lipid peroxidation in sepsis, thiobarbituric acid reactive products were analyzed, and increased MDA levels were reported to have effects such as impaired blood–brain barrier (Martins et al. 2003). In our study, we observed that MDA levels in brain tissue increased in the sepsis group compared with the control and LPS + bicuculline groups (P < 0.05). In experimental studies, MDA levels were reported to increase in the cortex, hippocampus, and cerebellum at hour 6 in sepsis-induced rats (Takezawa et al. 1983). A significant increase was shown in MDA levels in the sepsis group.
High serum levels were reported in cases of neuron-specific enolase (NSE) levels in the brain quantification of cardiac injury, cardiac arrest in the clinic, septic shock, brainstem, neuroendocrine tumors, and malignancy (Isgro et al. 2015). Researchers reported that NSE levels in patients with sepsis in ICUs could be used to predict the clinical course and might show high sensitivity and specificity (Weigand et al. 2000). Yao et al. reported that NSE and S100-β levels were significantly higher in patients with septic encephalopathy (Isgro et al. 2015; Yao et al. 2014). In our study, we found that NSE levels significantly increased in the sepsis group compared with the other experimental groups (P < 0.01). Our findings support the data in the literature.
We obtained non-focal epileptic wave-like recordings from our rats with sepsis under mild anesthesia in an investigation of non-invasive total brain electrical activity recordings. We found a higher degree of significance in the LPS group compared with the other groups in investigations of the minimum-maximum ratios of wave amplitudes between the groups. The other experimental groups had similar wave characteristics and minimum-maximum ratios to the control group. In our group, where we used bicuculline methiodide, we detected no convulsion waves, supporting that it might be a suitable agent in GABA blockade according to other GABA antagonists and bicuculline derivatives, in accordance with the literature (Sallam et al.2006).
In the immunofluorescence staining of the neurotransmitter through the synaptophysin antibody, which is a specific glycoprotein for the synaptic terminals, we observed little involvement in the brain tissue in the sepsis group compared with the other groups. This shows that neurotransmitter regulation was impaired in the sepsis group. These results indicate that the rats are also affected by sepsis. On the other hand, more intense synaptophysin involvement was observed in the control, bicuculline, and LPS + bicuculline groups compared with LPS groups. This suggests that bicuculline had a curative effect on the neurotransmitter regulation of sepsis.
S100-ß is one of the markers used to demonstrate neuronal damage in the brain. Studies have shown increased serum levels of S100-ß in sepsis, brain trauma, cerebral stroke, hypoxic ischemia, and encephalopathy. The exact mechanism for the secretion of S100-ß has not been fully elucidated; however, it was reported to cause neural tissue damage and was associated with oxidative stress (Isgro et al. 2015; Yao et al. 2014).
In the comparison of the number of damaged cells stained with S100-ß antibodies in the sepsis group and the number of cells stained with NeuN antibodies, which stain live neurons, we observed increased counts of live neurons in our study in the control, bicuculline, and LPS + bicuculline groups compared with the LPS group. Our findings suggest that LPS causes neuronal damage, and bicuculline reduces this damage. However, detailed studies are required to explain the mechanism [36,37].