Potential Neuroprotective Role of Melatonin in Sepsis-Associated Encephalopathy Due to Its Scavenging and Anti-Oxidative Properties

Round 1

Reviewer 1 Report

The topic is of interest to the readers. However, the paper requires revisions prior considerations, see below.

While the topic of sepsis and its clinical implication are were described, the section on melatonin is deficient.

Again, description of sepsis associated encephalopathy, clinical picture of SAE, epidemiology and mechanisms of action underlying pathology are comprehensively described.

However, melatonin section is based on outdated information, insufficient number of sometime random citations, and not fully comprehensive mechanistic approach.

For example, information on sites of production of melatonin is outdated and not informative.  Specifically, melatonin is widely produced in Nature in different species, and in humans it is produced in many peripheral organs, including barrier organs at concentration much higher than the serum concentration (Cell Mol Life Sci 74(21), 3913-3925, 2017). Particularly instructive in this matter is skin, which is continuously exposed to different stressors. 

Also, majority of anti-oxidative actions of melatonin are independent of MT1 and MT2 (Sci Rep 2017;7(1):1274, 2017). As relates to mechanisms of action, authors correctly point to NRF2, however, do not elaborate properly on it, and one citation is insufficient on this topic. Better mechanistic approach is suggested. Also, melatonin rapidly metabolized through enzymatic and non-enzymatic actions leading to production of equally potent metabolites with anti-oxidative properties (Exp Dermatol 26:563–568, 2017). This is not even mentioned.

In general, section on melatonin requires improvements and updating.

Adequate discussion of mechanism of action is expected.

The readers will also appreciate two instructive figures to illustrate the subject.

Typographical and English errors should also be corrected.

Some English editing is required

Author Response

Dear Reviewer,

Thank you for your effort in reviewing our manuscript and your suggestions aiming at improvement of our paper. We are very thankful for that. Please, find below responses to your comments.

1. For example, information on sites of production of melatonin is outdated and not informative.  Specifically, melatonin is widely produced in Nature in different species, and in humans it is produced in many peripheral organs, including barrier organs at concentration much higher than the serum concentration (Cell Mol Life Sci74(21), 3913-3925, 2017). Particularly instructive in this matter is skin, which is continuously exposed to different stressors. 

Thank you for this comment. Additional information on places of melatonin production was added (Section 1.5) :

Melatonin is a neurohormone widely spread in nature, found both in plant and animal species. The most important source of melatonin in humans is the pineal gland where this neurohormone is produced with circadian rhythm but multiple local sources of melatonin also exist, like retina [1], immune cells[2], skin [3] or epithelium of gastrointestinal tract[4].

2. Also, majority of anti-oxidative actions ofmelatonin are independent of MT1 and MT2 (Sci Rep 2017;7(1):1274, 2017).

Thank you for this comment. A information on that was added to text of section 3.1:

Nevertheless, it must be noted that most of anti-inflammatory and antioxidant actions of melatonin is independent from MT1 and MT2 and is exerted through activation of NRF2-related pathways, as it was shown by Janjetovic et al.

3. As relates to mechanisms of action, authors correctly point to NRF2, however, do not elaborate properly on it, and one citation isinsufficient on this topic. Better mechanistic approach is suggested.

Thank you for this remark, additional information about melatonin-NRF2 was added (section 4.1):

The melatonin is capable to activate NRF2 which in turn increases expression of antioxidative enzymes (like SOD) which may prevent multiorgan failure in sepsis. Kang et al. proved that melatonin may prevent development of lung injury through this pathway. [99] Melatonin-induced activation of NRF2 signaling pathway was also shown to exert neuroprotective action in a model of lipopolysaccharide-induced injury of neural tissue (analogous to models of septic encephalopathy). [100]

4. Also, melatoninrapidly metabolized through enzymatic and non-enzymatic actions leading to production of equally potent metabolites with anti-oxidative properties (Exp Dermatol 26:563–568, 2017). This is not even mentioned.

Thank you for this remark. A paragraph on that was added to section 3.3:

It is also noteworthy that metabolites of melatonin are potent antioxidants which increases therapeutical potential of the pineal neurohormone. Those metabolites may counteract environmental stresses, including oxidative stress, as it was described in a review by Slominski et al. . [97] .Main metabolites of melatonin, like N¹-acetyl-N²-formyl-5-methoxykynuramine (AFMK), N-acethyl-5-methoxykynuramine (AMK), 6-hydroxymelatonin, 4-hydroxymelatonin act as direct scavengers, metal chelating agents or as molecules capable of repairing oxidative damage as it was presented in a review by Galano and Reiter. [98]

5. In general, section on melatonin requires improvements and updating. Adequate discussion of mechanism of action is expected.

Thank you for this advice. Section on melatonin was improved and updated with discussion of mechanisms of action.

6. The readers will also appreciate two instructive figures to illustrate the subject.

Thank you for this suggestion. A Table with clinical trials with melatonin was added as well as figures showing mechanisms of septic encephalopathy and therapeutical potential of melatonin.

7. Typographical and English errors should also be corrected.

Thank you for this opinion, manuscript was spell-checked and corrected.

Reviewer 2 Report

The manuscript titled "Potential neuroprotective role of melatonin in sepsis-associated encephalopathy due to its scavenging and anti-oxidative properties" by Sieminski et al. presents a comprehensive review of the potential therapeutic role of melatonin in sepsis-induced brain injury, specifically focusing on sepsis-associated encephalopathy (SAE). The authors provide valuable insights into the neuroprotective effects of melatonin, attributed to its anti-inflammatory and antioxidant properties. The manuscript explores existing data from animal models and human clinical trials to support the hypothesis of melatonin's neuroprotective potential in SAE.

However, to strengthen the scientific contribution of the research, I provide constructive feedback and suggestions below:

–       While the introduction focuses on sepsis and SAE, it would be beneficial to include a brief background on melatonin and its well-known properties as a neurohormone responsible for controlling circadian rhythms. It could be a separated section from the one present now.

–       Include a concise statement in the introduction to clarify the specific objectives of the review. In the concluding paragraph of the introduction, clearly state that the review aims to explore the potential neuroprotective role of melatonin in the context of SAE. This will reinforce the central theme of the manuscript.

–       Expand on the connection between sepsis and central nervous system (CNS) dysfunction. Discuss the rapid development of CNS-related symptoms during sepsis, emphasizing the vulnerability of the CNS due to its circulatory and metabolic needs.

–       Provide a clear definition of sepsis-associated encephalopathy (SAE) and explain that it is characterized by brain failure resulting from sepsis without direct brain injury or CNS infection. Additionally, briefly discuss the lack of formal clinical diagnostic criteria for SAE and how it is currently diagnosed solely based on clinical grounds.

–       While the introduction mentions that the exact pathophysiological mechanisms of SAE remain unknown, it could benefit from briefly mentioning some of the potential pathways leading to SAE. These may include the role of brainstem dysfunction, frontal cortex, amygdala, and neuroendocrine centers in the development of SAE.

–       Highlight the impact of SAE on patients by discussing the broad spectrum of clinical symptoms it presents, ranging from cognitive deficits to delirium and coma. Emphasize that SAE has a significant effect on patient outcomes and quality of life.

–       Provide additional details about the significance of blood-brain barrier (BBB) disruption in SAE. Explain how increased permeability of the BBB allows pro-inflammatory cytokines and leukocytes to enter the central nervous system, leading to neuroinflammation. Emphasize the implications of this process on the progression of SAE and its clinical symptoms.

–       Expand on the interaction between neuroinflammation and microglial cells in the context of SAE. Elaborate on the activation of microglial cells by pro-inflammatory cytokines and their role in perpetuating the inflammatory response. Additionally, discuss the relevance of microglia activation to the development of delirium and other cognitive deficits.

–       Provide a clearer connection between sepsis-related hemodynamic instability, hypercoagulability, and ischemia in the brain. Explain how the disruption of cerebral blood flow and the formation of clots contribute to the ischemic conditions observed in septic patients.

–       Further highlight the effects of neuroinflammation and ischemia on neurotransmitter pathways, particularly the cholinergic and dopaminergic pathways. Discuss how the alterations in neurotransmission contribute to cognitive and consciousness disorders observed in SAE.

–       Elaborate on the relationship between the metabolic challenges faced by CNS cells during sepsis and the resulting oxidative stress and mitochondrial dysfunction. Explain how the increased energetic needs and decreased oxygen and glucose supply lead to the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) and, subsequently, cellular damage and apoptosis.

–       Include references to studies or experimental data that demonstrate increased production of ROS, reduced ATP production, and intense apoptosis in animal models of SAE.

–       Mention the potential therapeutic interventions aimed at reducing oxidative stress and scavenging reactive species, as discussed in animal models with molecular hydrogen or tetramethylpyrazine.

–       Part of section 3 could be moved to the introduction.

–       Begin section 3, by explicitly stating the significance of melatonin in the context of sepsis and SAE. Highlight the potential benefits of melatonin as a therapeutic agent for septic patients based on its neuroprotective, antioxidant, and anti-inflammatory properties.

–       Connect the discussion of melatonin's main clinical roles to its neurohormone functions, particularly its impact on the regulation of circadian rhythm and sleep promotion during the dark part of the day. Discuss how this rhythmic regulation can be disrupted during sepsis and SAE.

–       Elaborate on melatonin's potential to reduce sympathetic drive and restore sympathetic-parasympathetic balance in septic patients, as mentioned in the section. Discuss the implications of this effect on the overall pathophysiology of sepsis and its potential role in modulating systemic inflammatory responses.

–       Include references to relevant studies that support melatonin's role in reducing vasoconstriction and blood pressure and its protective effects against obesity and diabetes mellitus.

–       Elaborate on the mechanisms through which melatonin exerts its anti-inflammatory potential. Discuss the impact of melatonin on specific inflammatory mediators, such as interleukins and tumor necrosis factor alpha, and its role in preventing cell death caused by pyroptosis.

–       Further emphasize the importance of oxidative stress in the pathogenesis of SAE and explain how melatonin's ability to scavenge free radicals, chelate metal ions, and activate antioxidant enzymes can protect neural tissues from oxidative damage.

–       Whenever possible, incorporate relevant clinical studies that support the potential therapeutic use of melatonin in sepsis and SAE.

–       While section 5 mentions some mechanisms through which melatonin exerts its antioxidant effects, such as increasing antioxidant enzyme activity and chelating metal ions, it would be beneficial to expand on these mechanisms and provide more detailed explanations.

–       The section briefly mentions an interesting study that demonstrated different neurobehavioral effects depending on the timing of melatonin treatment in sepsis-related brain injury in a mouse model. It would be helpful to discuss the potential implications of this finding in the clinical context and how the timing of melatonin administration may impact its neuroprotective effects in human patients with SAE.

–       While the section highlights some promising results from small clinical trials, it would be beneficial to provide more details about these studies, such as the number of participants, study design, and outcomes assessed. Additionally, discussing the limitations of these trials and the need for larger multi-center studies with randomization would add depth to the discussion.

–       Given the diverse effects of melatonin observed in various studies, it would be valuable to discuss the potential dose-response relationships and optimal dosing regimens for melatonin in the context of sepsis and SAE. Addressing the safety profile of melatonin at different doses is also important, particularly when considering its potential use in critically ill patients.

–       Discuss the possible interactions between melatonin and other standard treatments for sepsis and critically ill patients. Since many septic patients receive multiple interventions, understanding the compatibility and potential synergy or antagonism with melatonin is crucial.

–       Conclude the section by summarizing the overall potential of melatonin as a therapeutic agent for sepsis and SAE. Emphasize the significance of preclinical evidence and initial clinical trial results in guiding future research and potential application in clinical practice.

–       To enhance the review and provide a more comprehensive understanding of the topic, the authors could consider including figures and tables. Here are some suggestions for potential figures and tables that could be included in the review:

o   Figure: "Pathomechanisms of Sepsis-Associated Encephalopathy (SAE)"

This figure could visually illustrate the key pathomechanisms of SAE, including blood-brain barrier disruption, neuroinflammation, oxidative stress, and ischemia. Arrows or connections could indicate the relationships between these mechanisms and their contribution to the development of SAE.

o   Table: "Clinical Studies on Melatonin in Sepsis and Sepsis-Associated Encephalopathy"

This table could provide a summary of relevant clinical studies that have investigated the therapeutic effects of melatonin in septic patients and those with SAE. Columns could include study design, patient population, intervention (dose and duration of melatonin treatment), outcomes measured (e.g., mortality, cognitive function), and key findings.

o   Figure: "Mechanisms of Melatonin's Neuroprotective Effects in Sepsis"

This figure could visually depict the various mechanisms by which melatonin exerts its neuroprotective effects in septic patients, such as its antioxidant actions, anti-inflammatory effects, regulation of blood-brain barrier integrity, and modulation of mitochondrial function.

o   Table: "Comparison of Melatonin with Other Antioxidants in Sepsis"

This table could compare melatonin with other antioxidant therapies that have been studied in sepsis. Columns could include the name of the antioxidant, its proposed mechanisms of action, evidence from animal and clinical studies, and potential advantages and limitations of each antioxidant.

o   Figure: "Potential Therapeutic Role of Melatonin in Sepsis and Sepsis-Associated Encephalopathy"

This figure could provide an overview of the potential therapeutic role of melatonin in sepsis and SAE, summarizing its effects on oxidative stress, neuroinflammation, blood-brain barrier integrity, and cognitive function. It could also include arrows indicating the points in the pathophysiological process where melatonin may exert its effects.

Author Response

Dear Reviewer,

Thank You for Your work on reviewing our manuscript and for your valuable comments helping us to improve quality of our paper. Please find below our responses to your comments.

1. –       While the introduction focuses on sepsis and SAE, it would be beneficial to include a brief background on melatonin and its well-known properties as a neurohormone responsible for controlling circadian rhythms. It could be a separated section from the one present now.

Thank you for this comment. A new section was added to introduction (Section 1.5), with general information about melatonin:

Melatonin is a neurohormone widely spread in nature, found both in plant and animal species. The most important source of melatonin in humans is the pineal gland where this neurohormone is produced with circadian rhythm but multiple local sources of melatonin also exist, like retina [1], immune cells[2], skin [3] or epithelium of gastrointestinal tract[4]. The most noticeable physiologic role of melatonin in humans is control of circadian rhythm [33][6,7] but it exerts numerous other effects. Melatonin is a potent antioxidant [8]and anti-inflammatory factor [9]. It also has an impact on energy metabolism [10] and on cardiovascular system [11]. 

2. –       Include a concise statement in the introduction to clarify the specific objectives of the review. In the concluding paragraph of the introduction, clearly state that the review aims to explore the potential neuroprotective role of melatonin in the context of SAE. This will reinforce the central theme of the manuscript.

Thank you for this suggestion. Such a statement was added (section 1.5) :

The above-mentioned properties of melatonin make it an important factor influencing inflammatory response. That is why so much attention is given recently to the role of melatonin in sepsis. The aim of our review is to analyze available literature on potential neuroprotective effect of melatonin in sepsis associated encephalopathy (SAE).

3. –       Expand on the connection between sepsis and central nervous system (CNS) dysfunction. Discuss the rapid development of CNS-related symptoms during sepsis, emphasizing the vulnerability of the CNS due to its circulatory and metabolic needs.

 Thank you for this comment. Information on that is given in section 1.2.

And central nervous system (CNS) is one of the most vulnerable organs due to its circulatory and metabolic needs. Systemic inflammatory process causes dysfunction of blood-brain barrier allowing influx of proinflammatory mediators to CNS resulting in spreading of inflammation across cerebral structure. Sepsis-related hypotonia decrease cerebral perfusion which leads to deficiency of metabolic substrates for neural tissue.   Therefore, symptoms of involvement of the CNS develop rapidly during sepsis being the first clinical symptoms of sepsis in some cases.

4. –       Provide a clear definition of sepsis-associated encephalopathy (SAE) and explain that it is characterized by brain failure resulting from sepsis without direct brain injury or CNS infection. Additionally, briefly discuss the lack of formal clinical diagnostic criteria for SAE and how it is currently diagnosed solely based on clinical grounds.

Thank you for this comment. Information on SAE definition and lack of formal diagnostic criteria is given in section 1.2:

Sepsis associated encephalopathy (SAE) is a term describing brain failure caused by sepsis in absence of direct brain injury or infection of central nervous system. It must be remembered that there is no formal clinical definition of SAE nor formal diagnostic criteria. Biochemical, neurophysiological or radiological markers of SAE are lacking as well. Therefore, diagnosis of SAE is made on clinical ground exclusively. The diagnosis is based upon clinical observation of cognitive deterioration temporarily related to symptoms of sepsis with exclusion of direct brain injury and neuroinfection.

5. –       While the introduction mentions that the exact pathophysiological mechanisms of SAE remain unknown, it could benefit from briefly mentioning some of the potential pathways leading to SAE. These may include the role of brainstem dysfunction, frontal cortex, amygdala, and neuroendocrine centers in the development of SAE.

 Thank you for this comment. The following statement is added to section 1.2:

The most important pathways leading to development of SAE are: dysfunction of BBB, neuroinflammation, oxidative stress and cerebral ischemia. Development of SAE is a consequence of dysfunction of brainstem, frontal cortex and amygdala as well as of failure of neuroendocrine centers: hypothalamus and pituitary gland

6. –       Highlight the impact of SAE on patients by discussing the broad spectrum of clinical symptoms it presents, ranging from cognitive deficits to delirium and coma. Emphasize that SAE has a significant effect on patient outcomes and quality of life.

Thank you for this comment. Information on that topis are presented in section 1.2 and 1.4:

 The clinical spectrum of SAE ranges from discrete cognitive deficits, e.g. reduced attention or diminished readiness for social interactions (so called sickness behavior) [12] through delirium  to coma [13]. Most commonly acute SAE is observed but its subacute (lasting for weeks) or chronic (lasting for months) forms may be observed.

Patients with encephalopathy were older, with no significant differences in gender distribution, had higher scores on APACHE II and SOFA scales and were at higher risk of in-hospital death. [14]. Those results are in concordance with those reported by Feng et al. Those authors found incidence of SAE of 42.3% septic patients and subjects with SAE had higher scores of APACHE II and SOFA and higher mortality rates [15]. Sonneville et al., performed a retrospective multi-center study and found slightly higher prevalence - 53% of septic patients had SAE [16]. Therefore, it may be assumed that about half of septic patients suffer from encephalopathy and that this condition is related to more serious course of sepsis and higher mortality.

A frequently neglected fact is that acute sepsis-related brain injury may transform into chronic form becoming a cause of neurological and cognitive decline. A prospective study performed by Iwashyna et al. showed that in 10% of sepsis survivors moderate an severe cognitive deficits are diagnosed de novo [17]. Another study shows that in population of elderly subjects who survived 3 years after diagnosis of sepsis there are approximately 15% of patients with moderate and severe cognitive deficit [18]. In other studies, the prevalence of cognitive disorders reached over 20% [19]. Recently it was also observed that in middle-aged population sepsis increases rate of cognitive decline [20].

7. –       Provide additional details about the significance of blood-brain barrier (BBB) disruption in SAE. Explain how increased permeability of the BBB allows pro-inflammatory cytokines and leukocytes to enter the central nervous system, leading to neuroinflammation. Emphasize the implications of this process on the progression of SAE and its clinical symptoms.

 Thank you for this comment. Details on the role of BBB are presented in section 2.1:

Sepsis-associated encephalopathy is diagnosed only in situation when central nervous system is not infected. The systemic inflammatory response carries on outside the brain which is hidden behind the blood-brain barrier (BBB). The BBB is a highly integrated „wall” built of endothelial cells, pericytes, astrocytes and microglial cells. Thanks to BBB most of molecules may get into the brain only through controlled transcellular transport - that also includes inflammatory mediators. BBB also limits migration of peripheral cells of the immune system to the CNS. [21] Therefore, increase in the permeability of BBB may be considered as the first step towards involvement of the brain into sepsis and development of SAE. Indeed, it has been found in post-mortem study that proteins (occludin, claudin-5, ZO-1) forming so called tight junctions between endothelial cells within BBB were practically absent in brains of septic patients. It means that during sepsis permeability of the BBB is significantly increased [22]. That allows influx of proinflammatory cytokines into the central nervous system which is followed by leukocyte infiltration - the systemic inflammatory response invades the brain.

8. –       Expand on the interaction between neuroinflammation and microglial cells in the context of SAE. Elaborate on the activation of microglial cells by pro-inflammatory cytokines and their role in perpetuating the inflammatory response. Additionally, discuss the relevance of microglia activation to the development of delirium and other cognitive deficits.

Thank you for this suggestion. The above-mentioned issues are discussed in section 2.1 of the manuscript:

The next step is activation of localized within CNS immunocompetent cells - microglia [23]. It has been shown in humans that activation of microglial cells is related to development of delirium [24]. Microglial cells activated by pro-inflammatory cytokines entering CNS through pathologically permeable BBB presents a pro-inflammatory phenotype [25] and start to produce their own pro-inflammatory cytokines - mainly tumor necrosis factor alpha (TNF alfa), interleukin -1 beta (Il-1b) and interleukin-6. Presence of those cytokines along with release of reactive oxygen species, nitric oxide and glutamate further stimulate the neuroinflammatory closing a vicious circle of neuroinflammation leading to progressing damage of neural cells and to cell death through mechanism called pyroptosis [26,27]. That injury of neural tissue leads to clinical symptoms of encephalopathy and delirium.

9. –       Provide a clearer connection between sepsis-related hemodynamic instability, hypercoagulability, and ischemia in the brain. Explain how the disruption of cerebral blood flow and the formation of clots contribute to the ischemic conditions observed in septic patients.

 Thank you for this note. Details on the relation between sepsis, hypercoagulability and brain ischemia are presented in section 2.1 of the manuscript:

Parallelly to neuroinflammatory process septic brain struggles with acute disturbances within the supply chain of metabolic substrates. Sepsis and septic shock are characterized by perturbances in systemic circulation, hypotonia being the most prominent of them [28]. Simultaneously it was shown that autoregulation of cerebral arteries is less efficient in septic patients [29–31].  Active inflammatory process is also a pro-thrombotic condition. Regardless of dysregulated blood flow the cerebral arteries get blocked with clots in septic patients.  Thus, another factor increasing severity of sepsis related brain injury is disseminated focal ischemia of the brain leading to cellular death or significant metabolic perturbances as oxygenation of the brain tissue is significantly reduced in sepsis [32]. Therefore, features of brain ischemia were present in all septic patient in one neuropathological study [33].  

10. –       Further highlight the effects of neuroinflammation and ischemia on neurotransmitter pathways, particularly the cholinergic and dopaminergic pathways. Discuss how the alterations in neurotransmission contribute to cognitive and consciousness disorders observed in SAE.

 Thank you for these suggestions. Those issues are addressed in section 2.1 of the manuscript:

Neuroinflammation, BBB disruption and ischemia lead to malfunction of neural cells which in turn is the cause of pathological neurotransmission. The most widely studied is the cholinergic pathway - its anti-inflammatory action is suppressed in sepsis [34]. Sepsis-related deficiency in dopaminergic transmission is also suggested by some publications [35,36]. Pro-inflammatory cytokine (Il-1B) may increase the biological effect of GABA-ergic transmission, leading to pathological somnolence or cognitive disorders [37].

11. –       Elaborate on the relationship between the metabolic challenges faced by CNS cells during sepsis and the resulting oxidative stress and mitochondrial dysfunction. Explain how the increased energetic needs and decreased oxygen and glucose supply lead to the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) and, subsequently, cellular damage and apoptosis.

Thank you for this valuable remark. The mechanism mentioned above are described in section 2.2 of the manuscript.

 In vivo models of lipopolysaccharide- evoked sepsis-associated encephalopathy show that neuroinflammation leads to mitochondrial damage in endothelial cells through pathological activation of dynamic-related protein 1 (Drp1) that was correlated with increasing mitochondrial oxidative stress and loss of mitochondrial membrane potential. Those phenomena were correlated with reduce expression of proteins responsible for formation of tight junctions between the endothelial cells (ZO-1 and occludin) and thus leading to increased permeability of BBB. The mitochondrial damage was also detected in neuronal cells with decrease in oxidative phosphorylation, increased glycolysis and reduction of the mitochondrial membrane potential and production of ATP. The final consequence of this process is death of neurons [38]. Other animal studies have also shown increased production of reactive oxygen species (ROS), reduced production of ATP and intense apoptosis [39]. Activated microglial cells also generates reactive oxygen species (ROS) and reactive nitrogen species (RNS) that finally lead to neuronal injury and death. [40]

12. –       Include references to studies or experimental data that demonstrate increased production of ROS, reduced ATP production, and intense apoptosis in animal models of SAE.

Thank you for this remark. Appropriate references are presented in section 2.2 of the manuscript. They are references numbered from [56] to [62].

13. –       Mention the potential therapeutic interventions aimed at reducing oxidative stress and scavenging reactive species, as discussed in animal models with molecular hydrogen or tetramethylpyrazine.

 Thank you. Those interventions are described in section 2.2 of the manuscript:

That is why any therapeutical intervention with potential of reducing the oxidative stress and scavenging the reactive species may be neuroprotective and limit the chronic cognitive deficits in survivors of sepsis. That was already shown in animal models with anti-oxidative therapies, e.g., with molecular hydrogen [41] or tetramethylpyrazine [39].

14. –       Part of section 3 could be moved to the introduction.

 Thank you for this suggestion. The Introduction is enriched in paragraph on melatonin.

15. –       Begin section 3, by explicitly stating the significance of melatonin in the context of sepsis and SAE. Highlight the potential benefits of melatonin as a therapeutic agent for septic patients based on its neuroprotective, antioxidant, and anti-inflammatory properties.

 Thank you for this comment. A following statement was added to the beginning of section 3 of the manuscript.:

Melatonin is frequently mentioned as potential drug in septic patients. The anti-oxidant, anti-inflammatory and neuroprotective properties of this hormone, that are described in details below, make it a very interesting potential therapeutic factor in patients with sepsis and SAE.

16. –       Connect the discussion of melatonin's main clinical roles to its neurohormone functions, particularly its impact on the regulation of circadian rhythm and sleep promotion during the dark part of the day. Discuss how this rhythmic regulation can be disrupted during sepsis and SAE.

 Thank you for this comment. Following sentences were added to the manuscript:

This role is crucial in terms of sepsis and development of SAE. Sleep/wake rhythm disorders are frequently reported in septic patients and their presence negatively impacts prognosis[42]. On the other hand, strong and probably causal relation between disrupted circadian rhythm and development of delirium/encephalopathy in critically ill patients is observed [43]. Low concentration of melatonin was discovered in septic patients with disordered circadian rhythm which suggest potential therapeutical role of this neurohormone at least as a circadian rhythm keeper[44] . It must be noted that usage of melatonin (as a hypnotic drug) in Intensive Care Units increases in some countries[45]

17. –       Elaborate on melatonin's potential to reduce sympathetic drive and restore sympathetic-parasympathetic balance in septic patients, as mentioned in the section. Discuss the implications of this effect on the overall pathophysiology of sepsis and its potential role in modulating systemic inflammatory responses.

 Thank you for this comment. An additional text was added:

This ability of harmonizing both parts of autonomic nervous system may be especially important in septic patients in which disruption of sympathovagal modulation may even precede development of systemic inflammatory response [46]. It may be speculated that use of melatonin may be parallel to stimulation of vagal nerve successfully implemented in animal models of sepsis[47] .   

18. –       Include references to relevant studies that support melatonin's role in reducing vasoconstriction and blood pressure and its protective effects against obesity and diabetes mellitus.

 Thank you for this comment. Relevant studies are included and numbered: 37; 71; 72; 73.

19. –       Elaborate on the mechanisms through which melatonin exerts its anti-inflammatory potential. Discuss the impact of melatonin on specific inflammatory mediators, such as interleukins and tumor necrosis factor alpha, and its role in preventing cell death caused by pyroptosis.

 Thank you for this suggestion. The mechanisms are presented in section 4.1 of the manuscript:

Melatonin influences also the interplay between pro- and anti-inflammatory cytokines. Carillo-Vico et al found that in rat model of septic shock melatonin was decreasing the concentration of the pro-inflammatory IL-10 and TNF-alpha, increasing simultaneously the concentration of anti-inflammatory IL-12. [48] Melatonin may also be a crucial player in modulating the inflammatory response through inhibition of action if the inflammasome NLRP3 [49].

20. –       Further emphasize the importance of oxidative stress in the pathogenesis of SAE and explain how melatonin's ability to scavenge free radicals, chelate metal ions, and activate antioxidant enzymes can protect neural tissues from oxidative damage.

 Thank you for this comment. Those properties of melatonine are presented in section 3.3 of the manuscript:

Melatonin has a significant antioxidant potential. First, it can scavenge some of the free radicals directly. It has been shown that melatonin can react with hydroxyl (OH) radical [50,51]. Melatonin may also serve as a substrate of reaction leading to elimination of peroxyl radicals [52,53] as well as nitric oxide [54].

Apart from direct scavenging of free radicals, melatonin is capable of suppressing their production through chelation of metal ions (e.g. Cu, Fe ) necessary for synthesis of oxidants. It was proven that melatonin chelates metal ions - melatonin was capable to create complexes with copper, iron, cadmium or aluminum, blocking participation of those ions in oxidant-producing reactions. [55]Therefore, melatonin is capable to protect tissues from oxidative, metal-catalyzed damage. [56,57]

Moreover, melatonin may participate in repairing of oxidative molecular damage. Colares et al. have recently proven that treatment with melatonin reduces damage of DNA in cirrhotic rats [58]. Therapy with melatonin also intensifies repair of DNA in neoplasmatic cells [59]. Melatonin was shown to reduce oxidative DNA damage in neural tissue caused by ischemia or trauma [60,61].

Last but not least, melatonin activates antioxidant enzymes and influences signaling pathways involved in generation of free radicals. Gou et al described protective effect of melatonin on neural tissue in hypoxic-ischemic brain damage exerted through modulation of pathway leading to activation of glutathione peroxidase - an antioxidant enzyme. [62] Melatonin-driven activation of antioxidant enzymes is a well-established fact [63].

21. –       Whenever possible, incorporate relevant clinical studies that support the potential therapeutic use of melatonin in sepsis and SAE.

 Thank you for this comment. Data on clinical trials are presented in section 4.1 of the manuscript:

Promising results of studies performed on animal models and observations of relation between low concentration of melatonin and sepsis-related mortality [64]  led to first human trials. Alamili and colleagues induced an endotoxemia in healthy human volunteers who were pre-treated with melatonin or placebo. The researchers observed that melatonin (compared with placebo) led to reduction of concentration of some pro-inflammatory cytokines (e.g. interleukin Il-1beta) during the day with no statistically significant effect during the night. [65,66] Galley et al performed a study with human whole blood model of sepsis proving that melatonin reduced oxidative stress and mitochondrial failure as well as production of pro-inflammatory cytokines. [67] Aisa-Alvares with co-workers performed a small, single-centre clinical trial in septic patients, assessing impact of various antioxidant molecules. Melatonin appeared to reduce concentration of procalcitonin and lipid per oxidation. [68] Melatonin was also shown to reduce the need for mechanical ventilation and for use of vasopressor in another small (n=40 patients), single-center clinical trial. [69] Mansilla-Roselló et al. has published very recently results of a clinical study comparing 5-day therapy of septic patients with iv infusion of 50 melatonin versus placebo. Author observed that patients treated with melatonin had significantly shorter hospital stay, lower values of SOFA score, lower concentration of procalcitonin and more intense antioxidant activity. [70] Clinical usefulness of antioxidant properties of melatonin was also shown in recent clinical study focusing on septic patients, which compared effectiveness of 50 mg of orally given melatonin with other antioxidants. It was shown that melatonin reduced SOFA score and lipid peroxidation while increasing total antioxidant capacity[71].

22. –       While section 5 mentions some mechanisms through which melatonin exerts its antioxidant effects, such as increasing antioxidant enzyme activity and chelating metal ions, it would be beneficial to expand on these mechanisms and provide more detailed explanations.

Thank you for this comment. Section 5 is a conclusive section summing up the presented facts. The mechanisms are described in earlier sections.

23. –       The section briefly mentions an interesting study that demonstrated different neurobehavioral effects depending on the timing of melatonin treatment in sepsis-related brain injury in a mouse model. It would be helpful to discuss the potential implications of this finding in the clinical context and how the timing of melatonin administration may impact its neuroprotective effects in human patients with SAE.

 Thank you for this valuable note. A clinical comment on that finding was added to section 4.2:

The potential (though speculative) explanation of this finding maybe that melatonin given immediately protects integrity of the BBB and thus prevents development of further neuroinflammatory process. Due to that sepsis-related brain injury is diminished and that decrease mortality with no visible neurobehavioral effect. Meanwhile melatonin given later may only have a neuroprotective effect in already inflamed brain and that explains the neurobehavioral benefit. This observation is very important in human and clinical context as it suggest that therapy with melatonin may be somehow beneficial regardless of moment it is started.   

24. –       While the section highlights some promising results from small clinical trials, it would be beneficial to provide more details about these studies, such as the number of participants, study design, and outcomes assessed. Additionally, discussing the limitations of these trials and the need for larger multi-center studies with randomization would add depth to the discussion.

 Thank you for this comment. A table presenting data of the clinical trials was added as well as a commentary on limitations of the trials. :

Details of all the above-mentioned clinical studies are presented in Table below. Although the results are very promising the limitations of the studies must be noted. All of them were single-center study with limited number of patients included. That does not allow generalization of the conclusions.  Multi-center studies with randomization of large group of patients are required.

25. –       Given the diverse effects of melatonin observed in various studies, it would be valuable to discuss the potential dose-response relationships and optimal dosing regimens for melatonin in the context of sepsis and SAE. Addressing the safety profile of melatonin at different doses is also important, particularly when considering its potential use in critically ill patients.

 Thank you for this comment. A following paragraph was added:

Human studies on melatonin in sepsis were performed with variety of dosages, ranging from 50 mg p.o. daily to 100 mg i.v. daily. Establishing the proper dosage of a neurohormone which is released in a specific circadian rhythm is challenging task. Galley et al. undertook it and performed a clinical study aiming at assessing clinical safety, tolerance and optimal dosing of melatonin. There were 10 patients with sepsis resulting from community-acquired pneumonia participating in the study. Five of them received 50 mg of melatonin p.o. daily and the other 5 was given 20 mg of the neurohormone daily. Clinical observation led to conclusion that therapy with melatonin did not led to any adverse event and was well tolerated. Twenty mg daily was found to be the optimal dose from pharmacological point of view.

26. –       Discuss the possible interactions between melatonin and other standard treatments for sepsis and critically ill patients. Since many septic patients receive multiple interventions, understanding the compatibility and potential synergy or antagonism with melatonin is crucial.

 Thank you for this comment. A suitable paragraph was added:

Implementation of melatonin into clinical practice must be performed cautiously as little is known about its potential interactions with other drugs, especially the ones used in critically ill subjects (e.g. sedatives, vasopressors, antibiotics or steroids). It is a consequence of sporadic use of melatonin in clinica practice. In the other hand, authors of clinical trials assessing melatonin in sepsis did not report any significant interactions with routine therapy.

27. –       Conclude the section by summarizing the overall potential of melatonin as a therapeutic agent for sepsis and SAE. Emphasize the significance of preclinical evidence and initial clinical trial results in guiding future research and potential application in clinical practice.

 Thank you for this comment. A following paragraph was added:

The facts collected in the previous paragraphs suggest that melatonin may be potent therapeutical agent in patient in sepsis and with SAE. It is strongly suggested by results of pre-clinical studies on animal models which found initial clinical confirmation in human studies performed so far. The collected data allowa us to plan more efficient protocols of clinical trials with melatonin in the upcoming future.

–       To enhance the review and provide a more comprehensive understanding of the topic, the authors could consider including figures and tables. Here are some suggestions for potential figures and tables that could be included in the review:

Thank you for this suggestion. Following Figures and Tables were added

o   Figure: "Pathomechanisms of Sepsis-Associated Encephalopathy (SAE)"

o   Table: "Clinical Studies on Melatonin in Sepsis and Sepsis-Associated Encephalopathy"

o   Figure: "Mechanisms of Melatonin's Neuroprotective Effects in Sepsis"

Round 2

Reviewer 1 Report

The authors were responsive to the critique and adequately revised the manuscript. There are some typos, specifically in references section, likely due to formatting, but these can be corrected at the proofs stage

Minor proof reading is suggested.

Reviewer 2 Report

Thank you for providing a detailed response to my comments and suggestions. After reviewing the revisions and the authors' explanations, it appears that the authors have made a genuine effort to address the concerns raised and have significantly improved the quality of their paper.

With the comprehensive changes and additions made to the manuscript, I believe it now offers a more in-depth and well-rounded perspective on the topic. I appreciate the authors' dedication to enhancing the content and ensuring that the paper is both informative and accurate.

Given the improvements and the thoroughness of the revisions, I believe the review manuscript can now be considered for publication.