Unclosing Clinical Criteria and the Role of Cytokines in the Pathogenesis of Persistent Post-COVID-19 Headaches: A Pilot Case-Control Study from Egypt

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors present a small case-control-study on the role of cytokines in post covid19 headache. 

There are a few comments:

- how did they match the HC, as to the patients with or without headaches

- There is quite a difference in age of 4 years and also sex distribution by 15% (although not significant?), would this explain part of your findings? please discuss

- Somehow I do not fully understand why the cytokine levels in the headache group are as high (although frequency was around 12). When were the samples taken in respect to headache days. Why are cytokine levels not elevated in the other post covid patients? please discuss with current literature.

Author Response

Ø Comment (1):

(How did they match the HC, as to the patients with or without headaches?)

Answer and correction:

Healthy controls were matched according to age and sex.  

Correction : We added this clarification in the methods section (lines 64).

Ø Comment (2):

There is quite a difference in age of 4 years and also sex distribution by 15% (although not significant?), would this explain part of your findings? please discuss?)

Answer and correction:

The higher incidence of post-COVID headaches in younger patients compared to those without headaches has been observed in various studies. This difference is thought to be due to a combination of biological, immune, and psychological factors that vary with age

1. Immune System Response in Younger Patients

• Stronger Immune Response: Younger individuals tend to have a more robust immune response to infections, which can result in stronger inflammatory reactions during and after the infection.
• Higher Cytokine Levels: Some studies suggest that younger patients may have higher levels of inflammatory cytokines (e.g., IL-6, TNF-α) in response to COVID-19, which could trigger or exacerbate neuroinflammation, leading to headaches.

2. Brain Sensitization:

• CNS Sensitization: The younger brain may be more sensitive to neuroinflammation, which is often seen in post-viral syndromes like post-COVID syndrome. Neuroinflammation can alter pain pathways and increase the likelihood of developing headaches.
• Higher Rates of Migraine in Younger Populations: Younger individuals, especially women, tend to have higher baseline rates of migraine  and COVID-19 infection may act as a trigger for these underlying conditions.

3. Hormonal Factors

• In younger women, hormonal fluctuations—such as those related to the menstrual cycle or the use of oral contraceptives—can make them more susceptible to headaches.

4. Psychological and Social Stress in Younger Patients

• Increased Stress: Younger patients, particularly those who are students or early in their careers, may experience heightened psychological stress.
• Increased Anxiety and Depression: Psychological symptoms such as anxiety and depression, which are prevalent in younger populations post-COVID, may also contribute to the development of headaches.

5. Long COVID and Younger Populations

• Long COVID Syndrome: Younger patients have been observed to experience a higher incidence of long COVID. Headaches are a common symptom of long COVID, and younger individuals seem to have a higher prevalence of this condition.

6. Increased Awareness and Reporting in Younger Populations

Younger individuals may be more aware of post-viral symptoms and more likely to report headaches to healthcare providers.

Also, the phenomenon of female predominance in post-COVID headache group has been observed in various studies and reports. Several factors, both biological and socio-environmental, are thought to contribute to this gender difference.

1. Hormonal Differences

• Estrogen's Role: Estrogen known to modulate the nociceptive system, which processes pain signals, and make women more susceptible to headaches. The fluctuation of estrogen, especially during menstrual cycles or postmenopausal or in women using hormonal contraceptives and in post-viral syndromes like COVID-19.

2. Autoimmune and Inflammatory Responses

• Immune System Differences: Women generally have stronger immune responses compared to men, which increase susceptibility to autoimmune conditions, including post-COVID headache.

3. Genetic Factors

• Genetic differences between men and women may influence the way the body responds to infections like COVID-19. Certain genes that regulate the immune system or pain pathways may be more active in women,.
• Psychological Factors

• Stress and Anxiety: Women are often more likely to report higher levels of stress, anxiety, and depression than men, which can amplify the severity and duration of headaches.
• 5. Pre-existing Migraines

• Many women are already predisposed to migraines, a common form of headache. COVID-19 infection can act as a trigger for individuals who already have a history of migraines,
• Sex Differences in the Nervous System

• Women generally have a higher pain sensitivity and may process headache-related signals more intensely than men. Post-COVID neurological symptoms might exacerbate these differences.

Corrections :  these differences although insignificant was highlighted in discussion section with related references  (lines 156-163) and references (6-8)

• Comment (3):

(Somehow, I do not fully understand why the cytokine levels in the headache group are as high (although frequency was around 12). When were the samples taken in respect to headache days. Why are cytokine levels not elevated in the other post covid patients? please discuss with current literature.

Answer and correction:

Cytokine level sample was taken during active headache days of the post covid headache group (was added to methodology section line number 76-77)

and the elevation of Interleukin-6 (IL-6) levels in post-COVID headache patients compared to those without headaches is an area of ongoing research, with several hypotheses based on the role of IL-6 in inflammation and immune responses. Here’s an explanation of why this difference is observed, with references:

1. Persistent elevation of IL-6 and its role post covid headache:

• IL-6 is a pro-inflammatory cytokine that plays in the acute-phase response to infections. During viral infections like COVID-19. In patients who develop post-COVID symptoms, including headaches, IL-6 levels remain elevated even after the acute infection phase, indicating ongoing inflammation.
• Headaches are thought to be a manifestation of neuroinflammation, which can be driven by cytokines like IL-6.

2. Cytokine Storm and Persistent chronic neuroinflammation in Post-COVID Syndrome:

• During the acute phase of COVID-19, some patients experience a cytokine storm, an exaggerated immune response involving high levels of inflammatory cytokines like IL-6.
• This chronic inflammatory state may lead to neurological symptoms, such as headaches, by affecting pain processing mechanisms in the brain and central sensitisation including hypothalamic affection.

3. Persistent blood brain barrier impairment:

• In post-COVID headache patients, it’s believed that IL-6 acts on the brain and interacts with the blood-brain barrier, potentially increasing its permeability, contributing to headache development.

4. Neuroimmune Interaction in Post-COVID Syndrome and immune system dysregulation

• Research suggests that IL-6 is involved in a bidirectional relationship between the immune system and the nervous system. It may not only reflect systemic inflammation but also exacerbate central nervous system (CNS) autoimmunity , contributing to persistent headache symptoms in post-COVID patients.
• We added a new paragraph in discussion section about this comment  (lines 198-201) and references (20-21)

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

This is a good study on an important topic. However, I have some suggestions, in my opinion, may improve this manuscript.

1. Please be more specific about the participant's screen. Did eligible participants have a history of vaccination? Were participants screened for previous virus infection? Individuals who had previously received a vaccine should be excluded. 
2. Demographic characteristics of the study population, the ethnicity, race and age range should be added.  

3. The percent match is way too high, 40%. 

Author Response

Ø Comment (1):

Please be more specific about the participant's screen. Did eligible participants have a history of vaccination? Were participants screened for previous virus infection? Individuals who had previously received a vaccine should be excluded.

Answer and correction:

We excluded all individuals with any recent vaccination and positive history of any recent viral infection including COVID 19 at least 1 month before the recruitment and it was confirmed by negative routine complete blood picture but no specific virology test was done

We highlighted this in methodology section (line 65-66)

Ø Comment (2):

Demographic characteristics of the study population, the ethnicity, race and age range should be added. 

Answer and correction:

We empathised all these details at methodology section (line 55-58)

Ø Comment (3):

The percent match is way too high, 40%.

Answer and correction:

We revised the manuscript writing again accurately to decrease this percent match .

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

comments have been adressed