Investigating the Neurological Impact of Post-COVID-19 Condition: Immune Activation and Pathological Insights

The COVID-19 pandemic, caused by the SARS-CoV-2 virus, has affected millions of people worldwide. While many people experience symptoms such as fever, cough, and difficulty breathing during the initial phase of the infection, known as acute COVID-19, some individuals also report neurological symptoms. These symptoms are related to the brain and nervous system and can include headaches, dizziness, loss of taste or smell, and difficulty thinking clearly.

Interestingly, even after recovering from the initial COVID-19 infection, some individuals continue to experience a range of symptoms that persist for weeks or even months. This condition is referred to as Post-COVID Condition (PCC), Post-Acute COVID Syndrome (PACS), or Neuro-Long-COVID-19 when the symptoms specifically affect the brain and nervous system. These long-term symptoms can significantly impact a person’s daily life and overall well-being.

Despite the growing number of cases, the exact reasons why some people experience these prolonged symptoms remain unclear. Scientists and doctors are working hard to understand the underlying causes and mechanisms of these post-COVID conditions. Recent research has shown that changes in the immune system, the body’s defense mechanism against infections, might play a significant role. These changes include alterations in immune cells, increased activity of the complement system (a part of the immune system that enhances the ability to clear microbes and damaged cells), and issues with blood clotting and coagulation. Additionally, an exaggerated immune response directed against the virus may also be involved.

The central nervous system (CNS), which includes the brain and spinal cord, is crucial for controlling most functions of the body and mind. Studies involving advanced imaging techniques, such as repeated magnetic resonance imaging (MRI) of the brain, have revealed significant changes in the brains of individuals previously infected with SARS-CoV-2. These changes include a greater reduction in the thickness of gray matter (the part of the brain involved in muscle control, sensory perception, memory, and emotions) and a decrease in overall brain size. Such changes are thought to be a result of inflammation in the brain caused by immune cells during the acute phase of COVID-19.

Other studies have shown that people with late-stage COVID-19 may have altered glucose metabolism (the process by which the body breaks down sugar to produce energy) and changes in the type I interferon response (a part of the immune system that helps defend against viral infections). Despite these findings, much remains unknown about the specific cellular and molecular processes that are affected in the brains of individuals with long-term post-COVID conditions.

To gain a better understanding of these processes, researchers have collected brain samples from individuals who recovered from COVID-19 but later died from unrelated causes. These samples were obtained from multiple medical centers and thoroughly analyzed to determine if there were any lingering effects of the virus on the brain. Additionally, cerebrospinal fluid (CSF), a clear fluid found in the brain and spinal cord that protects and nourishes these areas, was collected from living patients with Neuro-Long-COVID-19 for further analysis.

By examining these samples, scientists aim to identify any signs of persistent immune activation or inflammation in the brain. This includes studying specific immune cells, such as microglia, which are the brain’s primary immune cells. Microglia play a crucial role in protecting the brain from infections and maintaining normal brain function. However, in response to brain damage or infections, microglia can form clusters known as microglia nodules, which indicate an ongoing immune response.

Understanding the distribution and activity of these immune cells in the brain can provide valuable insights into the potential long-term effects of COVID-19 on the nervous system. Researchers use various advanced techniques, including immunohistochemistry (a lab technique to detect specific proteins in tissue samples) and imaging mass cytometry (a high-resolution imaging technique that uses metal-tagged antibodies to visualize different cell types), to study these brain samples in great detail.

In summary, while much progress has been made in understanding the immediate effects of COVID-19 on the brain and nervous system, more research is needed to fully grasp the long-term impacts and underlying mechanisms of Post-COVID Condition. By studying brain and cerebrospinal fluid samples from individuals affected by Neuro-Long-COVID-19, scientists hope to uncover important information that can lead to better treatments and support for those experiencing these prolonged symptoms.

The study…..

Acute COVID-19 is frequently accompanied by neurological symptoms, and a subset of individuals experiences persistent neurological post-COVID-19 manifestations, referred to as Post-COVID condition (PCC), Post-Acute COVID Syndrome (PACS), or Neuro-Long-COVID-19. However, the underlying pathogenesis for this phenomenon is largely unclear. Recent studies looking into the blood composition of patients with Long-COVID detected altered composition of immune cells, increased peripheral complement activation, dysregulation of markers related to blood clotting and coagulation, or exaggerated humoral response directed against SARS-CoV-2 with unclear relevance for the central nervous system (CNS). Regarding the central nervous system, some studies encompassed large clinical datasets or advanced imaging techniques.

For example, repeated magnetic resonance brain imaging revealed significant longitudinal effects in individuals previously infected with SARS-CoV-2, including a greater reduction in gray matter thickness and global brain size that may be a consequence of T-cell-mediated neuroinflammation previously described in acutely affected COVID-19 brains. Studies involving patients or patient samples of subacute, late-stage COVID-19 indicate an altered glucose metabolism and type I interferon response. Despite these proposed mechanisms, the cellular and molecular processes that are affected in the brains of long-term post-COVID-19 patients are largely missing.

Certainly! Below is a detailed table outline that explains the most important concepts reported in the document in simple and understandable terms for those without a medical background:

Formaldehyde-fixed paraffin-embedded (FFPE) central nervous system samples were obtained from autopsies at the University Medical Center in Freiburg, the Institute of Pathology, University of Basel, Institute of Legal Medicine at the University Medical Center Hamburg-Eppendorf and the Institute of Neuropathology at the University Medical Center Hamburg-Eppendorf. Post-COVID-19 patients had a COVID-19 unrelated cause of death, which was assessed by thorough assessment of the medical records, interviews with relatives, and meticulous autopsy findings. Patients had reported full recovery from COVID-19 and showed no signs of COVID-related symptoms before their passing. In particular, no long-term neurological deficits after exposure to SARS-CoV-2 have been reported. Patients were typically tested negative for SARS-CoV-2 after their COVID-19 infection during their lifetime. Additional testing for SARS-CoV-2 was performed for six patients during autopsy, and the test results were all negative. Patient characteristics are provided in Supplementary Table 1. Lumbar punctures of Neuro-Long-COVID-19 patients were performed at the Department of Neurology and Neuroscience at the University Medical Center Freiburg (Supplementary Table 2). Cerebrospinal fluid from patients with idiopathic intracranial hypertension served as controls. The analyses were performed with the approval of the institutional review boards (Ethics Committee of the University of Freiburg: 211/20, 10008/09; Ethics Committee of the Hamburg Chamber of Physicians: 2020-10353-BO-ff, PV7311; Ethics Committee of Northwestern and Central Switzerland: 2020-00629). The study was performed in agreement with the principles expressed in the Declaration of Helsinki and its amendments.

The immunohistochemical reactions (chromogenic immunohistochemistry) for CD8a, CD4, CD20, SARS-CoV spike glycoprotein, APP, and Alpha-Syn on 3 µm-thick FFPE sections were performed using the EnVision Flex Kit (DAKO, Agilent, cat. # K8000) and a DAKO Autostainer Link 48 system. EnVision low pH tissue pre-treatment was used for CD8a (Dako, cat. # IR623, RTU), APP (Millipore, cat. # MAB348, 1:2000), SARS-CoV spike glycoprotein (abcam, cat. # ab272420, 1:100), and Alpha-Syn (BioSB, cat. # BSB3291, RTU). EnVision high pH tissue pre-treatment was used for CD20 (DAKO, cat. # IR604, RTU) and CD4 (DAKO, cat. # IR649, RTU). EnVision Flex Mouse Linker (DAKO, cat. # K800221-2) was applied for CD4 immunohistochemistry.

Chromogenic Iba1 immunohistochemistry of 3 µm-thick FFPE sections was performed using the labeled streptavidin–biotin (LSAB) method as previously described. Slides were deparaffinized in Xylene and cooked in EnVision low pH antigen retrieval buffer for 40 min. Endogenous tissue peroxidase was quenched in 3% hydrogen peroxidase (Carl Roth, cat. # 8070.1) for 10 min. Samples were then blocked with 10% normal goat serum (SouthernBiotech, cat. # 0060-01), 1% Triton X-100 (Sigma, cat. # T8787-100ML) in TRIS buffer (EnVision Flex Wash Buffer, DAKO, cat. # K8000) for 1 h. The incubation with Iba1 primary antibody (abcam, cat. # 178846, 1:1000 in the blocking solution) was performed at room temperature overnight. After three washes with TRIS buffer, the slides were incubated with goat anti-rabbit secondary antibody (SouthernBiotech, cat. # 4050-08, 1:300 in the blocking solution) for 45 min. Slides were then washed three times. Streptavidin-HRP (SouthernBiotech, cat. # 7105-05) was diluted 1:1000 in TRIS buffer and added for 45 min. Specimens were then rinsed in TRIS buffer three times and incubated with DAB solution (1 drop EnVision Flex DAB Chromogen per 1 ml EnVision Flex Substrate Buffer).

All sections were counterstained with Gill’s Hematoxylin solution (Sigma, cat. 1051750500) and Vitro-Clud (R. Langenbrinck GmbH, cat. # 04-0001) was used as mounting medium. Imaging was performed on a Zeiss Axioscan 7 system equipped with a 20× objective.

Imaging Mass Cytometry was conducted as reported previously. In short, antibodies were conjugated to lanthanide metals using the Maxpar X8 antibody labeling kit. 4 µm thick formaldehyde-fixed paraffin-embedded (FFPE) sections were deparaffinized and cooked in EnVision FLEX Target Retrieval Solution High pH (DAKO, cat. # K8000) for 40 min. Sections were blocked using SuperBlock Blocking Buffer (ThermoFisher, cat. # 37581). The slides were then incubated with the antibody mix (Supplementary Table 3) in 0.5% BSA, 1% Triton-X-100 in TRIS at room temperature overnight. Iridium Cell-ID intercalator (Fluidigm, cat. # 201192A) was used to visualize DNA and applied for 30 min. The measurement was conducted using the Hyperion Imaging Mass Cytometry system (Fluidigm). For the IMC measurements, regions of interest (ROIs) were determined by Iba1 immunohistochemistry on a consecutive section. Areas with most prominent microglia nodules (if present) were selected.

Image segmentation was performed based on the IMCSegmentationPipeline. Image visualization was performed using MCD Viewer v1.0.560.6. An expression threshold of > = 2 and area > = 20 was used for the gating of Iba1 myeloid cells. For PhenoGraph clustering, the expression values of CD11c, CD162, CD163, CD204, CD206, CD64, CD68, FCERI, HLA-DR, HLA-DRA, HLA-DRB1, Iba1, INPP5D, Ki67, MX1, P2RY12, S100A9, SCAMP2, SLC2A5, TMEM119, and TYROBP were used. For a min–max scaling was performed for normalization. Expression values were normalized to Iba1 signal intensity. Compartments (parenchyma vs. nodule) were based on the microglia nodule index (defined as the coverage of Iba1 signal in a 15 µm radius) and a threshold >  = 0.5.Cerebrospinal fluid pre-treatment
Cerebrospinal fluid samples were centrifuged at 2000g for 10 min at + 4 °C. The cell-free supernatant was snap-frozen and kept at − 80 °C.

Enzyme-linked immunosorbent assays (ELISA) were performed on cerebrospinal fluid (CSF) samples. After thawing CSF samples on ice, enzyme-linked immunosorbent assays (YKL-40: Invitrogen, cat. # EHCHI3L1; TREM2: Invitrogen, cat. # EH464RB; CD14: Invitrogen, cat. # EHCD14; NF-light: Tecan, cat. # UD51001) were performed according to the manufacturer’s instructions. A Spark multimode microplate reader (Tecan) was used for measurements.

Targeted metabolomics analysis was conducted following established procedures outlined in the previous studies and involved the extraction of samples using a precooled extraction solution (80:20 methanol LC–MS grade: Milli-Q water). Quantification of targeted metabolites through LC–MS was performed on an Agilent 1290 Infinity II UHPLC coupled with an Agilent 6495 QQQ-MS operating in MRM mode. MRM settings were optimized individually for all compounds using pure standards

. Isotopically labeled yeast extract (ISOtopic Solutions, Vienna, Austria) was spiked into all samples as internal standard for identification of correct peaks and compensation of matrix effects. LC separation was performed as published previously. Briefly, a Waters Atlantis Premier BEH ZHILIC column (100 × 2.1 mm, 1.7 µm particles) was used, buffer A was 20 mM ammonium carbonate and 5 µM medronic acid in milliQ H2O and buffer B was 90:10 acetonitrile:buffer A and the solvent gradient was from 95 to 55% buffer B over 18 min. Flow rate was 150 µL/min, column temperature was 40 °C, autosampler temperature was 5 °C, and injection volume was 2 µL. Data processing was performed using the R package automRm. Spearman correlations were performed as reported previously.

Statistical analyses and visualizations were performed using GraphPad Prism 9.5.1. The statistical tests are mentioned in the figure legends. P values are stated in the figures.

In a multicenter study, brains were collected from 15 individuals that experienced a previously confirmed SARS-CoV-2 infection, recovered fully but died due to reasons unrelated to COVID-19 up to 27 months after viral infection (Supplementary Table 1). Brains from four healthy controls and 11 acute COVID-19 cases were included for comparison. First, all samples underwent a comprehensive COVID-19 centered neuropathological analysis by board-certified neuropathologists as previously described. Some samples (patients # 10, 11, and 12) were subsequently analyzed by single-cell-based immune phenotyping by cytometry-by-time-of-flight-(CyTOF)-based imaging mass cytometry (IMC) that allows detailed spatial profiling of single immune cells in the diseased CNS. Surprisingly, and in contrast to CNS specimens from acute cases, post-COVID-19 brain revealed only very few parenchymal CD8a+ T cells that were numerically almost compatible to controls. Additionally, CD4+ T cells and CD20+ B cells were examined and found no significant differences in parenchymal cell counts between controls and post-COVID-19 cases. In contrast, using high-dimensional, 40 marker-based IMC analyses of samples from the frontal cortex and the medulla, microglia cells with positive signal in the channels for the microglial markers P2RY12 and TMEM119, accompanied by signals in the channels for myeloid cell molecules CD11c, CD68, CD204, and SCAMP2, along with the induction of the MHC class II-related proteins HLA-DR, HLA-DRA, and HLA-DRB1, were identified.

A supervised machine learning approach was employed to segment the images and to extract single-cell expression data based on the intensity of each marker in the respective channel. After gating for the pan-myeloid marker ionized calcium-binding adaptor molecule 1 (Iba1), cells were clustered using the PhenoGraph algorithm. This approach identified a total of 18 clusters, as shown in a uniform manifold approximation and projection (UMAP) representing diverse myeloid cells, such as microglia (e.g., P2RY12+, TMEM119+ clusters 1, 2, 7, 8, and 11), perivascular macrophages (CD163+, CD204+, and CD206+ cluster 14), and monocytes (S100A9+ cluster 15). As depicted in mosaic plots, clusters enriched for specific stages following SARS-CoV-2 infection, such as cluster 11 that was enhanced in post-COVID-19, and cluster 7 that was prominent in acute COVID-19 patients, were identified.

Marker expression heat maps depict the varying marker expression profiles leading to distinct clusters. For example, microglia cells in cluster 1 exhibited strong positivity for the lysosomal activation marker CD68 and were mostly observed in samples from acute and post-COVID-19 patients, with minimal representation in control samples. Cluster 11, characterized by elevated integrin alpha x (CD11c) expression indicative of an activated microglial cell state, was mostly found in post-COVID-19 patients. Cluster 9 was also found in post-COVID-19 patients and was characterized by a moderate CD11c expression. Cluster 16 cells were primarily seen in acute cases, expressed the lysosomal activation marker CD68, and the MHC class-II-related molecules CD74, HLA-DRA, and HLA-DRB1. Myeloid cluster 14 that was shared across all patient groups expressed the markers CD206 and scavenger receptor cysteine-rich type 1 protein M130 (CD163) indicative of perivascular macrophages. Notably, Iba1+P2RY12+TMEM119+ microglia were found to be assembled in characteristic clusters, known as microglia nodules, that were defined as ≥ 50% coverage of the Iba1 signal in a 15 µm radius. Microglia nodules are usually considered morphological hallmarks of chronic neuropathological processes, such as viral encephalopathies, axonal damages, or neurodegenerative changes.

The spatial distribution of marker expression within single brain sections was explored. To achieve this, the dataset was compartmentalized into regions containing microglia nodules and non-microglia-nodule areas, utilizing a previously developed microglia nodule index. The normalized marker expression of cells within nodules was compared to those outside nodules in the medulla of post-COVID-19 patients. No statistically significant differences were observed for expression of P2RY12 or TMEM119 on microglia localized inside or outside the nodules allowing to unequivocally determine their cellular identity. Importantly, spatial analysis revealed a higher expression of CD11c, CD68, CD204, HLA-DR, HLA-DRA, HLA-DRB1, and SCAMP2 on P2RY12+TMEM119+ microglia within nodules. No differences were observed for CD74 and CD206. Collectively, these data identify the innate rather than the adaptive immune system as main functional player in post-COVID-19 brains and highlight the microglia nodule compartment as the key site of local tissue immune responses.

After having identified microglia nodules as the main immune feature of post-COVID-19 brains, the chronicity and the functional relevance of this phenomenon were assessed, as these structures are usually absent in healthy brain tissue. Microglia nodules were found to be widespread present among post-COVID-19 brains compared to controls. Upon quantification, the number of microglia nodules was significantly higher in post-COVID-19 patients when compared to controls, but less frequent compared to acute COVID-19 brains. SARS spike glycoprotein immunohistochemistry did not reveal positive signal in the brain parenchyma, indicating the absence of viral presence. To evaluate the consequences of chronic microgliosis, the extent of neuronal damage was assessed using immunohistochemistry for the amyloid precursor protein (APP), a surrogate marker for axonal damage.

Although a significant increase in APP deposits was evident in acute COVID-19 cases, only individual patients in the post-COVID cohort exhibited deposits without reaching statistical significance for this group. Because COVID-19 may predispose individuals to develop Parkinson’s disease later in life, the cohort was investigated for the presence of alpha-synuclein deposits. In post-COVID-19 brains, a significant increase of alpha-synuclein aggregates, a hallmark of several neuropathological conditions that show microglia nodules such as Parkinson’s disease (PD), dementia with Lewy Bodies (DLB), multiple system atrophy (MSA), and others, was not observed. In sum, obvious neuropathological correlates of neurodegeneration were absent from the investigated post-COVID-19 brains even at later stages.

To determine whether the histologically detectable persistent innate immune activation in post-COVID-19 brains is mirrored by any alterations in the cerebrospinal fluid (CSF), this fluid compartment from 31 living individuals with clinically confirmed Neuro-Long-COVID-19 and respective controls was analyzed. Neuro-Long-COVID-19 patients fulfilled the Post-COVID condition (PCC) criteria according to the WHO. A few proteins have emerged as robust markers to monitor neuroinflammation in Alzheimer’s disease (AD) or multiple sclerosis due to their reproducible relation to pathological features of the disease: soluble TREM2 (sTREM2) as a marker of microglial activation, YKL-40 as an astroglia stimulation molecule, CD14 as myeloid cell activation protein and neurofilament light chain (NF-light) as a correlate of neuronal damage. Notably, apart from the expected age-dependent increase, higher levels of these markers in the clinically affected cohort compared to controls were not observed. Given the fact that microglia are metabolically active cells that are extremely sensitive and versatile responders to minute changes of their microenvironment, high-dimensional targeted metabolomics of the CSF was performed and 67 metabolites in Neuro-Long-COVID-19 patients were analyzed. Although typical metabolites associated with microglial activation, such as tryptophan, kynurenine, or glutamine, were clearly detectable, no major differences in the analyzed metabolite levels between Neuro-Long-COVID-19 samples and controls were found using this highly sensitive method.

By combining high-dimensional histological CyTOF analyses with machine learning methods, the complexity of the brain immune landscape after systemic COVID-19 infections at the single-cell level was studied. In this study, autopsy cases from COVID-19 survivors at different time points after SARS-CoV-2 challenge were examined. Long-term neurological symptoms had not been reported in this cohort. Since an autopsy cohort

of Neuro-Long-COVID-19 patients was not available at this time, cerebrospinal fluid from living individuals with clinically confirmed Neuro-Long-COVID-19 was analyzed as the closest approximation. Patients in the autopsy cohort had reported full recovery from COVID-19. They were typically tested negative for SARS-CoV-2 after their COVID-19 infection during their lifetime. Based on neuropathological analyses of the autopsy tissue, typical neuropathological hallmarks of neuronal degeneration were not detectable in this patient cohort. Nevertheless, a clear shift from the T-cell linked adaptive immune activation during acute COVID-19 expositions toward a pronounced local innate immune stimulation in the CNS following virus resolution in this cohort was observed. Data further suggest a pervasive local pro-inflammatory milieu upon transient SARS-CoV-2 challenge mirrored by the presence and perseverance of microglia nodules.

In a parallel approach, a cohort of living patients with clinically confirmed Neuro-Long-COVID-19 according to the WHO’s Post-COVID Condition (PCC) criteria was analyzed. Using cerebrospinal fluid (CSF) samples from these patients, ELISA and targeted metabolomics were employed to investigate potential disease-specific patterns. However, a distinct disease-specific pattern in these analyses could not be detected.

A dysregulation of the innate immune system in the autopsy cohort of COVID-19 survivors who did not present with neurological symptoms during their lifetime was observed. This dysregulation might also be apparent in COVID-19 survivor with long-term neurological symptoms (Neuro-Long-COVID-19), potentially playing a role in the disease pathogenesis. However, establishing a definitive link remains challenging due to the absence of a dedicated autopsy cohort of patients with confirmed Neuro-Long-COVID-19 at this time. Further studies are required to explore this aspect in the future.

resource: https://link.springer.com/article/10.1007/s00401-024-02770-6

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