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. 2022 Aug 10;11(16):2480.
doi: 10.3390/cells11162480.

Neuro-Axonal Damage and Alteration of Blood-Brain Barrier Integrity in COVID-19 Patients

Maria Antonella Zingaropoli  1 , Marco Iannetta  2 , Lorenzo Piermatteo  3 , Patrizia Pasculli  1 , Tiziana Latronico  4 , Laura Mazzuti  5   6 , Laura Campogiani  2 , Leonardo Duca  3 , Giampiero Ferraguti  7 , Manuela De Michele  8 , Gioacchino Galardo  9 , Francesco Pugliese  10 , Guido Antonelli  5 , Massimo Andreoni  2 , Loredana Sarmati  2 , Miriam Lichtner  1   11 , Ombretta Turriziani  5 , Francesca Ceccherini-Silberstein  3 , Grazia Maria Liuzzi  4 , Claudio Maria Mastroianni  1 , Maria Rosa Ciardi  1
Affiliations

Neuro-Axonal Damage and Alteration of Blood-Brain Barrier Integrity in COVID-19 Patients

Maria Antonella Zingaropoli et al. Cells. .

Abstract

Neurofilament light chain (NfL) is a specific biomarker of neuro-axonal damage. Matrix metalloproteinases (MMPs) are zinc-dependent enzymes involved in blood-brain barrier (BBB) integrity. We explored neuro-axonal damage, alteration of BBB integrity and SARS-CoV-2 RNA presence in COVID-19 patients with severe neurological symptoms (neuro-COVID) as well as neuro-axonal damage in COVID-19 patients without severe neurological symptoms according to disease severity and after recovery, comparing the obtained findings with healthy donors (HD). Overall, COVID-19 patients (n = 55) showed higher plasma NfL levels compared to HD (n = 31) (p < 0.0001), especially those who developed ARDS (n = 28) (p = 0.0005). After recovery, plasma NfL levels were still higher in ARDS patients compared to HD (p = 0.0037). In neuro-COVID patients (n = 12), higher CSF and plasma NfL, and CSF MMP-2 levels in ARDS than non-ARDS group were observed (p = 0.0357, p = 0.0346 and p = 0.0303, respectively). SARS-CoV-2 RNA was detected in four CSF and two plasma samples. SARS-CoV-2 RNA detection was not associated to increased CSF NfL and MMP levels. During COVID-19, ARDS could be associated to CNS damage and alteration of BBB integrity in the absence of SARS-CoV-2 RNA detection in CSF or blood. CNS damage was still detectable after discharge in blood of COVID-19 patients who developed ARDS during hospitalization.

Keywords: MMPs; NfL; ddPCR; long-COVID; matrix metalloproteinases; neuro-COVID; neurofilament light chain; zymography.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Study design. (A) For COVID-19 patients, whole blood samples were collected in heparin tubes during routine clinical testing at two timepoints: on hospital admission (baseline) and after three months from discharge (Tpost). For the neuro-COVID group, CSF samples and heparin and EDTA whole blood samples were collected. RT-PCR and ddPCR were used for the detection and quantification of SARS-CoV-2 RNA in collected CSF and whole blood samples. For healthy donors (HD) heparin whole blood samples were collected. (B) According to clinical outcome, COVID-19 patients were stratified into ARDS and non-ARDS groups and the differences in plasma NfL levels were evaluated. According to disease severity, both neuro-COVID group and COVID groups were stratified into ARDS and non-ARDS groups and the differences in CSF NfL levels, plasma NfL levels, and CSF MMP-2 and MMP-9 levels were assessed. According to the detection of SARS-CoV-2 RNA in CSF and plasma samples, neuro-COVID group was stratified into positive and negative groups and the differences in CSF NfL levels as well as MMP-2 and MMP-9 levels were evaluated. Neuro-COVID group: COVID-19 patients with severe neurological symptoms; COVID group: COVID-19 patients without severe neurological symptoms; NfL: neurofilament light chain; MMP-2: matrix metalloprotease-2; MMP-9: matrix metalloprotease-9; SARS-CoV-2: severe acute syndrome coronavirus 2; RT-PCR: reverse transcription-polymerase chain reaction; ddPCR: droplet digital polymerase chain reaction; CSF: cerebrospinal fluid; HD: healthy donors; EDTA: ethylenediaminetetraacetic acid; ARDS: acute respiratory distress syndrome.
Figure 2
Figure 2
Evaluation of plasma NfL levels in COVID-19 patients and healthy donors. (A) Evaluation of plasma NfL levels in COVID-19 patients and HD. (B) Evaluation of plasma NfL levels in patients with ARDS (ARDS group), patients without ARDS (non-ARDS group) and HD. (C) Longitudinal evaluation of plasma NfL levels in COVID-19 patients. (D) Longitudinal evaluation of plasma NfL levels in COVID-19 patients who developed ARDS during hospitalization. (E) Longitudinal evaluation of plasma NfL levels in COVID-19 patients who did not developed ARDS during hospitalization. (F) Evaluation of plasma NfL levels in COVID-19 patients with severe neurological symptoms (neuro-COVID group), COVID-19 patients without severe neurological symptoms (COVID group) and HD. (G) Evaluation of plasma NfL levels in neuro-COVID and COVID groups stratified according to ARDS. Horizontal lines represent medians. COVID-19: coronavirus disease 2019; neuro-COVID group: COVID-19 patients with severe neurological symptoms; COVID group: COVID-19 patients without severe neurological symptoms; NfL: neurofilament light chain; HD: healthy donors; ARDS: acute respiratory distress syndrome; CSF: cerebrospinal fluid. ****: p < 0.0001; ***: 0.0001 < p < 0.001; **: 0.001 < p < 0.01; *: 0.01 < p < 0.05.
Figure 3
Figure 3
Evaluation of CSF NfL, MMP-2, and MMP-9 levels in neuro-COVID group. (A) Evaluation of CSF NfL levels in COVID-19 patients with severe neurological symptoms (neuro-COVID group) stratified according to ARDS development. (B) Evaluation of CSF MMP-2 levels in COVID-19 patients with neurological symptoms (neuro-COVID group) stratified according to ARDS development. (C) Evaluation of CSF MMP-9 levels in COVID-19 patients with neurological symptoms (neuro-COVID group) stratified according to ARDS development. Horizontal lines represent medians. Neuro-COVID group: COVID-19 patients with severe neurological symptoms; NfL: neurofilament light chain; HD: healthy donors; ARDS: acute respiratory distress syndrome; CSF: cerebrospinal fluid; MMP-2: matrix metalloprotease-2; MMP-9: matrix metalloprotease-9; OD: optical density. *: 0.01 < p < 0.05.
Figure 4
Figure 4
Detection of SARS-CoV-2 RNA on CSF and plasma samples and evaluation of NfL and MMPS levels in neuro-COVID group. (A) Among COVID-19 patients with neurological symptoms (neuro-COVID group), the detection of SARS-CoV-2 RNA on CSF and plasma samples was performed using RT-PCR and ddPCR. (B) Evaluation of CSF NfL levels in COVID-19 patients with severe neurological symptoms (neuro-COVID group) stratified according to ddPCR positivity on CSF and plasma. (C) Evaluation of plasma NfL levels in COVID-19 patients with severe neurological symptoms (neuro-COVID group) stratified according to ddPCR positivity on CSF and plasma. (D) Evaluation of CSF MMP-2 levels in COVID-19 patients with severe neurological symptoms (neuro-COVID group) stratified according to ddPCR positivity on CSF and plasma. (E) Evaluation of CSF MMP-9 levels in COVID-19 patients with severe neurological symptoms (neuro-COVID group) stratified according to ddPCR positivity on CSF and plasma. Horizontal lines represent medians. SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; RT-PCR: reverse transcription-polymerase chain reaction; ddPCR: droplet digital polymerase chain reaction; CSF: cerebrospinal fluid; NfL: neurofilament light chain; MMP-2: matrix metalloprotease-9; MMP-9: matrix metalloprotease-9.
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