Long-Term Sequelae of COVID-19 in Experimental Mice

Abstract

We recently reported acute COVID-19 symptoms, clinical status, weight loss, multi-organ pathological changes, and animal death in a murine hepatitis virus-1 (MHV-1) coronavirus mouse model of COVID-19, which were similar to that observed in humans with COVID-19. We further examined long-term (12 months post-infection) sequelae of COVID-19 in these mice. Congested blood vessels, perivascular cavitation, pericellular halos, vacuolation of neuropils, pyknotic nuclei, acute eosinophilic necrosis, necrotic neurons with fragmented nuclei, and vacuolation were observed in the brain cortex 12 months post-MHV-1 infection. These changes were associated with increased reactive astrocytes and microglia, hyperphosphorylated TDP-43 and tau, and a decrease in synaptic protein synaptophysin-1, suggesting the possible long-term impact of SARS-CoV-2 infection on defective neuronal integrity. The lungs showed severe inflammation, bronchiolar airway wall thickening due to fibrotic remodeling, bronchioles with increased numbers of goblet cells in the epithelial lining, and bronchiole walls with increased numbers of inflammatory cells. Hearts showed severe interstitial edema, vascular congestion and dilation, nucleated red blood cells (RBCs), RBCs infiltrating between degenerative myocardial fibers, inflammatory cells and apoptotic bodies and acute myocyte necrosis, hypertrophy, and fibrosis. Long-term changes in the liver and kidney were less severe than those observed in the acute phase. Noteworthy, the treatment of infected mice with a small molecule synthetic peptide which prevents the binding of spike protein to its respective receptors significantly attenuated disease progression, as well as the pathological changes observed post-long-term infection. Collectively, these findings suggest that COVID-19 may result in long-term, irreversible changes predominantly in the brain, lung, and heart.

Keywords: COVID-19; Long-term sequelae, mice; Mouse hepatitis virus-1; Multi-organ histopathology; SARS-CoV-2; Vascular defect.

Fig. 1

LongtTerm alterations in bodyweight post-MHV-1 infection. MHV-1-infected mice lost body weight in a biphasic manner. Acute loss was identified from 4 to 12 days post-inoculation of 8 weeks old mouse, and such loss was reduced from 3 to 12 weeks (graph 11–24 weeks). Furthermore, a gradual decrease was observed until 11 months and 21 days (51 weeks). Treatment of these mice with SPIKENET (SPK, 5 mg/kg, 3 doses on days 2, 4, and 6 post-MHV-1 inoculation) significantly reduced the weight loss. Data were subjected to ANOVA (n = 3). *p < 0.05 vs. control; †p < 0.05 vs. MHV-1 alone. Error bars, mean ± S.E

Fig. 2

Long-term clinical signs in MHV-1-infected mice. MHV-1-inoculated mice exhibit severe illness in the acute phase (stages IV–VI, from 7 to 12 days), while the animal sickness was significantly reduced to stages II after 12 months. Furthermore, SPIKENET (SPK, 5 mg/kg, 3 doses on days 2, 4, and 6 post-MHV-1 inoculation) prevented such effect in both acute phase and after long-term post-infection

Fig. 3

Acute/long-term changes in brain post-MHV-1 coronavirus infection. A Normal brain cortex. B Representative image from MHV-1-infected mouse brain cortex showed “perivascular cavitation, congested blood vessel, pericellular halos, darkly stained nuclei, vacuolation of neuropil, pyknotic nuclei and acute eosinophilic necrosis at 7 days (acute phase)” [1]. C MHV-1-infected mouse brain cortex (12 months post-infection). F–I Enlarged images of C showed widespread neuronal necrosis (long arrows), pyknotic nuclei/neuronal clearing (short arrows), vacuolation of neuropil (arrowhead), congested blood vessels (blue arrows), perivascular cavitation (yellow arrows, Virchow–robin space), darkly stained nuclei (green arrow), neuronophagia (red arrow, presence of necrotic neurons surrounded by invaded hypertrophic microglia (I). D and E Treatment of MHV-1-infected mice with the peptide drug SPIKENET (5 mg/kg) ameliorated the above-mentioned changes at day 7 and 12 months post-infection, respectively. These findings suggest that the changes observed in the brain post-long-term infection are more severe than in the acute phase and the peptide SPIKENET has therapeutic potential in reducing MHV-1 infection (n = 3). (H&E, original magnification 400 × (A–E), and F–I are enlarged images of C)

Fig. 4

Acute and long-term changes in lung post-MHV-1 coronavirus infection. A Normal mouse lung. B Representative image from MHV-1-infected mouse lung showed “inflammation (i.e., granular degeneration of cells, and migration of leukocytes into the lungs), along with proteinaceous debris filling of the alveolar spaces with fibrillar to granular eosinophilic protein strands caused by the progressive breakdown of the capillary wall and epithelial integrity, permitting leakage of protein-rich edematous fluid into the alveoli, and the presence of hemosiderin-laden macrophages (indicating pulmonary congestion with dilated capillaries and leakage of blood into alveolar spaces). Furthermore, peribronchiolar interstitial infiltration, bronchiole epithelial cell necrosis, necrotic cell debris within alveolar lumens, alveolar exudation, hyaline membrane formation, alveolar hemorrhage with red blood cells within the alveolar space, and interstitial edema, characteristic features of infected lungs in humans with SARS-CoV-2 infection are observed in MHV-1-infected mice at acute phase (at 7 days [1])”. C MHV-1-infected mouse lung (12 months post-infection). F–I enlarged images of C. Blue arrows, airspaces of alveolar ducts, and alveoli are lined by hyaline membranes; yellow arrow pulmonary edema located in bronchiolar and alveolar airspaces, along with congestion of capillaries in the septal wall, and in the perivascular interstitial spaces; white arrow, nuclear atypia and lack of polarity. Asterisks, intraluminal fibrosis. D and E Treatment of MHV-1-infected mice with the peptide drug SPIKENET (5 mg/kg) ameliorated the above-mentioned changes at day 7 and 12 months post-infection, respectively (n = 3). (H&E, original magnification 400 × (A–E), and F–I are enlarged images of C)

Fig. 5

Acute and long-term changes in heart post-MHV-1 coronavirus infection. A Heart from normal mice. B Representative image from MHV-1-infected mouse heart showed “severe interstitial edema, vascular congestion and dilation, and red blood cells infiltrating between degenerative myocardial fibers after acute infection at 7 days (acute phase)” [1]. C, D, and G–I MHV-1-infected mouse heart (12 months post-infection). G White arrow, enlarged myocytes; block arrows, widespread inflammation. H Red arrow, vacuolation; blue arrow, extensive degeneration of cardiac muscle; yellow arrows, foci of apoptotic cell debris; disorganization of the myofibrils with loss of striations. I Box showing loss of myocardial fibers. E and F Treatment of MHV-1-infected mice with the peptide drug SPIKENET (5 mg/kg) ameliorated the above-mentioned changes at day 7 and 12 months, respectively (n = 3). (H&E, original magnification 400 × (A, B, E, and F); and C, D, and G–I are enlarged images from 400 ×)

Fig. 6

Acute and long-term changes in heart post-MHV-1 coronavirus infection. A Liver from normal mice. B Representative image from MHV-1-infected mouse liver showed “hepatocyte degeneration, severe periportal hepatocellular necrosis with pyknotic nuclei, severe hepatic congestion, ballooned hepatocytes, vacuolation, and the presence of piecemeal necrosis, as well as hemorrhagic changes. Ground glass hepatocytes showed voluminous, abundant, granular cytoplasm, peripheral cytoplasmic clearing, and central nuclei, and apoptotic (acidophil) bodies, as well as absent hepatocytes replaced by abundant inflammatory cells. Condensation and dark staining of the cytoplasm, an absence of the nucleus, fatty changes, binucleated hepatocytes, and activated Kupffer cells were also identified at 7 days post-infection” [1]. C MHV-1-infected mouse liver (12 months post-infection). F–H Enlarged images of C. Note, the pale brown (yellow arrows in H) is lipofuscin pigment (indicative of oxidative stress) that has accumulated as the atrophic and dying cells likely due to hypoxia, undergo autophagocytosis; accumulation of small fat droplets in hepatocyte cytosol (short arrow in F); hepatic cells with ballooning degeneration (long arrows in F–H); dilated congested blood vessel (asterisk in F); presence of stellate cell (Ito cell or perisinusoidal lipocytes) lipidosis (blue arrows) and occasional fat-laden stellate cells showing multiple lipid vacuoles with indentation of the nucleus were also observed (green arrow in H). D and E Treatment of MHV-1-infected mice with the peptide drug SPIKENET (5 mg/kg) ameliorated the above-mentioned changes at day 7 and 12 months post-infection, respectively (n = 3). (H&E, original magnification 400 × (A–E), and F–H are enlarged images of C)

Fig. 7

Acute and long-term changes in kidney post-MHV-1 coronavirus infection. A Normal mouse kidney. B Representative image from “MHV-1-infected mouse kidney showed tubular epithelial cell degenerative changes, peritubular vessel congestion, proximal and distal tubular necrosis, hemorrhage in interstitial tissue, and vacuolation of renal tubules were observed in MHV-1 exposed mice kidneys at 7 days post-infection” [1]. (V). C MHV-1-infected mouse kidney (12 months post-infection). F–H, enlarged images of C, shows congested blood vessels (black arrow), distal tubular damage (yellow arrows), fibrosis and inflamed glomeruli (asterisks), necrosis (green arrows), loss of podocytes (white arrow), degenerating tubules (black asterisks), hyaline casts (pink arrows), loss of tubular epithelial cells (karyolysis) (dagger symbol) and Karyorrhexis (light blue arrow). D and E Treatment of MHV-1-infected mice with the peptide drug SPIKENET (5 mg/kg) ameliorated the above-mentioned changes at day 7 and 12 months post-infection, respectively (n = 3). (H&E, original magnification 400 × (A-E), and F–H are enlarged images of C)

Fig. 8

Increased cortical astrogliosis post-MHV-1 coronavirus infection. Representative image from MHV-1 infected mouse (12 months) brain cortex showed severe reactive astrocytes (astrogliosis, green, glial fibrillary acidic protein, GFAP, in MHV-1), as compared to normal mice brain cortex (control). Note, such an increase in astrogliosis was reduced when these mice were treated with the peptide drug SPIKENET (5 mg/kg) (MHV-1 + SPIKENET) (n = 3). Scale bar = 35 µm. Blue, nuclear stain DAPI

Fig. 9

Increased cortical reactive microglia post-MHV-1 coronavirus infection. Representative image from MHV-1 infected mouse (12 months) brain cortex showed severe reactive microglia (red, ionized calcium-binding adaptor molecule 1, Iba1, in MHV-1), as compared to normal mice brain cortex (control). Note, such an increase in reactive microglia was reduced when these mice were treated with the peptide drug SPIKENET (5 mg/kg) (MHV-1 + SPIKENET) (n = 3). Scale bar = 35 µm. Blue, nuclear stain DAPI

Fig. 10

Phosphorylated TDP-43 (p-TDP-43) level in the cerebral cortex of MHV-1-infected mice. Normal (control) brain illustrates the basal level of p-TDP-43 in the brain cortex. Note the marked increase in levels of p-TDP-43 in MHV-1-infected brains (MHV-1) and such increase in p-TDP-43 was reduced when these mice were treated with the peptide drug SPIKENET (5 mg/kg) (MHV-1 + SPIKENET) (n = 3). Scale bar = 35 µm. Blue, nuclear stain DAPI

Fig. 11

Phosphorylated Tau (p-Tau) level in the cerebral cortex of MHV-1-infected mice. Normal (control) brain illustrates the basal level of p-Tau in the brain cortex. Note the marked increase in levels of p-Tau in MHV-1-infected brains (MHV-1) and such increase in p-Tau was reduced when these mice were treated with the peptide drug SPIKENET (5 mg/kg) (MHV-1 + SPIKENET) (n = 3). Scale bar = 35 µm. Blue, nuclear stain DAPI

Fig. 12

Synaptophysin-1 (Syn-1) level in the cerebral cortex of MHV-1-infected mice. Normal (control) brain illustrates the basal level of Syn-1 in the brain cortex. Note the marked decrease in levels of Syn-1 in MHV-1-infected brains (MHV-1) and such decrease in Syn-1 was reduced when these mice were treated with the peptide drug SPIKENET (5 mg/kg) (MHV-1 + SPIKENET) (n = 3). Scale bar = 35 µm. Blue, nuclear stain DAPI

Fig. 13

NMDA mRNA in MHV-1-inoculated mice brain cortex. While there was a slight decrease in NR1 subunit of NMDA receptor mRNA at 7 days post-MHV-1, there was a significant loss at 12 months post-MHV-1 infection as determined by RT-qPCR. NMDA mRNA was normalized against glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Data were subjected to ANOVA (n = 3). *p < 0.05 vs. control. Error bars, mean ± S.E. D, days; M, months