Corneal innervation changes after Sars-Cov-2 Infection. An In vivo Confocal Microscopy Study

Purpose : To measure the innervation of the corneal subbasal nerve plexus of Covid-19 patients and compare the results with values of healthy patients. Methods : A prospective, observational study was conducted analyzing 39 eyes of patients who had overcome Covid -19 and 46 eyes of healthy volunteers included as a control group (verified by antibody analysis and negative qPCR result) which underwent in vivo confocal microscopy with Rodstock Cornea Module© attached to Heildelberg HRT3© . Ocular surgery procedures, previous ocular infections or systemic diseases that could cause alteration in corneal innervation were exclusion criteria. At least 5 non overlapping images of each eye were selected and only one eye of each patient was included in the study. Following sub basal nervous plexus parameters were measured with ACC Metrics © software: Corneal Nerve Fiber Density (CNFD), Corneal Nerve Branch Density (CNBD), Corneal Nerve Fiber Length (CNFL), Corneal Total Branch Density (CTBD), Corneal Nerve Fiber Area (CNFA), Corneal Nerve Fractal Dimension (CNFrD). Data analysis was performed with SPSS® software for Windows 22.0 (SPSS® Inc, Chicago, IL.). The differences of age and sex between groups were checked with T-test and chi-square tests. The normality of the sample was checked with the Shapiro-Wilk test and the results were compared with the T test or the Man-Whitney U test based on the distribution of the data. The differences were considered statistically significant for p<0.05. Results : There was no difference in the sex distribution between the groups (p= 0.248). The average age (± standard error) was 46.61±17.55 years for Covid-19 patients and 43.11±16.95 years for healthy control group (p=0.353) The mean of the analyzed variables (± standard error) from Covid-19 patients versus control group were CNFD: 16.09±6.92 and 23.03±8.31 fibers/mm2 (p=0,00008), CNBD: 21.93±15.37 and 28.93±17.84 branches/mm2 (p=0.064), CNFL: 11.61±3.61 and 14.05±3.71 mm/mm2 (p=0.002), CTBD: 38.48±20.02 and 43.29 ± 23.94 (p=0.41), CNFA: 0.0057±0.0017 and 0.006±0.0023 mm2 /mm2 (p=0.853), CNFrD: 1.46±0.041 and 1.47±0.037 (p=0.007). Conclusions : According to the data obtained, corneal subbasal nerve plexus is decreased in Covid-19 patients compared to the healthy control group, statistically significant for density, length, and fractal dimension. The results show the presence of possible small fiber neuropathy induced by Covid-19 disease.

OCULOMOTOR NERVE PALSY FOLLOWING COVID-19 INFECTION

CASE: Patient is a 60-year-old woman who works at a local hospital in billing department. She has a history of rheumatic fever, non ST elevation MI, osteoarthritis, Crohn's disease. Her husband was diagnosed with COVID-19 infection in November 2020. A Week later, patient developed myalgias, diarrhea and subsequent testing confirmed COVID-19 infection. Overall, her symptoms were mild and required no treatment or hospitalization. Six weeks following the infection she woke up one morning with diplopia and a large left pupil. She tried to manage this by covering one eye initially, but later visited with a neurologist, ophthalmologist, neuro-ophthalmologist. She was found to have fixed, dilated left pupil and horizontal diplopia with some diagonal component. There were no other neurological signs or meningismus. Laboratory tests showed hemoglobin of 12.5, White cell count 5.7, platelets 405. Electrolytes, kidney function, liver function tests were normal. ACH receptor antibodies were negative. Imaging studies included a negative CTA head, negative brain MRI, face, orbits and optic nerves. She was diagnosed with left third cranial nerve palsy possibly as a complication of COVID-19 infection. She was prescribed oral prednisone 60 mg with a slow taper. Her pupil size and vision gradually improved over the ensuing weeks and the recovery of the third cranial nerve was nearly complete. IMPACT/DISCUSSION: The third cranial nerve supplies the levator muscle of the eyelid, medial rectus, superior rectus, inferior rectus, and inferior oblique;constricts the pupil through its parasympathetic fibers. Patients with oculomotor cranial nerve palsy develop diplopia and droopy eyelid. Etiology for third cranial nerve palsy include many pathologies such as a structural lesion, infectious or inflammatory conditions, cerebrovascular disease and trauma. Our patient developed acute 3rd cranial nerve palsy 6 weeks following the COVID-19 infection. The workup was negative for any structural lesions, CVA or other known causes. This raised the possibility that her symptoms are possibly complications of COVID-19 infection. Neurological complications of COVID-19 infection have been well documented. These include encephalopathy, stroke, dysgeusia and anosmia. There were two case reports of oculomotor nerve palsy that occurred during the acute phase of COVID-19 infection. These were thought to be from direct invasion of the virus. Our patient however, had developed symptoms 6 weeks following the infection raising the possibility of immune mediated complication. She made near complete recovery with oral glucocorticoid treatment. However, it is not known whether the improvement is the result of the treatment. CONCLUSION: 1. Oculomotor cranial nerve palsy is potentially associated with COVID-19 infection. 2. Oculomotor cranial nerve palsy could present several weeks after the acute COVID-19 infection. 3. In patients presenting with 3rd cranial nerve palsy, it is important to obtain the history of past COVID-19 infection.

Distant Ocular Manifestations after New Coronavirus Infection

Purpose. Theoretically substantiate and practically identify ocular manifestations after the transferred SARS-CoV-2 virus. Patients and methods. For the study we were invited patients who had recovered from SARS-CoV-2 at the hospital of V.M. Buyanova, the age from 20 to 65 years old in period from 2020 to 2021. The total number of patients was 68 people. The patients had with them the results of PCR tests or ELISA tests, or an extract from the hospital confirmed COVID-19 case and also a clinical blood test and CT scan of the chest organs for the period of illness. Before the start of the study, the patients were asked to fill out a questionnaire “Questionnaire for patients who have had a new coronavirus infection” (Appendix 1). For a detailed study of this group of people, each underwent visometry, pneumotonometry, B-scan, a slit lamp study and also a slit lamp study with a 60D lens using 0.5 % Mydriacyl eye drops in the absence of contraindications and pupillography. Results. As a result of the work carried out, we concluded that the virus is capable of causing inflammation of the choroid of the eyeball, uveitis. Moreover, in our study, we identified patients with acquired intermittent divergent strabismus, anisocoria, ptosis, and accommodation disorder. And, in this regard, we came to the conclusion that the coronavirus belongs to the group of neurotropic, as it is able to affect the nervous tissue and cause the above clinical picture. In other words, the virus negatively affects the somatic and autonomic innervation of the oculomotor nerve. As a result of these lesions, we get the corresponding tetrad of symptoms: heterotropy, mydriasis, ptosis, accommodation paralysis. Conclusions. One of the extraordinary complications of coronavirus infection is damage effect to the fibers of the oculomotor nerve, the signs include: strabismus, mydriasis, ptosis and accommodation paralysis. Thus, this clinical picture is associated with the affinity of the virus to the nervous tissue. And as a result, this ability of the virus can probably infect various areas of the brain, which will lead to corresponding complications, not only from the oculomotor nerve, but also from other cranial nerves with the manifestation of the corresponding symptoms, which in theory can aggravate the patient's condition, causing deep disturbances of motor and sensory innervation.

Detection of SARS-CoV-2 within enteric neurons and in brain

SARS-CoV-2, the causative agent of COVID-19, typically manifests as a respiratory illness although extrapulmonary involvement, such as in the gastrointestinal tract and nervous system, are increasingly recognised. Through immunohistochemistry against the SARS-CoV-2 nucleocapsid protein (NP), we aimed to characterise the multisystem viral tropism of SARS-CoV-2. FFPE tissue was obtained from 16 PCR-confirmed post-mortem COVID cases. Of these cases, 10 were full-body, 5 were brain only and 1 was a brain biopsy. Brain regions studied included frontal cortex, medulla, cerebellum, pons and olfactory bulb. Neurological symptoms featured in the cohort included brainstem encephalitis, acute disseminated encephalomyelitis (ADEM) and brain infarction. Immunohistochemistry of digestive system tissues revealed presence of SARS-CoV-2 NP in neurons of the myenteric plexus, a site of high ACE-2 expression, the entry receptor for SARS-CoV-2 and one of the earliest affected cells in Parkinson's disease (PD). Within the brain, staining was widespread in all sampled regions but limited to endothelial cells only (including in the olfactory bulb). Furthermore, in the full-body post-mortem cases, positivity in brain endothelia was restricted to cases exhibiting multiorgan tissue positivity (3/9 cases). The average time from symptom onset to time of death was shorter in positively versus negatively stained postmortem cases (mean = 10.3 days vs mean = 20.3 days, p = 0.0416) suggesting NP detection was confined to the infectious period. Together, our findings provide evidence for enteric nervous system but not brain neuroinvasion of SARS-CoV-2 as well as potential insights into long-term complications of COVID-19 and PD pathogenesis.

Development of experimental model of ARDS in SHR strain old rats

Introduction. ARDS (acute respiratory distress syndrome) is a direct cause of death due to lung lesions of various origins including SARS-CoV-2 infection. Most lung and respiratory diseases are characterized by inflammation, which in turn causes occlusions, mucous hypersecretion, shortness of breath, cough and other symptoms of airway inflammation. Understanding the pathological processes involved in the regulation of the immune response may lead to the discovery of new mechanisms that support or suppress inflammatory processes in the lungs and respiratory tract. Methods. In order to develop the experimental model of ARDS we used precision pulmonary hyperventilation and intratracheal administration of poly I:C, which reproduces the body's response to viral infection (mimic viral infection). The severity of ARDS was estimated by the following parameters: the ratio of wet lung weight to dry lung (wet/dry lung weight), extensibility, impedance, lung stiffness, protein level and the number of neutrophils in the bronchial lavage, the level of proinflammatory cytokines in lung tissue. Moreover, we provide a method of simultaneous registration of the lungs vagal nerve activation parameters due to ARDS induction. It is known that the lungs have a massive innervation of the peripheral nervous system and such innervation has a powerful effect on the cells of the immune system. Results. The biologically active substances of neurons affect the activity of immune cells, and the activity of the immune system affects the functioning of the nervous system. That is why the investigation of neuro-immune interaction has a great potential in studying ARDS. Evaluation of dynamic changes in respiratory function due to simulated ARDS showed a significant increase in such parameters as Newtonian resistance, tissue stiffness, static elongation, and a decrease in elasticity and tissue dumping, which is fully consistent with the pathogenesis of ARDS in patients. Analysis of the dry/wet lung ratio showed a two-fold increase in pulmonary edema, a severe, life-threatening condition that develops as a result of ARDS. Significant increase of the protein content and concentration of neutrophils in bronchopulmonary lavage indicates an increase in the permeability of the pulmonary capillaries due to ARDS. The real time PCR identified significant increase of proinflammatory chemokine Cxl2 concentration. Also we observed an increase of neutrophil-activating protein 3 cytokine Gro1, which stimulates the migration of neutrophils. Electrophysiological registration of the activity of the vagal nerve innervating the lungs showed a significant increase in the activity of nociceptive and mechanosensitive fibers, especially in the last stage of acute respiratory distress syndrome, which confirms the role of the nervous system in this pathology. Conclusion. As a result we obtained a model that reproduces ARDS most relevant to human pathological condition.

SARS-COV-2 INDUCES EPITHYELIAL-NEURONAL CROSSTALK STIMULATING VASOACTIVE INTESTINAL PEPTIDE RELEASE AS A POTENTIAL MECHANISM OF COVID-19-ASSOCIATED DIARRHEA

Background: Up to 36.6% of COVID-19 patients have diarrheal symptoms and 48.1% test positive for SARS-CoV-2 via stool test. The mechanism of SARS-CoV-2-associated diarrhea remains poorly understood. We hypothesize that crosstalk between enterocytes and the enteric nervous system (ENS) plays a critical role in the pathogenesis of COVID-19-associated diarrhea. We studied the effects of SARS-CoV-2 on induction of endoplasmic reticulum (ER) stress and release of Damage Associated Molecular Patterns (DAMPs), which act on enteric neurons and stimulate the production of neurotransmitters. The influence of ER stress and enteric neuron-derived vasoactive intestinal peptide (VIP) on the expression of electrolyte transporter Na+/H+ exchanger 3 (NHE3) was also examined. Methods: SARS-CoV-2 (2019-nCoV/USA-WA1/2020) was propagated in Vero-E6 cells. Caco-2, a human colon epithelial cell line, expresses the essential SARS-CoV-2 entry receptor ACE2 and was thus used for infection (MOI, ~0.01). We used Western blotting to assess the expression of ER stress (phospho-PERK and Xbp1s) and DAMP (HMGB1) markers at 48 hours post-infection. Primary mouse enteric neurons were co-cultured with Caco-2 cells, pre-treated for 24 hours with 2 μM tunicamycin to induce ER stress. Supernatants from enteric neurons were used to assess the expression of VIP by ELISA. Primary enteric neurons were treated with HMGB1 or ATP (another form of DAMPs), and the expression of c-FOS, a marker of neuronal activity, was determined by Western blotting and immunofluorescence staining. Results: We found that SARS-CoV-2 infection of Caco-2 cells led to increased expression of phospho-PERK and Xbp1s. Compared to uninfected control, infected Caco-2 cells secreted HMGB1 into culture media, indicating epithelial production of DAMPs in response to SARS-CoV-2 infection. Tunicamycin was used to induce ER-stress and secretion of HMGB1 by Caco-2, mimicking SARS-CoV-2 infection. Importantly, enteric neurons co-cultured with tunicamycin-treated Caco-2 cells secreted significantly higher levels of VIP. Treating Caco-2 cells with tunicamycin or VIP on the basolateral side led to decreased surface NHE3 expression, suggesting a potential impairment of intestinal electrolyte/fluid absorption. More-over, HMGB1 and ATP both increased the expression of phospho-c-FOS in cultured enteric neurons, indicating DAMP-induced neuronal activation. Conclusions: Our findings demon-strate that enterocytes infected by SARS-CoV-2 release DAMPs with the capacity to induce VIP secretion by the enteric neurons, which in turn acts on enterocytes and inhibits apical localization of NHE3. These findings establish basic mechanisms relevant to diarrheal disease in COVID-19 patients and identify potential targets for the treatment of SARS-CoV-2 infection of the gastrointestinal tract.

Autonomic innervation of the carotid body as a determinant of its sensitivity: implications for cardiovascular physiology and pathology.

The motivation for this review comes from the emerging complexity of the autonomic innervation of the carotid body (CB) and its putative role in regulating chemoreceptor sensitivity. With the carotid bodies as a potential therapeutic target for numerous cardiorespiratory and metabolic diseases, an understanding of the neural control of its circulation is most relevant. Since nerve fibres track blood vessels and receive autonomic innervation, we initiate our review by describing the origins of arterial feed to the CB and its unique vascular architecture and blood flow. Arterial feed(s) vary amongst species and, unequivocally, the arterial blood supply is relatively high to this organ. The vasculature appears to form separate circuits inside the CB with one having arterial venous anastomoses. Both sympathetic and parasympathetic nerves are present with postganglionic neurons located within the CB or close to it in the form of paraganglia. Their role in arterial vascular resistance control is described as is how CB blood flow relates to carotid sinus afferent activity. We discuss non-vascular targets of autonomic nerves, their possible role in controlling glomus cell activity, and how certain transmitters may relate to function. We propose that the autonomic nerves sub-serving the CB provide a rapid mechanism to tune the gain of peripheral chemoreflex sensitivity based on alterations in blood flow and oxygen delivery, and might provide future therapeutic targets. However, there remain a number of unknowns regarding these mechanisms that require further research that is discussed.

Bone Pain in Multiple Myeloma (BPMM)-A Protocol for a Prospective, Longitudinal, Observational Study.

Multiple myeloma (MM) is a bone marrow neoplasia that causes bone pain in 70% patients. While preclinical models of MM have suggested that both nerve sprouting and nerve injury may be causative for the pain, there is a lack of clinical data. Thus, the primary aims of this clinical study are: (1) to provide a deep characterization of the subjective experience of pain and quality of life in MM patients; (2) to investigate disturbances in the bone innervation of MM patients. Secondary aims include exploring correlations between pain and serum inflammatory and bone turnover biomarkers. In a prospective, observational study (clinicaltrials.gov: NCT04273425), patients with suspected MM requiring a diagnostic iliac crest biopsy at Sheffield Teaching Hospital (UK) are invited to participate. Consenting patients answer seven standardized questionnaires assessing pain, quality of life and catastrophizing. Bone turnover biomarkers and inflammatory cytokines are measured in fasting serum samples, and bone innervation is evaluated in diagnostic biopsies. MM patients are invited to a follow-up upon completion of first line treatment. This will be the first deep characterization of pain in MM patients and its correlation with disturbances in bone innervation. Understanding how bone turnover and inflammation correlate to pain in MM is crucial to identify novel analgesic targets for this condition.