Viral replication, immune hyperactivation and post-acute sequelae or Long Covid is a multi-phasic stage characterization currently used for COVID-19 following SARS-CoV-2 virus infection (30). According to this three-stage view, SARS-CoV-2 virus enters the host body and replicates itself. Following an incubation period of approximately five days, a clinical phenomenology of an upper respiratory system infection, fever, muscle fatigue and pain appears. The organism initially reacts with innate and adaptive immune responses and, in non-severe COVID-19, symptoms are commonly resolved within a four-week period (e.g., 30). Conversely, in severe COVID-19, SARS-CoV-2 virus can escape immunity and eventually a second phase of immune hyperactivation can take place. This second phase has been associated principally with an abnormal response of immune cells of the host such as macrophages and natural killer cells, which can function abnormally and promote a dysregulated release of interferons and proinflammatory cytokines such as IL-1b, IL-6 and IL-12 resulting in PAN-optosis and eventually in hypercytokinemia or cytokine storm (e.g., 30–33). This sequence of events usually leads to overwhelming systemic inflammation and multiorgan failure manifested as stroke, lung injury, cardiac, liver and kidney injuries as well as vasculopathy, secondary infections and sepsis with high mortality (30, 32, 34, 35). Although in COVID-19 survivors, symptoms would usually resolve within one to four weeks from their initial appearance, a number of patients would continue reporting symptoms, such as fatigue, post-exertional malaise, headache, dyspnea/shortness of breath, anosmia and cognitive dysfunction beyond this period of time (36). If these symptoms persist “for at least two months occurring within three months after COVID-19 infection which cannot be explained by an alternative diagnosis” the World Health Association (WHO) has termed this clinical condition as post-COVID-19 condition (PCC), which is synonymous with “Long COVID” or Post-Acute Sequelae of COVID-19 (PASC) (34, 37). Histopathologically, it seems that SARS-CoV-2 virus infection produces an endotheliopathy and that the pulmonary capillary endothelium is the most common entry in the body for viral replication and, eventually, for the virus to get access in the blood stream (35, 38–40). Endothelial damage is diffuse and extends beyond the respiratory system impairment underlying such multi-system, multi-organ clinical manifestations of COVID-19 (19, 20, 22) as those present in the cardiovascular system, the liver, the kidney and the brain (e.g., 21, 35).

Neurological complications of acute COVID-19 involve the brain, cranial nerves and peripheral nerves (41) with clinical manifestations that include thromboembolic strokes, intracranial hemorrhages as well as encephalitis, meningoencephalitis and neuropathy. It is not uncommon that acute neurological phenomenolgy may persist for weeks and also for a more prolonged period, which can last from months to years after recovery from the initial infection (42). Mechanistically, the neuropathogenesis of acute COVID-19 remains unclear (41, 43), and the elucidation of whether the neurological effects in COVID-19 are a) mediated directly by SARS-CoV-2 virus or b) an indirect effect of the virus itself (namely, hypercoagulopathy) or an immune-mediated/autoimmune-mediated (such as the cytokine storm) neuroinflammation (41) is a matter of great importance biologically and clinically. Neurohistopathological studies have demonstrated acute hypoxic ischemic injury in the cerebrum and cerebellum in brain tissue of patients who have undergone autopsy (e.g., 44–46). More specifically, Solomon and colleagues (2020) reported that microscopic examination has shown neuronal loss in the frontal lobe, hippocampus, and Purkinje cell layer of the cerebellum. Another study on a post-mortem case series by Matschke and colleagues (2020), showed pronounced neuroinflammatory alterations in the brainstem such as in the upper medulla oblongata (44 with microscopic details in Figures 1 – 3 ). Schurinck and colleagues (2020) by contrast, in a prospective autopsy study investigating a cohort of 21 patients with lethal COVID-19, showed “a severe innate inflammatory state” with massive activation of microglia in the brain. Based on their findings and in light of other histopathological studies reporting the presence of SARS-CoV-2 infected cells in the brain (46, 53), they suggested that a large inflammatory response may lead swiftly to viral clearance “shifting the pathology towards an autonomous immune-mediated reaction.” Schurinck et al. also emphasized the extensive presence of inflammation in the medulla oblongata, which is critically important in regulatory respiratory functions, which could well contribute to the respiratory failure occurring in these patients (45). Thus, it seems likely that while direct viral effects cannot be easily estimated in acute COVID-19, the clinical profile of brain inflammation is more likely produced by immune-mediated responses or autoimmune reactions (54). Furthermore, the intense virus infection-related systemic inflammatory response can lead to breakdown of the blood-brain barrier (BBB), which could allow entry into the central nervous system (CNS) of peripheral inflammatory molecules such as cytokines producing autoimmune encephalitis (55, 56). Besides short-term or acute consequences, there are also important long-term or post-acute consequences of COVID-19 that also go beyond the respiratory system and constitute the post-acute sequelae of the Long COVID condition. Long COVID occurs in at least 10% of patients, which had severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection (57). By 2022, more than 65 million individuals worldwide are estimated to have long COVID, a number that is currently rising (58). This illness is multisystemic affecting seriously the nervous system with such new onset manifestations as myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) and dysautonomia (especially postural orthostatic tachycardia syndrome (POTS) (59, 60), which can become lifelong conditions (61). While the picture of Long COVID’s causes remains unclear, several hypotheses have been suggested regarding its pathogenesis. As reviewed recently by Davis and colleagues (2023) these mechanisms include “immune dysregulation with or without reactivation of underlying pathogens (such as Epstein–Barr virus and human herpesvirus-6), microbiota and virome disruption (including SARS-CoV-2 persistence), autoimmunity and primed immune cells from molecular mimicry, microvascular blood clotting with endothelial dysfunction, and dysfunctional neurological signaling in the brainstem and/or the vagus nerve” (modified from Figure 3 in Davis, 57). Long COVID is characterized by a plethora of symptoms and bears remarkable similarities to such viral-onset illnesses as ME/CFS and POTS (57). More specifically, frequent neurological symptoms associated with Long COVID are cognitive impairment (i.e., brain fog), memory loss, fatigue, unfreshening sleep, pain and post-exertional malaise (11). A principal condition underlying these behaviors is neuroinflammation, the cause of which may be a chronic or relapsing neuroinflammatory process initiated by initial SARS-CoV-2 virus infection, which can lead to increased permeability of the blood-brain barrier (11, 12, 57). Neuroinflammation seems to be widespread (25, 62) and involves important nervous centers and fiber pathways that play a key role in several biobehavioral functions. Given the importance of endotheliopathy and BBB dysfunction in understanding similarities and differences in pathological mechanisms in acute and Long COVID-19 and in other neurodegenerative diseases, as well as ways in which these disorders may be interrelated (e.g., comorbidity), we will elaborate further in this regard in section 3. We will review neurologically based functions in more detail in sections 4 and 5, addressing autonomic, neuroendocrine, affective or limbic, and cognitive aspects. Furthermore, we will emphasize the role of the known neuroanatomical structures underlying brain-immune interactions, namely the neuroimmune network in the CNS in sections 4 and 5. Finally, in section 6 how we will review how to investigate the neuroimmune circuitry using current neuroimaging.

Endotheliopathy and BBB dysfunction are considered key underlying pathophysiological processes in acute and Long COVID-19 (63). Endotheliopathy has been reported to result from SARS-CoV-2 protease activity, whereas complement activation can be involved in hypoxic neuronal injury and blood–brain barrier (BBB) dysfunction (63, 64). The BBB is a complex structure comprising endothelial cells that form tight junctions to protect the neural tissue. These cells impede the entry of different substances, cells or molecules that, if penetrating the BBB, would induce CNS pathology. The permeability of the BBB is regulated by various perivascular structures such as microglia, pericytes, astrocytes and the basement membrane, which form a physical barrier (see e.g., 11, 65). At a mature stage, the BBB is stabilized by highly specialized perivascular structures. BBB restriction of paracellular and transcellular transport of solutes is affected by a number of processes, as detailed by Zhao and colleagues (66). BBB permeability is altered following the addition of extracted SARS-CoV-2 spike protein, resulting most likely from a pro-inflammatory response producing endothelial damage (63). In a recent study of the pathophysiologic mechanism of encephalopathy and prolonged comatose or stuporous state in severally ill patients with COVID-19, Dalakas and colleagues (67) indicated that a) high levels of circulating proinflammatory cytokines due to SARS-CoV-2 infection possibly disrupt the BBB allowing antibodies and other inflammatory mediators to enter the brain parenchyma, as recently shown by other investigators (68); b) systemic effects related to multiorgan failure may be additional factors facilitating BBB disturbance; and c) endothelial cells, which are a structural part of the BBB, can also be directly affected by the virus or the circulating cytokines resulting in endotheliaitis, which further compromises BBB integrity. Thus, as concluded by Dalakas and colleagues “the cause and consequence of disturbed BBB needs to be elucidated” (67). The effects of endotheliopathy and BBB leakage can be quite serious, given their contribution in acute and/or chronic neural inflammation and neuronal injury as well as associated neurological clinical manifestations (63). It should be pointed out that breakdown of the BBB can also be present in clinical conditions other than COVID-19, including acute disorders such as traumatic brain injury, stroke, and seizures or neurodegenerative diseases such as Alzheimer’s disease (AD), vascular dementia, small vessel disease and multiple sclerosis (MS) (66, 69, 70). Importantly, we are able to detect BBB leakage using neuroimaging. The most commonly applied technique is a T1-weighted dynamic contrast-enhanced (DCE)-MRI, during which a gadolinium-based contrast agent is injected intravenously; leakage of the contrast agent can be detected when it crosses the damaged BBB (e.g., 70, 71). It should be noted that there is substantial comorbidity between COVID-19 and AD (72, 73) and the chronic neurological sequelae in Long COVID are also present in AD clinical phenomenology. Furthermore, there is evidence indicating APOE e4e4 genotype as a potential genetic determinant for the development of severe COVID-19 (74), and ApoE e4e4 homozygotes are more likely to be COVID-19 test positive (75). Furthermore, ApoE e4e4 homozygotes were found to be at the highest risk of sporadic AD (73, 76). However, the mechanism underlying this association remains unclear (74). Another lesson learned by studying neurodegeneration in MS, AD and Parkinson’s disease (PD) that may bear insight in approaching Long COVID and myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is the relevance of assessing in the cerebrospinal fluid (CSF) inflammatory biomarkers (11) or other potential biomarkers for COVID-19 such as tau (77). This may also be important information in reasoning on diagnosis and prognosis as well as such aspects as disease activity and response to treatment (11). Moreover, the relationship between COVID-19 and tau pathology remains an unsolved problem and topic of ongoing research. Although in experiments using brain organoids, SARS-CoV-2 virus affects neurons showing altered distribution of tau throughout the neurons from axons to soma, hyper-phosphorylation and neuronal death, it remains unclear whether these effects are produced directly by the virus or other factors such as neuronal stress (78). Furthermore, it has been shown in experimental animals that pathogenic tau can “seed” synaptically connected cells with further spreading of the disease (79, 80). This process is carried out by extracellular micro-vesicles (EVs) or “exosomes”. Autopsy studies have shown tau deposits in brain tissue of COVID-19 patients (77). In addition, EVs containing tau are also present in cerebrospinal fluid and plasma of patients with mild AD and frontotemporal dementia (FTD) (77, 81). Nevertheless, regarding the relationship between the two clinical conditions, as reported by Wang and colleagues, “whether COVID-19 might trigger new-onset Alzheimer’s disease or accelerate its emergence is unclear” (82). That said, a highly important topic in any clinical condition including viral infections and neurodegenerative disorders is how the brain and the immune system are interrelated.

It was not until breakthroughs in experimental animal research in the 1990s (e.g., 2–4, 6) provided evidence of direct immune-brain interactions that the immune and nervous systems would be considered strongly and dynamically related (83). A critical discovery in this regard has been the demonstration of the cholinergic anti-inflammatory pathway as a means of direct neural modulation in suppressing inflammation by the motor branch of the vagus nerve (84, 85), as well as the conceptualization and demonstration of the neuroinflammation reflex as a reflexive hard-wired selective neural pathway with inflammation-sensing and inflammation-suppressing functions (7). Since then, the topic of nervous system (NS) modulation of immunity has been a highly active area of research and has provided considerable insights into the anatomical and molecular mechanisms underlying the brain-immune dynamics during inflammation. Modulation of inflammation by the NS appears to be done by way of neural and humoral pathways. In COVID-19, the pathological process is commenced by initial SARS-CoV-2 virus infection. The NS involvement seems to be triggered either indirectly by the inflammatory peripheral response in the body or directly by the virus per se or any inflammatory molecules that gain access to the CSF due to increased BBB permeability. According to this model, the afferent component of the vagus nerve, which innervates various viscera such as the airways, lungs, heart, liver, spleen, stomach and intestines, is stimulated by pro-inflammatory cytokines produced by the peripheral inflammatory process in SARS-CoV-2 virus infection (7, 86, 87). Peripheral inflammatory status information is thus conveyed to the nucleus of the solitary tract (NTS) in the medulla. The connections of the NTS are numerous and complex, nevertheless its connection with the dorsal motor nucleus of the vagus (DMN) seems to be one of the most relevant in the brain for immune system interactions in response to inflammation. The NTS-DMN ensemble (or dorsal vagal complex (DVC)) seems to be where the coordination of vagal afferent and efferent responses takes place (7, 88). Subsequently, through efferent outflow, the vagus inhibits activated tissue macrophages in releasing pro-inflammatory cytokines. Thus, through this neural pathway, called the “cholinergic anti-inflammatory pathway” (84, 85), the vagus is able to suppress inflammation in real time (7, 88). The afferent and efferent branches of the vagus together with the NTS and DMN constitute the neuroanatomical substrate of the ‘inflammatory reflex’, a hard-wired neural circuit that aims to inhibit acute inflammation in real time. The discovery of the inflammatory reflex has been a fundamental stepping-stone in the understanding of brain-immune interactions; nevertheless, the complete neural circuitry involved in immune modulation remains to be identified with clarity. Although it is conceivable that circuitries including the DVC should be part of a larger distributed neuroimmune network, the exact definition of a comprehensive neuroimmune system beyond the inflammatory reflex that modulates the immune system is still not well understood. This is due, in part, to the fact that the autonomic nervous system (ANS) is arguably one of the most enigmatic aspects of the NS anatomically and functionally. Consistent with this view, the neuroimmune network would involve centers and fiber pathway connections across the CNS, encompassing the cerebral cortex, subcortical forebrain structures, the cerebellum and the brainstem. Following the overall anatomical organization of the ANS structural architecture (89), in the efferent or motor path of the system, there should likely be a hierarchical process with the involvement of antecedent neurons to premotor and motor neurons innervating preganglionic neurons, which in turn act upon postganglionic neurons to affect the target organs. In addition to the structural aspect of the neuroimmune network, there is also the humoral side that has to be equally considered. Furthermore, the pain system plays a role in this schema as well (7). Realizing the complexity of a comprehensive neuroimmune network, in which a multitude of structures and circulating substances are involved, we can appreciate that our current knowledge is scant and that any attempt to specify a precise and complete blueprint of the neuroimmune network would lack clarity. Nevertheless, in an effort to schematize the neuroimmune network, we could attempt an approach based on consolidated basic principles and known facts, namely the inflammatory reflex and the organizational architecture of the ANS. Thus, at a first approximation, the NTS and the DMN of the vagus, and their direct connections supplied principally by fiber pathways such as the medial forebrain bundle (MFB) and the dorsal longitudinal fasciculus of Schutz (DLF) with the cortico-limbic system, seem to be implicated in the structural brain-immune connectome. We must keep in mind that this is a simplistic and hypothetical view of the neuroimmune network blueprint and that humoral and pain processing pathways seem to be involved as well. Based on available literature and neuroanatomical knowledge of the ANS (e.g., 89, 90), we illustrate in Figure 1A simplified blueprint of neuroimmune network anatomy involving the NTS, the DMN of the vagus and the hypothalamus, in particular the paraventricular nucleus (PVN), as well as the white matter fiber system that interconnects these structures. Moreover, the humoral pathway involving the adrenocorticotropic hormone (ACTH) and the hypothalamic-pituitary-adrenal (HPA) axis associated with stress and pain has been considered an integral part of the neuroimmune network given the adrenergic and noradrenergic inhibitory effect on macrophage activation resulting in suppression of the synthesis of tumor necrosis factor (TNF) and other cytokines, which leads to an anti-inflammatory effect (7, 86, 87, 91–93).

The clinical phenomenology of COVID-19 and its schematic characterization as a multi-phase sequence of events, namely viral replication, immune hyperactivation and Long COVID (e.g., 30), is useful for a clearer understanding of the pathophysiological process of the disease as well treatment approaches. More specifically, as Sapir and colleagues (2022) denoted and summarized, the initial viral invasion and replication produces an upper respiratory tract infection, fever, muscle fatigue, pain and, given the viral active presence, antiviral agents can be used to decrease viral load, transmission, and prevent disease progress. In the phase of immune hyperactivation, pneumonia, vasculopathy, and multiorgan pathologies such as acute cardiac and renal damage, thromboembolic strokes, sepsis and secondary infections can occur. Given the systemic nature of the disease, monoclonal antibodies, anti-coagulants, immunosuppressants, oxygen, and antiviral drugs need to be used for treatment. Finally, in Long COVID where fatigue, headache, dyspnea, and anosmia are the common symptoms, a therapeutic approach using immunosuppressants and convalescent plasma therapy may be warranted. Histopathologically, a diffuse endotheliopathy is produced by the virus with consequent multi-organ injury, which can also affect the brain with several neuropathological alterations that we reviewed previously. Herein we focus on neuroinflammation, which can occur in the acute as well as the post-acute chronic phase. There are several hypotheses on the mechanisms by which neuroinflammation arises and establishes itself, although several aspects of these processes remain to be clarified (e.g., 94, 95). It has been hypothesized that the SARS-CoV-2 virus infection-related systemic inflammatory response can lead to increased permeability of the blood-brain barrier (BBB), through which peripheral inflammatory molecules such as cytokines can access the central nervous system to produce neuroinflammation (55, 56). Furthermore, the virus could have direct access to and effect on the CNS through such circumventricular organs (CVOs) as the area postrema (AP), the subforniceal organ (SFO) and the organum vasculosum of the lamina terminalis (OVLT), which are characterized by high permeability in the BBB and fenestrated capillaries and thus are easily reachable by the SARS-CoV-2 virus via the bloodstream (e.g., (96, 97). Although, to our knowledge, there is currently no published evidence supporting this idea, it is conceivable that the SARS-CoV-2 virus could enter the CNS through the AP, the SFO and the OVLT. One of the reasons for studying the CVOs in COVID-19 is that the AP, SFO, OVLT and PVN show high expression of angiotensin-converting enzyme 2 (ACE2), which is a binding site of entry in the cells for SARS-CoV-2 virus (e.g., 96, 97). In addition, the AP is connected with the NTS, whereas the SFO, OVLT and PVN form a pathway leading to the secretion of vasopressin or antidiuretic hormone (ADH), given that circulating angiotensin II (Ang II) readily reaches the SFO and the OVLT (98, 99). Ang II in turn stimulates the PVN via direct axonal connections to secrete ADH (e.g., 100, 101). In another pathway critical for central autonomic regulation, the AP is involved via its structural connectivity with the nucleus of the solitary tract (NTS), the dorsal motor nucleus of the vagus (DMN) as well as with the nucleus ambiguus, the rostral ventrolateral medulla and the PVN (e.g., 89, 102–104). Thus, both pathways involving the AP, SFO, OVLT and PVN are important in arterial blood regulation and cardiac function; consequently, their dysfunction could well lead to cardiovascular disease (e.g., 104–108). Furthermore, the AP affects the dorsal vagal complex (DVC), which is the basis of the inflammation reflex circuitry (7). Reflecting on these considerations with histopathological observations from brain autopsies of COVID-19 victims in our mind, we believe that there is a dire paucity of specific histopathological information targeting critical brain structures associated with the neuroimmune network as described herein. We believe it is crucial to accrue this essential information, especially in Long COVID where neuroinflammation seems to be an established chronic condition, and clarify whether the fundamental member structures of the neuroimmune network, in particular the NTS, DMN of the vagus and PVN are actually affected by an inflammatory process. Nevertheless, it already has been hypothesized that neuroinflammation affects the paraventricular nucleus of the hypothalamus, which leads to an array of biobehavioral disturbances associated with a chronic alteration of the HPA axis (11). The latter question could be addressed using current neuroimaging.

Histopathologically, neuroinflammation is characterized by the activation of the astrocytes and microglia, which represent the brain’s innate immune system (29, 109). For instance, pro-inflammatory cytokines that reach the brain through a damaged BBB can activate microglia, which in turn produces cytokines that initiate a cascade of events leading to a neuroinflammatory response. When this response is short-term, it may be beneficial and contribute to tissue repair; however, if it is prolonged, it may produce damage to the neighboring tissue. More specifically, a long-term neuroinflammatory response, lasting for weeks to years, could destroy the cellular and extracellular matrix of the brain tissue, such as neurons and oligodendrocytes including the axonal myelin sheaths. Ultimately, long-term neuroinflammation would lead to brain tissue atrophy and neurodegeneration, and consequently to damage of the brain circuitry in its entirety (28, 29, 110–114). A key to understanding the involvement of neuroinflammation with respect to the neuroimmune circuitry is our ability to localize and to monitor neuroinflammation with respect to where and when it begins and to maintain a record of these parameters as the neuroinflammatory process progresses. However, monitoring neuroinflammation in vivo in the brain using CSF markers is challenging and does not provide us with specific information on its location and extent in gray matter structures and fiber pathways (29, 115). Importantly, current neuroimaging provides us with a number of techniques that are demonstrated to be a valuable aid in addressing and, eventually, solving this problem. It should be pointed out that the technological advances in imaging during the past four decades have generated tremendous innovation in medicine. Indeed, MRI was rated by general practitioners in the USA as the principal contributor to medical practice in “the decade of the brain” (i.e., 1991–2000) (116). Neuroimaging caused a revolution in neurology and neurosurgery for diagnosis and therapeutic strategies, and it is an undisputed fact that clinical neurosciences and neuroimaging are mutually benefiting from a thriving symbiosis (117). A critically important factor underlying the applicability of imaging technology is that these techniques can be performed non-invasively and in vivo, and thus in clinical settings. Current neuroimaging approaches can contribute substantially in studying the neuroimmune network in normality and pathological conditions such as neuroinflammation. Brain structure analysis is commonly done using such imaging modalities as anatomical and quantitative magnetic resonance imaging (MRI) including different types of morphometric analysis by T1/T2-weighted MRI, structural connectivity using diffusion MRI (dMRI) tractography, and neurochemically using magnetic resonance spectroscopy (MRS). In current neuroimaging clinical research, brain circuits are usually studied using structural and functional connectivity imaging techniques, namely a combination of T1/T2-weighted MRI, diffusion MRI tractography and functional MRI connectivity. Using a combination of imaging techniques enables the performance of various complex studies, which can be structural and functional or of other nature such as metabolic and vascular ( Figure 2 ). T1/T2-weighted MRI is optimal for gray matter analyses, whereas dMRI is used for white matter analysis and fiber tract delineation (e.g., 29). Functional analyses, by contrast, are performed using fMRI and functional connectivity analysis (fCONN), whereas metabolic studies are carried out using positron emission tomography (PET) (e.g., 29). Importantly, these neuroimaging techniques can also detect neuroinflammation, and to this end PET is the technique of preference (e.g., 29). PET enables us to detect neuroinflammation by using tracers such as Isoquinoline ligand 1-[2-chlorophenyl]-N-methyl-N-[1-methyl-propyl]-3-isoquinoline carboxamide (PK11195) (118), a ligand specific for the translocator protein-18-kd (TSPO) of the mitochondrial membrane (119). TSPO is highly expressed in activated microglia and to a lesser extent in reactive astrocytes and is thus detectable by PET in inflamed neural tissue (29, 120). With respect to T1-weighted imaging, there are several variants that can be of relevance in studying the neuroimmune network. T1-weighted imaging is a MRI technique used for anatomical characterization of gray matter brain structures and for gross delineation of white matter as well. Thus, T1-MRI is commonly used for MRI-based morphometric analysis (see e.g., 29, 121–123). With respect to neuroinflammation, there are two T1-weighted imaging techniques, namely gadolinium-enhanced T1, and magnetization transfer (MT) useful for the identification of neuroinflammation (28). By contrast, T2-weighted imaging is routinely used for edema detection, which appears hyperintense as compared to healthy brain tissue (124) because water has higher T2 than brain tissue and water content is increased in brain regions affected by an inflammatory process (125, 126). Fluid attenuation inversion recovery (FLAIR) and quantitative T2 can further highlight such hyperintensities, thus these techniques are also useful for the study of neuroinflammation (28, 127). Furthermore, because of edematous excessive water production in neuroinflammation, geometrical changes occur in the extracellular space of the affected area and are detectable by diffusion imaging. Thus dMRI can be used as an indirect method to measure neuroinflammation (27, 128, 129). Importantly, using a particular dMRI model called the “free-water model,” we are able to detect directly the specific contribution of extracellular water that is “free” or far away from brain tissue membranes (129). Finally, MRS allows us to detect specific metabolites, such as N-acetyl-aspartate (NAA), myo-inostitol (MI), choline compounds (Cho) and total creatine (tCr), some of which are involved in neuroinflammation (130–132). The data obtained by these techniques in vivo allow us to observe anatomical-clinical and other types of correlations, which provide us with valuable and unique information that cannot otherwise be obtained clinically, i.e., by traditional anatomical, histopathological, physiological or metabolic tests. Specifically, in investigations of neuroimmune network neuroinflammation, we would need to obtain an array of data that provide us with information regarding the neuroanatomy of the component structures of this circuitry as well as the structural, physiological and pathological status of these tissues. In essence, it is the “what, where, which and how” questions we need to answer, namely “what” is the anatomy, i.e., the individual structures of the circuitry we are investigating including the degree or extent of the pathology involving these structures; “where” is this circuitry in the individual brain under investigation; “which” is the pathological process affecting this circuitry, and “how” the mechanistic aspect of the pathological process affects this circuitry. Multimodal neuroimaging is an important instrument to address these key questions. Herein we illustrate a multimodal neuroimaging approach combining T1- weighted MRI morphometric analysis techniques and dMRI tractography (50, 51) to reconstruct the gray and white matter structures of the neuroimmune network in normal and clinical datasets as described in previous reports of our group (47, 49, 52, 133) ( Figure 3 ).

Recent neuroimaging studies in COVID-19 have shown the presence of neuroinflammation across several brain areas. More specifically, these studies have corroborated previous findings derived by histopathology in acute COVID-19, in which microglia activation and neuroinflammation have been shown in several brain areas such as the frontal lobes, olfactory bulbs, hippocampus, cerebellum and brainstem. PET studies in particular, have shown alterations in the superior and middle frontal cortex, the anterior, middle and posterior cingulate cortex, the thalamus, hippocampus, cerebellum and brainstem (see e.g., 23–25, 62, 134, 135). Diffuse alterations in the brain, involving the neocortex, thalamus, striatum, hippocampus, cerebellum and brainstem have also been reported recently in a PET study combined with histopathology in macaque monkeys (136). Importantly, these brain areas include also the principal component structures of the neuroimmune network, such as the NTS, DMN, PVN and the dorsal vagal complex (DVC)-corticolimbic connections ( Figure 4 ). The diffuse pathological alterations across brain regions such as the frontal lobe, cingulate gyrus, diencephalon and brainstem indicate that autonomic, limbic and cognitive systems should be compromised, which is reflected in the clinical and behavioral phenomenology of acute COVID-19 and also Long COVID. Among the most frequent PASC symptoms that have been reported are fatigue and brain fog characterized by altered cognitive functions, such as attention, memory and executive function in particular (137) as well as vegetative symptoms, including increased temperature, heart rate, and breathing difficulties. The assessment of these symptoms is addressed by using an array of behavioral and clinical tests and batteries. The most frequently used clinical assessment batteries are the Trial Making Test (138) and the Montreal Cognitive Assessment (139) for cognitive evaluation, and the Fatigue Severity Scale (140) for the assessment of fatigue. Finally, to evaluate vegetative symptoms, a checklist of symptoms is used including questions regarding subjective feelings of confusion, drowsiness, cold or heat sensations, nausea as well as assessment of breathing function, sleep and heart rate.