**Brain Injury and Neurodegeneration: Molecular, Functional, and Translational Approach**

Editors

**Kumar Vaibhav Meenakshi Ahluwalia Pankaj Gaur**

MDPI • Basel • Beijing • Wuhan • Barcelona • Belgrade • Manchester • Tokyo • Cluj • Tianjin

*Editors* Kumar Vaibhav Augusta University Augusta GA USA

Meenakshi Ahluwalia Augusta University Augusta GA USA

Pankaj Gaur Georgetown University Medical Center Washington DC USA

*Editorial Office* MDPI St. Alban-Anlage 66 4052 Basel, Switzerland

This is a reprint of articles from the Special Issue published online in the open access journal *Biomedicines* (ISSN 2227-9059) (available at: https://www.mdpi.com/journal/biomedicines/special issues/Brain Injury Neurodegeneration).

For citation purposes, cite each article independently as indicated on the article page online and as indicated below:

LastName, A.A.; LastName, B.B.; LastName, C.C. Article Title. *Journal Name* **Year**, *Volume Number*, Page Range.

**ISBN 978-3-0365-8384-6 (Hbk) ISBN 978-3-0365-8385-3 (PDF)**

Cover image courtesy of Kumar Vaibhav

© 2023 by the authors. Articles in this book are Open Access and distributed under the Creative Commons Attribution (CC BY) license, which allows users to download, copy and build upon published articles, as long as the author and publisher are properly credited, which ensures maximum dissemination and a wider impact of our publications.

The book as a whole is distributed by MDPI under the terms and conditions of the Creative Commons license CC BY-NC-ND.

## **Contents**


Reprinted from: *Biomedicines* **2021**, *9*, 1216, doi:10.3390/biomedicines9091216 ........... **151**


### **About the Editors**

#### **Kumar Vaibhav**

Kumar Vaibhav (MS, PhD, Asst. Prof.) is a Neuroscientist, Educator, Mentor, and Director of the Brain injury, Senescence and Translational Neuroscience laboratory in the Department of Neurosurgery, Medical College of Georgia at Augusta University, Augusta, GA, USA. Dr. Vaibhav is also affiliated with the Department of Oral Biology and Diagnostic Sciences, Center for Excellence in Research, Scholarship and Innovation, Dental College of Georgia, Augusta University, Augusta, GA and the Transdisciplinary Research Initiative in Inflammaging and Brain Aging (TRIBA), Augusta University, Augusta, GA. His main research focus is on the outcomes of traumatic brain injury and intracerebral hemorrhages with emphasis on immune interaction, the systemic influence of diseases, and alteration in the endocannabinoid system in acute and chronic pathology. His lab is further investigating pathology related to brain injury-induced senescence, neurodegeneration, and dementia.

#### **Meenakshi Ahluwalia**

Meenakshi Ahluwalia (MS, PhD) is a senior post-doctoral fellow in the Department of Pathology, Medical College of Georgia, and is currently working in the Georgia Cancer Center at Augusta University, Augusta, GA. She has expertise in traumatic brain injury, hemorrhagic stroke, glioma, and genetics. Her research interest is to develop non-invasive therapy for brain insult and oncolytic treatment for the repression of glioma.

#### **Pankaj Gaur**

Pankaj Gaur (MS, PhD—Neuroscience) is a highly accomplished researcher and scientist who has made significant contributions to the fields of neuroscience, immunology, and cancer research. Dr. Gaur is affiliated as a Research Instructor (Faculty) at Lombardy Cancer Center, Georgetown Medical College, Georgetown University, Washington DC. Dr. Gaur's primary research interest revolves around identifying innovative strategies to enhance specific immune responses against cancer, autoimmune diseases (such as multiple sclerosis and systemic lupus erythematosus), as well as various brain disorders including Parkinson's disease, epilepsy, traumatic brain injury, and neurodegeneration. His aim is to unravel the intricate mechanisms underlying immune responses, ultimately leading to the development of novel therapeutic approaches.

## **Preface to "Brain Injury and Neurodegeneration: Molecular, Functional, and Translational Approach"**

We are glad to introduce this Reprint of the Special Issue "Brain Injury and Neurodegeneration: Molecular, Functional, and Translational Approach" for the readers. This Special Issue includes 19 articles, including 1 editorial, 10 research articles, and 8 reviews on current topics of brain injury, pathology, and neurodegeneration. This collection has shed light on many aspects of brain pathologies, related meta-analysis, and alternative medicines. This Issue will be a good read for scientists, medical professionals, students, and for the general public.

We would like to acknowledge our gratitude to the authors for publishing in our Issue, and the reviewers for doing a wonderful job of promptly assessing the articles. We are also thankful to our Editor in Chief, Prof. Dr. Shaker A. Mousa, our Assistant Editor, Ms. Dora Xie, and the entire *Biomedicines* editorial staff for the support we received throughout this journey. Last but not least, I would like to applaud our team of guest editors, Drs. Meenakshi Ahluwalia and Pankaj Gaur, who helped and supported the cause.

> **Kumar Vaibhav, Meenakshi Ahluwalia, and Pankaj Gaur** *Editors*

### *Editorial* **Brain Injury and Neurodegeneration: Molecular, Functional, and Translational Approach**

**Meenakshi Ahluwalia 1,2,3, Pankaj Gaur <sup>4</sup> and Kumar Vaibhav 3,5,6,\***


Recently, we have achieved substantial progress in our understanding of brain injury and neurodegeneration. We have enhanced our knowledge about different brain pathologies or insults (accidental or non-accidental), such as hemorrhages, traumatic brain injury (TBI), ischemia, hypoxic/hypobaric insults, and neurological disorders such as Parkinson's disease and Alzheimer's disease (PD and AD). Brain pathology is multifactorial, encompassing a cascade of inflammation, necrotic, and apoptotic pathways. It is well known that brain insults or injuries to the brain may lead to neurological disorders over time, and genetic or environmental factors play essential roles in the progression of a brain disease. The absence of a specific cure to limit injury progression after an insult has persuaded the scientific community to study the mechanisms behind brain injury and degenerative cascade and to explore different therapeutic strategies.

This Special Issue, entitled "Brain Injury and Neurodegeneration: Molecular, Functional, and Translational Approach", has addressed various important aspects of brain injury and neurodegeneration, such as TBI, cerebral hypoxia, epilepsy, AD, and SARS-CoV-2-mediated brain damage. This Special Issue has received an enthusiastic response globally, resulting in the publication of 18 peer-reviewed articles, including 10 research articles and 8 reviews.

The recent SARS-CoV-2 pandemic caused more than 3 million deaths globally [1]. Similarly to past coronavirus outbreaks, including SARS and MERS, SARS-CoV-2 infections were associated with fever, dry cough, pneumonia, fatigue, and acute respiratory distress syndrome (ARDS) [2]. However, each host–pathogen interaction leaves a footprint on the health of different organs [3]. Clinical studies on COVID-19 patients have revealed atypical symptoms and neurological signs, including headaches, anosmia, nausea, dysgeusia, damage to respiratory centers, and cerebral infarction [2,4]. Extreme cytokines release (cytokine storm) occurs due to aberrant immune pathways, and microglial activation propagates CNS damage in COVID-19 patients [1–4]. Moreover, elderly with neurological problems such as PD and AD showed a higher incidence of COVID-19-associated complications [4]. In this Special Issue, an Editor's Choice article by Donegani et al. [5] provided proof of CNS damage via an [18F]-FDG PET scan. Twenty-two SARS-CoV-2 patients underwent wholebody [18F]-FDG PET examination, including a dedicated brain acquisition between May and December 2020 after their recovery from SARS-CoV-2 infection, and fourteen patients were found to have persistent hyposmia in bilateral fusiform gyri and parahippocampal, and in left insula, as compared to the controls [5].

**Citation:** Ahluwalia, M.; Gaur, P.; Vaibhav, K. Brain Injury and Neurodegeneration: Molecular, Functional, and Translational Approach. *Biomedicines* **2023**, *11*, 1947. https://doi.org/10.3390/ biomedicines11071947

Received: 4 July 2023 Accepted: 6 July 2023 Published: 10 July 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Our Special Issue has a large number of TBI-related articles, which includes five research articles and one review. TBI is a global health concern as it results in substantial death and disability [6–11]. In light of this, our group [12] has provided a comprehensive review on TBI. In this Editor's Choice review, we have provided a broad understanding of TBI pathology, mechanisms, inflammation, and immune interactions. Understanding these mechanisms and exploring potential targets for neuroprotective treatments are crucial for advancing new therapies. This review delves into the molecular events that occur following TBI, encompassing inflammation and programmed cell death, and offers an overview of the current literature and therapeutic approaches, contributing to a deeper understanding of secondary injuries caused by TBI. Cheng et al. [13] have shown that stretch injury (an in vitro model of TBI) in SH-SY5Y neuroblastoma cells altered the mitochondrial membrane potential and triggered oxidative DNA damage at 24 h. Stretch injury increased neuronal stress via reducing brain-derived neurotrophic factors (BDNFs) and increasing amyloid-β. Mechanistically, neuronal injury was exaggerated through the loss of the insulin pathway and via increased glycogen synthase kinase 3β (GSK-3β)S9/p-Tau protein levels [13]. In addition, Puhakka et al. [14] reported that small non-coding RNAs (sncRNAs) play a crucial role in modulating post-TBI neuroinflammation. Further, increased expression of the miR-146a profile and of 3- tRF-IleAAT and 3- tRF-LysTTT was found to be associated with behavioral deficits in animals with chronic TBI-induced neuroinflammation [14]. Another study by Vorn et al. [15] profiled the plasma exosomal microRNAs from young adults with mild TBI and from healthy individuals, and identified 25 dysregulated exosomal miRNAs in the chronic mTBI group 4.48 mean years after injury. These miRNAs are associated with pathways of neurological disease, organismal injury and abnormalities, and psychological disease, and can be useful to be diagnostic markers for chronic mTBI [15].

A higher percentage of patients with TBI die from secondary pathological processes despite the application of preventative measures and the provision of medical supervision [8–11]. Post-traumatic epilepsy (PTE) is one of the most common debilitating implications of TBI [16]. Post-traumatic seizures (PTS) are experienced in increasing TBI individuals who also display resistance to traditional anti-seizure medications (ASMs). Hentig et al. [17] identified an upregulated sonic hedgehog (Shh) signaling pathway in zebrafish after CNS injury that helps in regeneration. Shh signaling increases excitatory amino acid transporters (Eaat)2a to inhibit TBI-induced glutamate excitotoxicity and subsequent seizure sequelae [17]. Further, Ghosh et al. [18] provided an account for epilepsy, the roles played by various neurotransmitters and their corresponding receptors in the pathophysiology of epilepsy. One of the forefront areas of epilepsy research is drug-resistant epilepsy (DRE), which is the focus of this review. The authors mentioned that drug-resistant epilepsy (DRE) remains a prominent focus of research due to its link to psychosocial complications and premature mortality, and to a high prevalence among epileptic patients [18]. The review examines various hypotheses relating to DRE and explores unconventional therapeutic strategies and combination therapy. Additionally, recent studies supporting modern treatment approaches for epilepsy are discussed, with specific emphasis on the mTOR pathway, blood–brain barrier breakdown, and inflammatory pathways.

There are other factors, CNS insults and diseases, such as hypoxia, alcohol, viral infections, ischemia, and neurodegeneration, which contribute towards acute and chronic deficits in CNS function. Our collection also highlights the interesting findings in these particular areas. Both chronic alcoholism and human herpesvirus-6 (HHV-6) infection may cause movement-related disorders and promote neuroinflammation. Jain et al. [19] observed decreased perivascular CD68+/Iba1+ microglia in the postmortem brain from alcoholic individuals as compared to the dominant CD68+/Iba1<sup>−</sup> microglial subpopulation in the control brains. All the control brains were HHV-6 negative. Further, HHV-6 infection in alcoholics elevated microglial dystrophic changes with higher Iba1+ cells and compounded the microglia-mediated neuroinflammation. Another research article by Baltanas et al. [20] explored the rare, biallelic variants of the AGTPBP1 gene that caused its loss of function, and led to childhood-onset neurodegeneration with cerebellar atrophy

(CONDCA). Mutations in AGTPBP1 led to the substantial loss of cerebellar Purkinje cells in the mouse model of cerebellar ataxia, and might be used for CONDCA modeling in mice. Asik et al. [21] provided a review of Alzheimer's disease in relation to Amyloid-β (Aβ). They have provided a comprehensive view of Amyloid-β (Aβ)-related pathology and mechanisms and of the current clinical status of anti-amyloid therapy. They emphasized that the relationship between dysfunctional mitochondrial and the progression of AD required further research. Yoshida et al. [22] reported that higher oxidative stress in hippocampal mitochondria leads to cognitive impairment in a 5xFAD mice model of Alzheimer's disease. They further mentioned that age can be a vital factor in elevated oxidative stress in AD pathology, and preventing mitochondrial oxidative damage may be important to protect cognitive function.

Hypoxia as a result of the deprivation of oxygen, temporary or chronic, can either be adaptive or pathological. A systematic review by Stoica et al. [23] utilized the "Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA)" filtering method, and probed five internationally renowned medical databases. Using this method, they identified 45 eligible papers and provided information on pathophysiology, mechanisms, and consequent clinical conditions following hypoxic episodes. Ischemia occurs as a result of transient or permanent interruption blood supply in a given region, which leads to poor oxygenation, inflammation, and oxidative stress. Fatty acid-binding proteins (FABPs) mediate lipid metabolism and regulate the dynamics of fatty acids. Following transient MCAO, the levels of FABP3, FABP5, and FABP7 were found to be upregulated in the brain. Guo et al. [24] reported that the FABP inhibitor, i.e., FABP ligand 6 [4-(2-(5-(2-chlorophenyl)- 1-(4-isopropylphenyl)-1H-pyrazol-3-yl)-4-fluorophenoxy)butanoic acid] (referred to here as MF6), minimized the prostaglandin E2 (PGE2)-mediated inflammation in ischemic brain. Flavonoids, icariin (ICA), and icaritin (ICT) derived from *Herba epimedii* have been identified as neuroprotective phytochemicals. Wu et al. [25] reported that both ICA and ICT treatment improved neuronal cell apoptosis, minimized oxidative stress, and countered extracellular matrix (ECM) accumulation in mice brains post-acute cerebral ischemia. Demyanenko et al. [26] reviewed the role of histone deacetylases and their inhibitors in ischemic stroke.. The authors showed that ischemic stroke generally reduces gene expression via suppression of the acetylation of histones H3 and H4. Inhibitors to histone deacetylases promoted functional recovery post-cerebral ischemia by inducing neurogenesis and angiogenesis in the injured areas of brain. This review aimed to explore neuroprotective activities of histone deacetylase inhibitors in ischemic stroke. In line with neuroprotection, Haider et al. [27] investigated the role of mitoquinone in chronic neuroprotection post-TBI, and found that mitoquinone reduced gliosis, decreased oxidative stress, limited neuroinflamamtion, and improved axonal integrity and neuronal survival in an open-head CCI mouse model of moderate TBI.

With the increase in the expectancy of the life span of humans, incidences of accidental injury and neurodegenerative diseases (NDs) have risen and have imposed a considerable burden on the family, society, and the nation. The review by Khan et al. [28] explores the mechanisms of action of the phytochemicals and nutraceuticals available to date for various NDs. The group has reviewed clinical and pre-clinical studies involving phytochemicals in neurodegeneration. Despite phytochemicals showing a robust effect in animal studies, mixed results were observed in several clinical trials, and therefore, the authors stressed a need to reassess their efficacies in more robust clinical studies [28]. While traditional medicines and chemical inhibitors are being actively studied in pre-clinical and clinical settings, non-invasive methods such as exercise [29,30], whole-body vibration [31], or ischemic conditioning [32–34] can enhance endogenous protection against many diseases. Nhu et al. [35] reviewed treadmill exercise (TE) on neural mitochondria in PD. Parkinson's disease is the second most common neurodegenerative disorder [36,37], and TE has been widely applied in its rehabilitation [30,38]. For this systematic review [35], the CAMARADES checklist was used to assess the methodological quality of the studies. The review findings supported the hypothesis that treadmill exercise could attenuate neuronal

mitochondrial dysregulation and respiratory deficiency in PD and could slow down the progression of PD.

In conclusion, this Special Issue represents a novel and exciting perspective on brain injury, hypoxia, ischemia, and neurodegeneration, and it represents a comprehensive research resource for readers on disease-related pathology, mechanisms, and translational approach. However, we acknowledge that this field of neuroscience is under active research, and several new findings are being made daily. Therefore, this Special Issue of articles, along with new discoveries, will be an interesting and substantial read for scholars in this field.

**Funding:** Authors' research has been supported by grants from the National Institutes of Neurological Diseases and Stroke (NS114560), National Institutes of Child Health and Development (HD094606), and AURI Research support to K.V.

**Conflicts of Interest:** The authors declare no financial or competing conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; in the decision to publish the results; or in the editing of articles.

#### **References**


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### *Article* **Brain Metabolic Correlates of Persistent Olfactory Dysfunction after SARS-Cov2 Infection**

**Maria Isabella Donegani 1,2, Alberto Miceli 1,2, Matteo Pardini 1,3, Matteo Bauckneht 1,2,\*, Silvia Chiola 4,5, Michele Pennone 1,2, Cecilia Marini 1,6, Federico Massa 1,3, Stefano Raffa 1,4, Giulia Ferrarazzo 1,4, Dario Arnaldi 1,4, Gianmario Sambuceti 1,4, Flavio Nobili 1,3 and Silvia Morbelli 1,2**


**Abstract:** We aimed to evaluate the brain hypometabolic signature of persistent isolated olfactory dysfunction after SARS-CoV-2 infection. Twenty-two patients underwent whole-body [18F]-FDG PET, including a dedicated brain acquisition at our institution between May and December 2020 following their recovery after SARS-Cov2 infection. Fourteen of these patients presented isolated persistent hyposmia (smell diskettes olfaction test was used). A voxel-wise analysis (using Statistical Parametric Mapping software version 8 (SPM8)) was performed to identify brain regions of relative hypometabolism in patients with hyposmia with respect to controls. Structural connectivity of these regions was assessed (BCB toolkit). Relative hypometabolism was demonstrated in bilateral parahippocampal and fusiform gyri and in left insula in patients with respect to controls. Structural connectivity maps highlighted the involvement of bilateral longitudinal fasciculi. This study provides evidence of cortical hypometabolism in patients with isolated persistent hyposmia after SARS-Cov2 infection. [18F]-FDG PET may play a role in the identification of long-term brain functional sequelae of COVID-19.

**Keywords:** 18F-FDG PET; anosmia; COVID-19; SARS-CoV-2; olfactory dysfunction

#### **1. Introduction**

Coronavirus disease 2019 (COVID-19) due to SARS-CoV-2 infection was initially thought to be mainly restricted to the respiratory system, but it has become evident that this disease also involves multiple other organs, including the central and peripheral nervous system. Indeed, neurological complications such as stroke, encephalopathy, delirium, meningitis, seizures, and cranial nerve deficits have been reported in patients with COVID-19 [1]. Besides these more severe manifestations and complications, other frequent symptoms of COVID-19 are loss of smell (anosmia) and taste (ageusia) which can occur as first symptoms of infection or in the absence of any other clinical features [1]. In a European study including more than 400 COVID-19 patients, olfactory dysfunction and ageusia were reported in 86% and 82% of patients, respectively [2]. Different underlying mechanisms have been advocated to explain the presence of anosmia in patients with COVID-19. These include olfactory cleft syndrome, direct damage of olfactory sensory

**Citation:** Donegani, M.I.; Miceli, A.; Pardini, M.; Bauckneht, M.; Chiola, S.; Pennone, M.; Marini, C.; Massa, F.; Raffa, S.; Ferrarazzo, G.; et al. Brain Metabolic Correlates of Persistent Olfactory Dysfunction after SARS-Cov2 Infection. *Biomedicines* **2021**, *9*, 287. https://doi.org/ 10.3390/biomedicines9030287

Academic Editor: Kumar Vaibhav

Received: 13 January 2021 Accepted: 9 March 2021 Published: 12 March 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

neurons, postviral anosmia syndrome, cytokine storm, and/or impairment of the olfactory perception centers in the brain [3]. Indeed the olfactory bulb might represent a potential route of entry of SARS-CoV-2 in the CNS, and the investigation of the pathophysiology of olfactory dysfunction might help to further understand the pathogenesis and long-term implications of CNS involvement in COVID-19 [4]. In this framework, the availability of sensitive biomarkers tracking disease substrates might speed up the investigation of CNS involvement in patients with SARS-CoV-2 infection and might be used to monitor and better predict the risk of long-term effects. To date, there are few, partially conflicting, results on magnetic resonance imaging (MRI) abnormalities in patients with COVID-related anosmia [5,6]. Indeed normal, transiently increased, and even reduced volume of the olfactory bulb has been reported in patients with isolated or persistent anosmia [5,6]. Similarly, [ 18F]-Fluorodeoxyglucose ([18F]-FDG) PET data of COVID-19 patients with anosmia have to-date been made available only through case reports and small case series of patients with a self-reported reduction in smell [7–11]. This very preliminary evidence has been mainly acquired at the time of viral infection or just after recovery in patients affected by moderate to severe disease, thus complicating data interpretation [11].

[ 18F]-FDG PET may represent a sensitive tool to further confirm SARS-CoV-2 neurotropism through the olfactory pathway. Furthermore, given the potential functional and cognitive sequelae of COVID-19 and the established role of PET to support differential diagnosis of cognitive impairment, [18F]-FDG PET can represent a suitable tool to identify the concomitant involvement of cortical structures potentially relevant for subsequent persistent cognitive, sensory or emotion disturbances [8]. Moreover, an increasing number of patients showing persistent symptoms (such as fatigue, dyspnea, anosmia/dysgeusia, memory impairment, and pain) have been described after recovery from SARS-CoV-2, defining an emerging chronic syndrome, so-called Long Covid [12,13]. Given these premises, we aimed to evaluate the presence of regional brain hypometabolism in patients with persistent isolated and objectively-assessed olfactory dysfunction after recovery from SARS-CoV-2 infection.

#### **2. Material and Methods**

#### *2.1. Patients*

Patients with anosmia after SARS-CoV-2 infection were recruited among subjects who underwent whole-body [18F]-FDG PET, including a dedicated brain acquisition for clinical reasons other than SARS-CoV-2 infection in our institution between May and December 2020 (following their recovery after infection). The main inclusion criteria were previous SARS-CoV-2 infection, confirmed by polymerase chain reaction (PCR) at the time of initial symptoms, PET examinations performed during the recovery phase of SARS-CoV-2 infection, and an olfactory test still demonstrating olfactory dysfunction. The recovery phase was defined as when at least one negative swab test after infection was available. Exclusion criteria were demonstration of brain lesions on MRI, previous diagnosis of encephalopathy/encephalitis or cerebrovascular disorders due to or concomitant with the SARS-CoV-2 infection, or any other previous or current neurological or psychiatric disease. Patients that previously required mechanical ventilation or showed severe respiratory distress syndrome due to SARS-CoV-2 infection were also excluded, given the potential independent effect of these clinical scenarios on brain metabolism. Patients with a history of anosmia before SARS-CoV-2 infection, as well as those treated with chemotherapy in the last 3 months or previous radiotherapy in the head and neck district for oncological reasons, were also excluded. The study was approved by the Regional Ethical Committee (CER Liguria code 671/2020), all procedures and informed consent collection were in accordance with the ethical standards of the 1964 Helsinki declaration.

#### *2.2. Olfactory Test*

Olfaction was assessed by means of the Smell diskettes olfaction test [14] on the same day of PET examination. In fact, while self-reported newly onset loss of smell is important from an infection control perspective, self-reporting may result in misdiagnosis. The test was based on reusable diskettes as applicators of 8 different odorants. Using a questionnaire with illustrations, the test was designed as a triple forced multiple-choice test resulting in a score of 0 to 8 correct answers. Hyposmia was defined as making at least 2 mistakes on the questionnaire; the number of correct answers was recorded.

#### *2.3. [18F]-FDG Brain PET Acquisition and Image Processing*

A dedicated [18F]-FDG Brain PET acquisition was performed in all recruited patients according to the European Association of Nuclear Medicine (EANM) guidelines on two Siemens Biograph PET/CT systems (16 and mCT Flow 40, respectively) in the same center [15]. Images preprocessing was conducted using Statistical Parametric Mapping software version 8 (SPM8; Wellcome TrustCenter for Neuroimaging, London, UK) [16]. See Supplementary Materials for further details.

#### *2.4. Voxel-Wise Analysis of Hypometabolic Signature of Olfactory Dysfunction after SARS-CoV-2 Infection*

After preprocessing, smoothed images underwent a whole-brain voxel-wise group analysis to identify regions of relative hypometabolism with respect to a control group of 61 subjects consisting of 48 healthy controls acquired on the Biograph 16 system and previously recruited in our laboratory without any neurologic or psychiatric disease as detailed elsewhere [17] and thirteen subjects with smoldering multiple myeloma with both normal body and brain scans acquired on Biograph mCT Flow 40 PET/CT system (age 61.1 ± 11.1; 10 males). Patients with smoldering myeloma had no present or previous history of neurologic or psychiatric diseases and were never submitted to chemotherapy. Age, gender, and scanner type were included as nuisance variables in the analysis. We set a height threshold of family-wise error (FWE)-corrected *p* < 0.05 for multiple comparisons, at both the peak and cluster levels. Details on SPM analysis are included in the Supplementary Materials.

#### *2.5. Structural Connectivity of Regions of Hypometabolism in Patients with Olfactory Dysfunction*

The hypometabolic clusters in patients with hyposmia with respect to controls (hyposmia clusters) which had been obtained by means of the whole brain voxel-based analysis in SPM8, were saved as a volumetric region of interest (VOI). To assess the structural connectivity of metabolic correlates of hypo/anosmia after SARS-CoV-2 infection, we used the "Brain Connectivity and Behaviour" (BCB\_ toolkit (18, http://www.toolkit.bcblab.com (accessed on 10 December 2020)), which included diffusion MRI data from healthy control subjects. Moreover, using the disconnectome pipeline in the BCB toolkit we computed structural connection maps of all voxels included in the hyposmia clusters by tracking fibers passing through them to identify their structural connectivity with other brain areas [18,19]. Briefly, the hypometabolic clusters present in patients with hyposmia with respect to controls (hyposmia clusters) and obtained by means of the whole brain voxelbased analysis in SPM8 were saved as VOI [16]. First, using the Tractotron pipeline, we evaluated the probability of the major white matter tracts crossing the hyposmia clusters and considered as significant only those voxels with a probability of at least 0.5. Moreover, using the disconnectome pipeline of the BCB toolkit [18,19], we computed the structural connection maps of all voxels included in the hyposmia clusters by tracking fibers passing through them to identify their structural connectivity with other brain areas as previously described [20]. Details about this procedure are detailed elsewhere [21].

#### **3. Results**

#### *3.1. Patients*

Twenty-two consecutive patients (12 males and 10 females; mean age 64 ± 10.5 years, range 35–79) in the recovery phase of SARS-CoV-2 infection were submitted to wholebody [18F]-FDG PET in our center from 1 May and 1 December 2020. [18F]-FDG PET was performed between 4 and 12 weeks after the first positive RT-PCR nasopharyngeal swab for SARS-CoV-2. Only nineteen of these patients met our inclusion criteria and were submitted to Smell diskettes olfaction test, which indicated the presence of hyposmia in fourteen of them who have been ultimately included in the present analyses. Figure 1 and its notes report the steps that narrowed the final study group. Further details on reasons for patients' exclusion are reported in the Supplementary Materials. Characteristics of the 14 analyzed patients are detailed in Table 1.


#### **Table 1.** Patients' Characteristics.

Values are shown as mean ± standard deviation (range). \* None of these patients was complaining of other known possible sequelae of COVID-19 such as fatigue, chest pain, dyspnea, or reported any other focal neurological signs both at the time of SARS-CoV-2 infection and at the time of PET. † None of the patients had proven previous COVID-related lung involvement or previously received steroids, hydroxychloroquine, or other medication specifically aimed to support patients' response to COVID-19 (other than paracetamol).

#### *3.2. Hypometabolism in Patients with Isolated Persistent Hyposmia after SARS-CoV-2 Infection*

With respect to the controls, patients with hyposmia after SARS-CoV-2 infection were characterized by relative hypometabolism in parahippocampal (Brodmann area (BA) 36), fusiform (BA 20 and 37) gyri in both hemispheres and in the insula in the left hemisphere (BA 13). Clusters of significant hypometabolism in patients with hyposmia after SARS-CoV-2 are reported in Figure 2. Details on coordinates and z-score are reported in Table 2.

**Figure 2.** Hypometabolism with respect to controls in patients still presenting with hyposmia during early recovery after SARS-CoV-2 infection was highlighted in parahippocampal and fusiform gyri in both hemispheres (BA 20, 36, 37) and in the insula in the left hemisphere (BA 13). Height threshold of significance was set at *p* < 0.05 FWE-corrected at the cluster level. Regions of significant difference are shown color-graded in terms of Z values. Talairach coordinates and further details are available in Table 2.


< 0.05, corrected for multiple comparisons with the Family-Wise error option both at peak and cluster level were accepted as statistically significant. In the 'cluster level' section on the left, the corrected *p*-value and the brain lobe with hypometabolism are reported. In the 'peak level' section on the right, the Z score and peak coordinates, the corresponding cortical region, and Brodmann area (BA) are reported. L, left; R, right.

*p*

#### *3.3. Hyposmia Clusters Tractography and Connectivity*

The hyposmia cluster was found to be included in the bilateral longitudinal fasciculi (ILF) with a probability 0.82 and 1 for the left and right ILF, respectively. The tractography results for the hyposmia cluster are shown in Figure 3.

**Figure 3.** Structural connectivity of regions of hypometabolism in patients with olfactory dysfunction generated through the Brain Connectivity and Behaviour (BCB) toolkit (http://www.toolkit.bcblab.com (accessed on 10 December 2020)), which includes diffusion MRI data from healthy control subjects. Panel (**A**): The connectome map indicated a significant probability of connection of the hyposmia cluster with the inferior longitudinal fasciculus; Panel (**B**): tractography results of the hyposmia cluster.

#### **4. Discussion**

The present brief communication provides a demonstration of brain hypometabolism, namely in the bilateral limbic cortex, in a group of patients with isolated persistent hyposmia proven by olfactory test more than four weeks after SARS-CoV-2 infection. The highlighted area of hypometabolism also encompassed the insula in the left hemisphere and included the bilateral ILF.

One of the ongoing hypotheses to explain the anosmia of patients with COVID-19 (in the absence of nasal congestion) is that the virus enters the CNS through the first neurons of the olfactory pathway located in the olfactory mucosa [1]. Post-infectious olfactory dysfunction is thought to be caused by damage to the olfactory epithelium or central olfactory processing pathways [22]. The present evidence of hypometabolism in two symmetric, similar regions within the limbic cortex may support the occurrence of a distal involvement of the olfactory pathway. Moreover, the bilateral involvement of key cortical structures is sound from the pathophysiological point of view. In fact, hyposmia might not be subjectively perceived in case of unilateral involvement of the olfactory pathway [23]. To-date one case report provided [18F]-FDG PET data in a patient with mild COVID-19 and isolated and persistent anosmia (in absence of any other COVID-related symptom) [10]. In fact, Karimi-Galougahi and colleagues reported the case of a 27-year-old woman with persistent anosmia for six weeks presenting hypometabolism of the left orbitofrontal cortex but with preserved metabolism in temporal cortex [10]. However, the evaluation of images was mainly based on visual inspection without observed-independent analysis or comparison with a control database which might have helped to more accurately evaluate a small region such as the medial temporal lobe. Evidence about the involvement of the limbic cortex after recovery from COVID-19 was also provided by two well-documented cases of patients submitted to whole body PET to assess metabolic activity of residual lung lesions just after COVID-related pneumonia [8]. Hypometabolism of the olfactory/rectus gyrus was present

in both patients with additional hypometabolims within the amygdala, hippocampus, parahippocampus, cingulate cortex, pre-/post-central gyrus, thalamus/hypothalamus, cerebellum, pons, and the medulla in only one of them (who was not reporting anosmia). However, at the time of infection both these patients required hospitalization in intensive care unit and in one case, mechanical ventilation was needed. These more severe presentations and in particular mechanical ventilation may, at least in theory, have played a role on [ 18F]-FDG PET hypometabolic regions especially during early recovery [9,24,25]. However, this limitation does not apply to our patients' group. Recently, Guedj and colleagues provided the first brain FDG PET data in Long COVID patients and again demonstrated bilateral hypometabolism in the bilateral rectal/orbital gyrus, amygdala and the hippocampus, brainstem and bilateral cerebellum. Thus, our findings largely confirm the topography of brain hypometabolism in patients in long COVID patients although associated with persistent hyposmia or with other persistent functional complaints [26]. The presence and the topography of hypometabolism after recovery in all the mentioned case reports, in previous small group studies and in our hyposmic group repetitively highlighted an involvement of limbic regions and might point to the risk of developing long-term neurological (possibly cognitive) sequelae, a hypothesis requiring studies in patients with a much longer recovery from infection [8,27,28]. Indeed, olfactory cortical area feeds into multimodal integration relevant for cognition control and the hippocampal regions is known to exchange input for storage of olfactory memory (also relevant for working memory [29]). Almeria et al. evaluated the impact of COVID-19 on neurocognitive performance in thirty-five patients with confirmed COVID-19 infection and found that the presence of anosmia and dysgeusia at the time of infection were among the main risk factors for cognitive impairment related with attention, memory and executive function [28].

Of note, the present group of patients showed hypometabolism also in the insula in the left hemisphere. The insula is densely interconnected with orbitofrontal and anterior cingulate cortices, amygdala, and hippocampus [30] and plays a key role in processing selfawareness. Indeed, olfaction aims to provide critical information about the environment subsequently directed at the cortical level also for multisensory integration.

Regarding the connectivity data, the involvement of the ILF is in line with observations of its role in hyposmia in Parkinson's Disease [31]. Interestingly, the ILF has been shown to be affected early on in viral infections, such as in HIV [32] and hepatitis C virus (HCV) [33].

Finally, it should be noted that MRI cortical signal has been evaluated in patients with COVID-19 and anosmia in few small studies. However, abnormalities have been substantially reported in the very early phase of infection [5,6,34,35]. In this framework, while the presence of hypometabolism at [18F]-FDG PET cannot prove the direct spread of the virus along the olfactory pathway and cortex, the high sensitivity of FDG PET for cortical deafferentation may act as a measurable biomarker of persistent impairment of the transmission along the olfactory pathway [36]. Further investigation might help to understand if [18F]-FDG PET data could be used to predict the prognosis of olfactory function recovery also at the single patient level [34]. The present study has some limitations, mainly related to its naturalistic observational nature and to the small group of patients being submitted to [18F]-FDG PET for other clinical reasons, including the suspect or follow-up of oncological diseases. Brain lesions were radiologically excluded in all patients, and to reduce the potential confounding effect of comorbidities, we also excluded patients submitted to chemotherapy in the last three months or who underwent radiotherapy in the head and neck district. Indeed despite the small number of included patients, the present study provides a group analysis on brain metabolism of patients with persisting olfactory dysfunction after SARS-CoV-2 infection for the first time proven by olfactory test. The demonstration of hyposmia by means of an olfactory test (that was not possible in larger epidemiologic studies) and the exclusion of patients who suffered from COVID-related pneumonia, requested COVID-oriented treatment or mechanic ventilation is a further strength of the present study [37].

#### **5. Conclusions**

The COVID-19 outbreak has impacted clinical neurology in previous months, and other challenges might come in the next future. For several reasons, a not negligible number of neurological and cognitive complaints might emerge once the acute phase of the pandemic crisis is overcome. It will be of great scientific and clinical relevance to describe COVID-19 related cognitive symptoms (likely to be reversible or in any case not progressive) and to identify and characterize biomarkers that will help us to support clinical differential diagnosis with respect to cognitive impairment caused by neurodegenerative disease. [18F]-FDG PET might play a role in this clinical setting. To this aim, we will need to be aware of the confounding effect of subtle sequelae of SARS-COV2 infection and on their reflection on PET and other biomarkers as demonstrated in the present study.

**Supplementary Materials:** Descriptive Details on patients' selection and further details on image preprocessing and analysis are available online at https://www.mdpi.com/2227-9059/9/3/287/s1.

**Author Contributions:** Conceptualization, S.M. and M.I.D.; methodology, S.M., M.I.D., A.M., and M.P. (Matteo Pardini); software, S.M., M.P. (Matteo Pardini); validation and formal analysis, S.M., M.I.D., and M.P. (Matteo Pardini); investigation, all authors.; data curation, all authors; writing original draft preparation, S.M., M.I.D., and M.P. (Matteo Pardini); writing—review and editing, all authors; visualization, all authors; funding acquisition, S.M. and M.B.; All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by a grant from the Italian Ministry of Health—Rete Italiana di Neuroscienze.

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Review Board.

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study. Written informed consent for publication was obtained from all participants.

**Data Availability Statement:** The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

**Acknowledgments:** Authors are grateful to radiologic technologists, nurses, and administrative personal of the Nuclear Medicine Unit of the IRCCS Policlinico San Martino for their contribution to the acquisition of brain PET data of the patients included in this study. We also would like to thank them for their constant presence and support in the assistance of patients who underwent [ 18F]-FDG PET for oncologic and neurologic clinical reasons at our institution during the pandemic emergency. This work was supported by a grant from the Italian Ministry of Health—Rete Italiana di Neuroscienze.

**Conflicts of Interest:** Silvia Morbelli and Flavio Nobili have received speaker honoraria from G.E. Healthcare. All other authors declare no conflict of interest.

#### **References**

