*Article* **Innovating Care in Multiple Sclerosis: Feasibility of Synchronous Internet-Based Teleconsultation for Longitudinal Clinical Monitoring**

**Nima Sadeghi 1,2, Piet Eelen 2, Guy Nagels 1,3,4,5, Corinne Cuvelier 2, Katinka Van Gils 2, Marie B. D'hooghe 1,2,3, Jeroen Van Schependom 3,6,† and Miguel D'haeseleer 1,2,3,\*,†**

**Abstract:** The 'coronavirus disease of 2019' crisis has recently forced an expedited adoption of teleconsultation (TC) in most medical domains. Short-term digital interventions have generally been associated with feasibility, clinical benefits, user satisfaction, and cost-effectiveness in patients with multiple sclerosis (MS) but outcomes after repeated utilization over extended periods need to be further evaluated. In this feasibility study, 60 subjects with MS were 1:1 randomized to receive standard care augmented by four TCs using an audiovisual Internet platform (intervention) versus standard care alone (controls), over a period of 12 months. Effects on functional status, medical costs, and satisfaction were explored as secondary outcomes. Eighty-nine out of 108 scheduled TCs (82.4%) were completed, and 26 patients could complete at least one TC (86.7%), meeting our prespecified feasibility target of 80%. The intervention did not lead to significant differences in functional status (with the potential exception of fatigue) nor medical costs. Most interventional patients declared themselves to be (very) satisfied about the quality of care and technical aspects associated with the TCs. Our results demonstrate that longitudinal clinical monitoring using real-time audiovisual TC over the Internet is feasible and well-received by patients with MS. Such an approach can be a promising new care strategy.

**Keywords:** multiple sclerosis; teleconsultation; internet; feasibility; digital health

#### **1. Introduction**

Modern society, including the way we practice medicine, has been shaped by the technological progress inherited from the three industrial revolutions that have taken place since the mid-eighteenth century. An extension of the third phase is currently unfolding, characterized by a shift towards digital electronics, augmenting computing power, artificial intelligence, and a dominant role for the Internet. Telemedicine (TM)—sometimes also referred to as e-health or digital medicine—can be defined as the exchange of medical information between patients and healthcare providers, who are in separate locations, by means of electronic communication technology [1]. Phone calls and video conferencing are prototypical examples of directly interactive sessions in real-time, whereas their asynchronous

**Citation:** Sadeghi, N.; Eelen, P.; Nagels, G.; Cuvelier, C.; Van Gils, K.; D'hooghe, M.B.; Van Schependom, J.; D'haeseleer, M. Innovating Care in Multiple Sclerosis: Feasibility of Synchronous Internet-Based Teleconsultation for Longitudinal Clinical Monitoring. *J. Pers. Med.* **2022**, *12*, 433. https://doi.org/ 10.3390/jpm12030433

Academic Editor: Cristina M. Ramo-Tello

Received: 2 January 2022 Accepted: 4 March 2022 Published: 10 March 2022

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**Copyright:** © 2022 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/).

counterparts (e.g., texting, email, self-scoring devices, wearable sensors, instructive tools) rely on store-and-forward transmission of medical data and/or advice [2,3]. TM applications have originally been developed to enable medical services that are difficult to deliver face-to-face, mainly due to time-sensitive requirements or geographic hurdles, but are now increasingly and more widely solicited as a complementary support in conjunction with the more traditional customs [4,5]. Especially in 2020, digital medicine has undergone an expedited adoption, as the 'coronavirus disease of 2019' (COVID-19) rapidly evolved into a global crisis, for which unprecedented social isolation and mobility restrictions have been installed as key measures of constrain [6,7], with teleconsultation (TC) becoming an essential escape scenario for a vast proportion of our health system [8–10].

Studies that have explored the impact of TM in patients with multiple sclerosis (MS), a frequently occurring, chronic inflammatory demyelinating and degenerative disorder of the central nervous system [11], were heterogeneous in terms of intervention type, methodology, and objectives (i.e., disability assessment, disease management, remote treatment, rehabilitation/exercise) but have generally shown that the applied procedures provide clinical benefit, user satisfaction, time gain, and/or cost-effectiveness [3,12]. Notably though, scientific data on synchronous communication for TC purposes remain scarce in this field. Two independent pilot studies have recently demonstrated that such single sessions, using an audiovisual Internet platform, were feasible and well-received in patients with MS [13,14]. The outcomes after repeated utilization over extended periods of time, however, are still unknown and form the subject of our current paper, hereby addressing an important and actual knowledge gap in the anticipation that TC—once we have reached the post-COVID-19 era—will consolidate its newly obtained place in the MS clinic [15].

#### **2. Materials and Methods**

#### *2.1. Study Design, Objectives and Ethics*

We performed a single-center prospective study exploring the feasibility (primary endpoint) of multiple planned synchronous TCs, using an audiovisual Internet platform, in the longitudinal clinical monitoring of patients with MS. Effects on functional status, medical costs estimates, and satisfaction were assessed as secondary outcomes; a randomized control trial design was adopted because most of these exploratory analyses required a comparison to standard care. Our study was approved by the ethics committee of the Nationaal Multiple Sclerose Centrum (NMSC) Melsbroek (local; internal reference: AvN/AVDZ) and the Universitair Ziekenhuis Brussel (leading; internal reference: 2018/269, Belgian Unique Number:<PHONE_NUMBER>97). Written informed consent was obtained from all participants prior to inclusion. Recruitment and teleconsulting procedures were similar to a preparatory pilot, in which one digital visit was planned in twenty patients with MS [14], who all agreed to be involved in this longitudinal project as well.

#### *2.2. Participants*

Sixty French- and/or Dutch-speaking patients with MS, according to the 2017 revised McDonald criteria [16], were recruited at the NMSC Melsbroek, which is a specialized Belgian MS center, during routine medical follow-up. Home access to the Internet with a webcam-equipped device was mandatory for study participation. Inclusion of individuals with a high suspicion of moderate to severe cognitive impairment (based on common sense judgement of the medical record and/or initial patient contact; any known cognitive dysfunction defined as scoring less than 21 on the Mini Mental State Evaluation, if present in the medical record, was used as an exclusion criteria) was actively avoided. Eligible candidates were 1:1 randomized to receive standard care (control group) versus standard care, augmented by four scheduled TM visits (intervention group) over the following 12 months, using "Research Randomizer" (https://randomizer.org; last accessed on 22 February 2020).

#### *2.3. Teleconsultations*

The study period started with an inclusion visit and terminated with a close-out evaluation (both face-to-face), conducted by the study supervisor (MiD), with an interval of 12 ± 2 months between both. Subjects in the intervention group received the date and time of their first TC after randomization, and subsequent appointments were planned at the end of each digital contact, striving for an equal distribution over the study period (i.e., one TC every 3 months). The rescheduling of digital appointments was permitted as this regularly occurs with standard visits as well. All TCs were performed by the same MS nurse or neurologist—derived from a pool of three consulting nurses (PE, CC, KVG) and one neurologist (MiD)—for each individual patient, using an Internet-based audiovisual communication platform obtained from Zebra Academy [17]. The main goal of the TCs was to explore the patient's current global health status during a routine clinical consult. A checklist (with questions about e.g., general and neurological health, medication, and life-style factors—see Table in Ref. [14]), similar to the usual content of a regular face-to-face MS consultation, was used as the backbone for the conversation, but deviations at the patient's initiative were allowed. Patients were provided with a unique hyperlink by email in advance, leading them directly to the virtual waiting room where they could see and accept our incoming call (see Figure in Ref. [14]). Access was possible from any device with a webcam (i.e., laptop, desktop, tablet, smartphone). Google Chrome (Google LLC, Mountain View, CA, USA) was used as the web browser on both sides of the connection, as advised by Zebra Academy. All subjects had 30 min to respond to the call, starting from the scheduled time, with maximum of three attempts. A written report was forwarded by the study team to the treating neurologist after each TC.

#### *2.4. Evaluations*

#### 2.4.1. Feasibility

Our TC approach was considered feasible if at least 80% of the patients in the intervention group could complete at least one digital visit and if at least 80% of the total number of scheduled digital visits could be completed (a priori defined).

#### 2.4.2. Functional Status

Clinical relapses during the study period were recorded, as self-reported by the patients, at the close-out visit. Disability, clustered symptomatology, and health-related quality of life were assessed at both the inclusion and close-out visit. The following variables were recorded: general disability with the Expanded Disability Status Scale (EDSS), mobility with the Timed 25-Foot Walk Test (T25FWT), dexterity with the Nine-Hole Peg Test (9HPT), information-processing speed with the Symbol Digit Modalities Test (SDMT), fatigue with the Fatigue Severity Scale (FSS), depression with the Beck Depression Inventory (BDI) and the Hospital Anxiety and Depression Scale (HADS), anxiety with the HADS, sleep quality with the Pittsburgh Sleep Quality Index (PSQI), and overall health-related quality of life with the Multiple Sclerosis Impact Scale (MSIS-29).

#### 2.4.3. Medical Costs

The number of emergency room visits, days of hospital admission (not including the study site) and visits to the general practitioner over the study period, as self-reported by the patients, were recorded at the close-out visit, as a proxy of medical costs.

#### 2.4.4. Satisfaction

The satisfaction with the study trajectory was enquired about for all patients, and their respective digital caregiver if applicable, at the close-out visit by means of 5-point Likert scales containing the following categories: very unsatisfied, unsatisfied, neutral, satisfied, very satisfied. Assessments were independently carried out for (a) global quality of care, (b) technical quality of the TCs, (c) convenience of the TCs, (d) quality of care of the TCs, and (e) added value of the TCs to medical care. Only item (a) was relevant

for participants of the control group. Quantification per item was performed by giving a score of one for responding 'very unsatisfied' and increasing by one point for each higher response category.

#### *2.5. COVID-19 Interference*

Patients were enrolled in this project from 26 August 2019 to 21 February 2020. Due to unexpected COVID-19 measures, non-urgent medical appointments had to be postponed in Belgium on multiple occasions during the course of the study (particularly March–June and October–November 2020). This resulted in (a) two close-out evaluations falling outside the foreseen time frame (15 and 16 months after the inclusion visit, respectively) and (b) the need for scheduling several TCs sooner than initially intended. Timelines containing inclusion, close-out and all TC visits for each participant of the intervention group are displayed in the Figure 1.

**Figure 1.** Timeline of study visits for each participant of the intervention group. M: month, P: patient. \*\* close out visit, ◦◦ drop-out. Successful teleconsultations are indicated in green, unsuccessful attempts in red. Corrections: 1 the successful teleconsultation was performed at M6, 2 two successful teleconsultations were performed at M6, 3 two successful teleconsultations were performed at M10.

#### *2.6. Statistics*

All statistical procedures were performed with GraphPad Prism version 9.0.0 (Graph-Pad Software; San Diego, CA, USA). Data are expressed as mean ± SD for reasons of uniformity. Group differences were assessed by means of unpaired Student *t* tests or Mann–Whitney *U* tests, where appropriate, depending on the data distribution revealed by Shapiro–Wilk testing. All reported *p* values are two-tailed and were considered statistically significant at the 0.05 level.

#### *2.7. Data Availability*

Anonymized data will be shared upon reasonable request from any qualified investigator.

#### **3. Results**

#### *3.1. Participants*

Baseline demographical data of all participants are shown in Table 1. Twenty-four of the patients randomized to the intervention group were under disease-modifying treatment at study inclusion (interferon beta: 1; teriflunomide: 1; dimethylfumarate: 3; fingolimod: 1; ocrelizumab: 4; natalizumab: 8; alemtuzumab: 6), versus 26 of the controls (interferon beta: 5; glatiramere acetate: 4; teriflunomide: 3; dimethylfumarate: 3; fingolimod: 1; ocrelizumab: 4; natalizumab: 6). Four subjects in each group dropped out during the study; reasons were loss of follow-up (one intervention subject who cancelled his first TC versus three controls), loss of interest (two intervention subjects versus one control), and a suitable device being no longer available (one intervention subject). Timing of drop-out in the intervention group can be deduced from the Figure 1.

**Table 1.** Baseline demographic of the study participants.

MS: multiple sclerosis, RR: relapsing-remitting, SP: secondary progressive, PP: primary progressive, ES: elementary school, HS: high school, HE: higher education, U: unemployed, E: employed (active), S: sick leave (temporary), D: disability leave (permanent), R: retired. \* Data expressed as mean (SD).

#### *3.2. Feasibility*

Eighty-nine out of 108 scheduled TCs (82.4%) were successfully completed during the study while 26 patients could successfully complete at least one TC (86.7%). Failures were due to patients not responding (14/19) and technical issues (5/19). The non-responders were contacted at a later time by telephone and advised us that they either had forgotten the appointment or did not want to participate any longer (two subjects). Technical issues included no notification of the incoming call (three occasions), difficulties maintaining the Internet connection (one occasion), and insufficient quality of sound (one occasion). Isolated success rates were 21/29 (72.4%) for TC-1, 24/27 (88.9%) for TC-2, and 22/26 (84.6%) for TC-3 and -4.

#### *3.3. Functional Status*

The mean number of patient-reported relapses during the study period did not differ between participants of the intervention and control groups (0.3 ± 0.5 each, *p* = 0.54) who completed the study. Results of all other functional outcome measures are shown in Tables 2 and 3. Mean changes were not statistically significant except for the FSS scores, which decreased by 0.3 ± 1.2 in patients of the intervention group, compared with an increase of 0.4 ± 1.0 in those of the control group (*p* = 0.03).

#### *3.4. Medical Costs*

No significant differences were observed in the mean number of emergency room visits (0.3 ± 0.5 versus 0.2 ± 0.5, *p* = 0.91), days of hospital admission (0.7 ± 2.7 versus 2.7 ± 8.0, *p* = 0.18), and the number of visits to a general practitioner (3.1 ± 4.5 versus 2.1 ± 2.1, *p* = 0.77) over the study period between patients of the intervention and control groups, respectively, who completed the study.

#### *3.5. Satisfaction*

Quantified results of the satisfaction enquiry are demonstrated in Table 4. The proportion of patients in the intervention group—who completed the study—declaring themselves to be satisfied or highly satisfied was 26/26 for global quality of care, 19/26 for technical quality of the TCs, 24/26 for convenience of the TCs, 24/26 for quality of care of the TCs, and 23/26 for added value of the TCs to medical care; results for the health professionals who performed the TCs were 25/26, 16/26, 21/26, 25/26, and 25/26, respectively.

**Table 2.** Functional outcomes in patients randomized to the intervention group who completed the study (*N* = 26) \*.

EDSS: Expanded Disability Status Scale; T25FWT: Timed 25-Foot Walk Test; 9HPT-dom: Nine-Hole Peg Test for dominant hand; 9HPT-ndom: Nine-Hole Peg Test for non-dominant hand; SDMT: Symbol Digit Modalities Test; FSS: Fatigue Severity Scale; BDI: Beck Depression Inventory; HADS-anx: Hospital Anxiety and Depression Scale for anxiety; HADS-dep: Hospital Anxiety and Depression Scale for depression; PSQI: Pittsburgh Sleep Quality Index (PSQI); MSIS-29-psy: Multiple Sclerosis Impact Scale for psychological impact; MSIS-29-phy: Multiple Sclerosis Impact Scale for physical impact. \* Scores expressed as mean (SD) [missing values]; *p* value for comparison with the respective change in the control group, as expressed in Table 3.

**Table 3.** Functional outcomes in patients randomized to the control group who completed the study (*N* = 26) \*.

EDSS: Expanded Disability Status Scale; T25FWT: Timed 25-Foot Walk Test; 9HPT-dom: Nine-Hole Peg Test for dominant hand; 9HPT-ndom: Nine-Hole Peg Test for non-dominant hand; SDMT: Symbol Digit Modalities Test; FSS: Fatigue Severity Scale; BDI: Beck Depression Inventory; HADS-anx: Hospital Anxiety and Depression Scale for anxiety; HADS-dep: Hospital Anxiety and Depression Scale for depression; PSQI: Pittsburgh Sleep Quality Index (PSQI); MSIS-29-psy: Multiple Sclerosis Impact Scale for psychological impact; MSIS-29-phy: Multiple Sclerosis Impact Scale for physical impact. \* Scores expressed as mean (SD) [missing values].

**Table 4.** Satisfaction as quantified from 5-point Likert scales with regard to patients who completed the study \*.

HCPs: healthcare providers; TCs: teleconsultations; QoC: quality of care. \* Scores expressed as mean (SD); ◦ *p* value not significant (0.58).

#### **4. Discussion**

We present the first ever study, to our knowledge, demonstrating the feasibility of synchronous TC using an audiovisual Internet platform, when repeatedly applied for the clinical monitoring of individuals with MS over a substantial time period (i.e., one year). In addition, the appraisal of general care, technical quality, and convenience, as related to the digital visits, was excellent in the majority of patients and healthcare providers involved in the interventional arm. These results are in line with a recent and similarly designed trial among subjects with Parkinson disease—representing another frequently occurring chronic disorder of the central nervous system—living throughout the United States of America, in which 98% of the 97 participants randomized to the intervention group completed at least one digital visit, and 91% of the 388 planned digital visits were completed as scheduled. Here, the authors calculated that each virtual session would have saved patients a median of 88 min (95% CI 70–120; *p* < 0.001) and 38 miles per visit (95% CI 36–56; *p* < 0.001), compared to their usual care [18]. Feasibility was considered as a binary outcome measure in both studies (i.e., either the TC works or not) and the prespecified success target of at least 80% corresponds well to previously reported 'no-show' rates associated with in-person visits at neurology clinics [19,20].

Our TC approach did not lead to significant differences in parameters reflecting functional status (with the exception of fatigue) and medical costs. These secondary analyses were incorporated mainly for exploratory purposes, accepting a possible risk of a type II error. The likelihood of inducing a clinically relevant benefit with our intervention was considered low during study preparation, since it was never the intention to offer specific nor standardized treatment programs. Nonetheless, retaining the status quo can also be valuable as we, a priori, did consider the possibility of (increased) virtual attention inducing or aggravating MS-related symptomatology, such as anxiety and/or sleep difficulties. The positive impact of the intervention on fatigue levels should be interpreted with caution because the effect size was small and would not have survived a statistical (Bonferoni) correction for multiple comparisons. It is worth noting, however, that fatigue and physical activity levels showed improvement with other web- and telephonebased interactive sessions primarily based on educational and motivational coaching [21,22]. Surrogates of medical costs did not significantly differ between our two groups and were preferred above direct values because TC still occurred in addition to standard care. Gain in that domain, though, can be expected in future studies which actually replace face-to-face with digital visits in routine follow-up.

COVID-19 has deeply disrupted human socialization and forced our health system towards a large-scale adoption of TC on very short notice. Our findings can help solidify the scientific basis for continuing such practice, using the Internet as the flagship of modern-day communication, within a complementary hybrid model for next-generation MS care [15]. A number of reasons can be given to explain why patients with MS may be particularly suitable candidates to benefit from digitalization of neurological care. First, diagnosis is typically established during young adulthood [11], when time availability for medical attention is limited due to other priorities in life. Second, MS may lead to cumulative physical disability [11], complicating access to neurological facilities, even in areas offering sufficient and dense resources. Third, over the past two decades, we have seen a spectacular growth of the disease-modifying and symptomatic treatment armamentarium for individuals with MS, which is expected to ameliorate at least short- to medium-term prognosis but also increases the complexity of routine follow-up. Fourth, a significant proportion of affected patients (i.e., 30%) appears to completely miss out on neurological care [23], likely decreasing their chance of receiving state-of-the-art disease management and jeopardizing a favorable clinical outcome. Simultaneously, there seems to be a high interest among individuals with MS for using the Internet as a health information source and for online interaction with medical specialists [24]. As a final, general, and perhaps most convincing argument, we can state that nervous system disorders are currently the leading source of disability, affecting over one billion people worldwide [25], and represent a burden that is expected to at least double over the next two decades, mostly because of a growing elderly population [26]. In parallel, we have recently witnessed an increased prevalence and life-expectancy in patients with MS [27,28]. Access to neurological care is indigent already, as expressed by several health authorities in statements that do not solely apply to remote or low-income regions [4]. Consequently, the pressure on our classic health model will naturally rise to levels necessitating at least some form of digital redesign in order to avoid a total overflow, a rationale that has recently been put to the forefront by COVID-19 but survives even in complete abstraction of this crisis.

The most important limitations of this study were caused by the heterogeneity of the healthcare provider pool and the absence of a clinical neurological exam in the TC protocol. Neurology has long remained a very bedside-orientated 'hands-on' specialty, and the fear of missing subtle yet critical clinical details during a remote physical evaluation likely forms the Achilles heel of teleneurology in general. Supportive proposals are starting to be published [3,29], and it is of interest to mention that Bove and colleagues recently reported an agreement within one point between in-person and televideo-enabled EDSS scores for 88% of the cases, which is similar to the in-person inter-rater reliability described earlier by others [30]. The NMSC Melsbroek is a highly specialized hospital specifically focusing on the neurological management, multidisciplinary care, and/or rehabilitation of individuals with MS. All participants were allowed to use the ambulatory and in-hospital rehabilitation services of the center as a part of standard care during the study, if deemed necessary by their treating physicians. This decision was based on ethical considerations, but it cannot be excluded that such rehabilitation activities have influenced our secondary outcomes. As explained above (see Section 3.5), a priori unforeseen COVID-19 measures may have resulted in reduced access to the clinic and/or delay of standard care on multiple occasions during the study, which could have created a disproportionately positive welcoming of digital solutions. Furthermore, there might be other reasons to be cautious when generalizing our findings to the full MS community, as it cannot be ruled out that disease-specific, geographic, cultural, and/or social differences may lead to less positive outcomes. Previous studies have disclosed that patients with cognitive or visual impairment experienced more difficulties while using home-based TM systems [31,32], whereas we have actively avoided recruitment of participants with apparent cognitive dysfunction. We also have to acknowledge that there was a greater proportion of participants with a higher education level in our interventional group, as compared to the controls. TM has generally been praised for its potential to facilitate access to medical care, but recent reports have, somewhat surprisingly, warned of the persistence or even aggravation of ethnical and other disparities [33–35]. Possible solutions include wide-spread promotional campaigns, mobile paramedical teams, assistance by (educated) caregivers, and fully equipped community centers, of which most examples can also help with the more challenging aspects of the clinical exam. These factors related to background variability should not be forgotten when designing future research aimed at demonstrating the non-inferiority of replacing face-to-face with digital visits in the MS clinic, compared to the classic approach.

In conclusion, our study demonstrated the technical and practical feasibility of live audiovisual TC over the Internet for routine neurological follow-up in patients with MS. The digital approach was well-appreciated by both participating patients and healthcare providers. Future trials can now be designed in which the effect (e.g., non-inferiority) of replacing classic face-to-face visits with such TM modalities can be assessed for multiple purposes. We believe that this will be the phase where their full potential will come to expression but also one in which we must factor in the abovementioned systemic pitfalls and tailor the interventions to the individual patient needs.

**Author Contributions:** M.D. and G.N. conceptualized the study. The statistical analyses were conducted by M.D., G.N. and J.V.S.; N.S. and M.D. wrote the first draft of the paper, while all authors were involved in the critical reading and revision process. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external competitive funding.

**Institutional Review Board Statement:** Our study was approved by the ethics committee of the Nationaal Multiple Sclerose Centrum (NMSC) Melsbroek (local; internal reference: AvN/AVDZ) and the Universitair Ziekenhuis Brussel (leading; internal reference: 2018/269, Belgian Unique Number:<PHONE_NUMBER>97).

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Data Availability Statement:** Anonymized data will be shared upon reasonable request from any qualified investigator.

**Acknowledgments:** The authors thank Zebra Academy for offering their technology free of charge for this study. We would also like to express our gratitude toward Gert Ooms and Ann Van Remoortel (NMSC Nursing Deparment), Thomas Van Oosthuysen (NMSC Information and Communications Technology Department) and the Zebra Academy crew for providing logistic and/or technical support.

**Conflicts of Interest:** This study was supported by a non-competitive research grant from Roche (Basel, Switzerland). G.N. is a shareholder of Zebra Academy. The authors have no potential conflicts of interest to report.

#### **References**

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ISBN 978-3-0365-5256-9


**4. Clinical presentation**

Trachoma presents in two phases: the active phase and the cicatricial or scarring phase; however, both phases can coexist concurrently [6]. Active trachoma is characterized by a mucopurulent keratoconjunctivitis [36]. The conjunctival surface of the upper eyelid shows a follicular and inflammatory response. The cornea may have limbal follicles, superior neovascularization also known as pannus, and punctate keratitis [31]. *C trachomatis infection* may occur concurrently with that of other extraocular mucous membranes, e.g. the nasopharynx, leading to a nasal discharge.

Most patients with active trachoma are relatively asymptomatic [11].

The cicatricial phase has characteristic clinical features, which can lead to definitive diagnosis in most cases [7, 37].


*Edited by Mansour Al Qubeissi*

Amongst concerns about climate change, energy security decline and depletion of fossil fuels, this book explores the high importance of and interests in alternative energy resources. Many studies have shown that biomass fuels are sustainable, environmentally friendly and can be the most appropriate replacement to the depleting crude oil. Additionally, they can expand green landscapes, create new job opportunities, be directly utilised in standard power systems and improve combustion performance. Biomass fuels can be limited due to production cost and competition with food. Therefore, plant and food wastes play an important role in reducing these costs and recycling dump bio-materials. Production of biofuels from non-food biomass has emerged as a sustainable option to tackle the problems associated with growing demands for energy.

Published in London, UK © 2019 IntechOpen © Sharon McCutcheon / Unsplash

Biofuels - Challenges and opportunities

Biofuels

Challenges and opportunities


*Review* **The Eco-Immunological Relevance of the Anti-Oxidant Response in Invasive Molluscs**

**Davide Malagoli 1,2,\*, Nicola Franchi 1 and Sandro Sacchi 1**

**Abstract:** Reactive oxygen species (ROS) are volatile and short-lived molecules playing important roles in several physiological functions, including immunity and physiological adaptation to unsuitable environmental conditions. In an eco-immunological view, the energetic costs associated with an advantageous metabolic apparatus able to cope with wide changes in environmental parameters, e.g., temperature range, water salinity or drought, could be further balanced by the advantages that this apparatus may also represent in other situations, e.g., during the immune response. This review provides an overview of molluscs included in the IUCN list of the worst invasive species, highlighting how their relevant capacity to manage ROS production during physiologically challenging situations can also be advantageously employed during the immune response. Current evidence suggests that a relevant capacity to buffer ROS action and their damaging consequences is advantageous in the face of both environmental and immunological challenges, and this may represent a trait for potential invasiveness. This should be considered in order to obtain or update information when investigating the potential of the invasiveness of emerging alien species, and also in view of ongoing climate changes.

**Keywords:** ecoimmunology; stress; immunity; haemocytes; biodiversity; *Pomacea canaliculata*; *Achatina fulica*; *Mytilus galloprovincialis*; *Dreissena polymorpha*; alien species

#### **1. Introduction**

Climate change and invasive species are among the most relevant threats to biodiversity [1]. Invasive alien species are demonstrating an outstanding capacity to adapt to the new environment they colonize. Beside the adaptations to new physical-chemical environmental aspects, invasive species must also present a high degree of metabolic plasticity and an outstanding capacity of adaptation to new antigenic ecospaces [2,3]. In the IUCN list of the worst invasive species [4], numerous invertebrate species are retrievable, presenting hypervariable defence-related molecules and specific anti-microbial peptides [5,6]. In addition to the immune features and mediators that evolved or differentiated along specific evolutionary lineages, one widespread function of the immune anti-pathogen responses is represented by the controlled production of reactive oxygen species (ROS) [7,8]. These volatile and short-lived molecules are of extreme importance both in invertebrate and vertebrate immune response and, thanks to their non-specificity, they can be directed against a plethora of potential pathogens. Beside this, environmental conditions can also create oxidative stress in organisms [1,9]. In this last case, the ROS become a menace for the organism itself, and oxidative stress must be managed in order to avoid permanent damage.

Oxidative stress is a ubiquitous phenomenon that can be studied at cellular, tissue, organ and organism level. It occurs when the production or presence of ROS exceeds antioxidant defence capabilities [10,11]. ROS-mediated oxidative stress can be promoted either from the environment [12,13] or from the activity of endogenous enzymes, such as

**Citation:** Malagoli, D.; Franchi, N.; Sacchi, S. The Eco-Immunological Relevance of the Anti-Oxidant Response in Invasive Molluscs. *Antioxidants* **2023**, *12*, 1266. https:// doi.org/10.3390/antiox12061266

Academic Editors: Marcelo Hermes-Lima, Daniel Carneiro Moreira and Tania Zenteno-Savín

Received: 7 May 2023 Revised: 1 June 2023 Accepted: 3 June 2023 Published: 13 June 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/).

those involved in cellular respiration or various forms of oxydase [14]. The main cellular effects of oxidative stress are unspecific and include lipid peroxidation, protein misfolding and nucleotide alteration [15] although other more complex and less investigated effects cannot be excluded [16].

As cellular respiration and metabolism are natural and constitutive sources of ROS [14], all cells have evolved mechanisms of defence that include enzymes, e.g., catalases, peroxidases and superoxide dismutase and molecular scavengers, e.g., glutathione. More complex molecular chaperones, such as heat shock proteins (HSPs), can also intervene in recovering the protein misfolding promoted by oxidative stress [17]. While the molecular bases of oxidative stress and their association with the immune defences are well conserved, differences exist among organisms in the ability to manage ROS production during the immune response [18] and physiologically challenging situations such as those experienced as a consequence of a sudden change in oxygen availability, e.g., changes in water level or arousal after estivation/hibernation periods [12,17,19,20].

#### **2. The Oxidative Stress Response against Immune or Environmental Challenges in Highly Invasive Mollusc Species**

The resistance to oxidative stress introduces a further level of complexity in the proper management of invasive species. It has already been documented that on some occasions the usage of biocides may positively affect the spread of invasive species. An experiment performed by exposing native and invasive ant species to sublethal doses of a neonicotinoid, a class of highly diffused chemical biocides, demonstrated that it could either increase or decrease the probability of invasive ant survival according to the exposure status of the native ants [21]. Neonicotinoids may have important effects in non-target organisms. These effects include DNA damage, protein oxidation and lipid peroxidation that are largely the consequence of the increased ROS concentration that follows the altered mitochondrial Ca2+ homeostasis and the hindered mitochondrial respiration [22]. The invasive species endowed with a high resistance to ROS-mediated insults are less likely to suffer consequences from those pesticides provoking ROS-mediated oxidative stress; either the invasive species are the direct target of the treatment or they are non-target species. This further restricts the number of the compounds available for pest control, pushing researchers to investigate the effects of biological control [23] or biopesticides [24]. However, as is described in more detail below, biopesticide effects can also be limited by highly efficient ROS detoxification.

The 2013 IUCN list of the worst invasive species [4] includes bivalves, e.g., *Mytilus galloprovincialis*, *Dreissena polymorpha* and *Potamocorbula amurensis* and gastropods, e.g., *Achatina fulica*, *Euglandina rosea* and *Pomacea canaliculata*. Although a different level of information is available for these species, the retrievable data point towards a common trait of an efficient immune response and a relevant resistance to oxidative stress.

The Mediterranean mussel *Mytilus galloprovincialis* presents circulating haemocytes (immunocytes) that support efficient cell-mediated innate immune functions [25–28]. ROS species have been demonstrated to intervene during immune response and after the tissue injury of *M. galloprovincialis* [29,30]. As a filter feeder and a species of economic relevance, several studies have investigated the effects of environmental pollutants on Mediterranean mussel health and anti-oxidant stress response [13,30]. As the increase in water temperature may represent a threat for mussel cultivation, the possibility to improve *M. galloprovincialis* heat stress response has been studied at physiological and molecular levels. It has been demonstrated that a brief pre-exposure to thermal stress (a procedure labelled as "hardening") significantly increases the capability of mussels to cope with a further and more prolonged exposure to high temperatures [31]. Among the numerous parameters taken into account, oxidative stress and redox signalling were studied by measuring the expression of different superoxide dismutases, glutathione-Stransferase and catalase, the enzymatic activities of superoxide dismutases, catalase and glutathione reductase and the protein levels of HSP70 isoforms (i.e., HSP72 and HSP73). Superoxide dismutase and catalase expression during the prolonged exposure to high temperatures were increased, and they were significantly higher in hardened mussels with respect to stressed but non-hardened animals. For some molecules, the increase in enzymatic activities followed a similar temporal profile in hardened and non-hardened mussels, but it was higher in hardened ones. Glutathione reductase acted similarly to superoxide dismutases and catalase. Consistently, HSP72 and HSP73 protein levels were higher in hardened animals, and the increased expression of electron transport system elements seemed synchronized with that of the other components of the anti-oxidant response [31]. The complex and adaptive anti-oxidant response of hardened mussels to the experimental thermal stresses suggested that hardened mussels are able to manage and coordinate both the metabolic and the oxidative stress responses in the face of sudden temperature increase.

The zebra mussel *D. polymorpha* is an invasive species that can also be studied as a sentinel organism in eco-physiological studies. As frequently reported in molluscs [32], the haemocytes can be divided into two main morphologies, namely agranular and granular haemocytes. Agranular haemocytes may include cells with different morphologies and, possibly, functions, i.e., blast-like cells and hyalinocytes. In the zebra mussel, the latter exhibited the highest oxidative activity, also in comparison with granular haemocytes [33]. In ex vivo experiments, *D. polymorpha* haemocytes exposed to chemical, physical and biological stresses elicited a complex response that included the expression of anti-oxidant enzymes and they changed significantly on the basis of the stressor considered [34]. Numerous studies included the effects of cadmium, as the zebra mussel has been proposed as a sentinel organism in freshwater environments for the evaluation of water pollutant effects [35]. The oxidative activity of *D. polymorha* haemocytes changed in different haemocyte sub-populations, as the granular cells were the less affected by the treatment and the hyalinocytes seemed to be influenced only at the highest doses of cadmium ions, indicating an important stability of mitochondrial activity and ROS management for these two cell populations [36], even in the presence of relevant stresses.

To our knowledge, no information comparable to that reported for *M. galloprovincialis* and *D. polymorpha* is available for the Asian clam *P. amurensis*. This notwithstanding, experiments correlating the HSP protein levels to water salinity values, as well as studies comparing the aerobic-fermentative metabolism rates in relation to water salinity and seasonal temperature changes, have been presented [37–39], suggesting that the Asian clam could also finely adjust stress response and metabolism as a consequence of environmental changes.

The African giant snail *A. fulica* has been labelled as the most widely distributed invasive pest land snail [40]. Its large haemocytes were described a long time ago; they displayed phagocytic activity, were able to synthesise superoxide anion radical, but did not present endogenous peroxidase activity [41]. As the intermediate vector of the human pathogenic nematodes of the genus *Angiostrongylus*, the immune response of *A. fulica* to these parasites has been investigated. In snails infected with *Angiostrongylus vasorum* larvae, increased phenoloxidase (PO) activity and consequently a ROS-mediated immune response were observed, with a PO activity peak in the immediacy of the infection. The activation of PO was also followed by melanisation. Beside PO activation, the immediate increase in nitric oxide (NO) production was also observed via Griess reaction, confirming that the immune response against the nematode included a significant ROS-mediated component, especially during the first phases of the infection [42]. Ex vivo experiments performed by confronting withdrawn haemocytes with the axenic larvae of diverse metastrongyloid lungworms also demonstrated that *A. fulica* haemocytes can release their nuclear content, thus forming extracellular traps (ETs). Beside containing histones that exert a well-known antimicrobial function [43], the *A. fulica* ETs also contained molecules similar to myeloperoxidase, an enzyme involved in the production of hypohalous acids and exerting a cytotoxic function especially by means of oxidative stress [44]. The exposure of *A. fulica* to the causative agent of human eosinophilic encephalitis, *Angiostrongylus cantonensis*, promoted

the metabolic shift towards oxidative activity by increasing the glycolytic pathway and the activity of lactate dehydrogenase in order to keep the redox balance, at least in the haemolymph [45]. As an invasive snail, *A. fulica* has also been the subject of experiments aimed at controlling its diffusion without damaging other species. In this regard, the efficacy of a nematode, *Phasmarhabditis hermaphrodita*, also used as a component of a biobased molluscicide, has been assessed on the giant African snail. Juvenile snails are highly resistant to the nematodes as they are able to entrap them into the inner layer of the shell, apparently without further metabolic or immune responses [46] (Figure 1).

**Figure 1.** Highly invasive molluscs of different Classes present common traits of resistance versus oxidative stress that could be originated either from environmental stimuli or from immune challenges. The advantage of managing ROS increase is shared among diverse physiological responses, thus limiting the associated energy expenditure and allowing efficient energy trade-offs [47]. Created with BioRender.com (accessed on 2 May 2023).

While no specific information is retrievable about the immune defences and oxidative responses of the rosy wolfsnail that, however, can be vehicle of *A. cantonesis* [48], more data can be retrieved for the snail *P. canaliculata*. The circulating haemocytes of *P. canaliculata* have been described [49–51] and their proteome is available [52]. In addition to the haemolymph, the haemocytes have also been recognized within organs and in regenerating tissues [53,54], increasing the number of potential sites where the haemocyte-mediated immune response can take place and haemocytes can replicate. The immune system of *P. canaliculata* has been the target of numerous studies focused on the control of its spread. The vegetal pesticide pedunsaponin A modified the haemocyte number, membrane potential and morphology and promoted ciliary loss in ciliated tissues, thus affecting snail respiration and excretion [55,56]. The effects of the pesticide were increased after silencing the expression of HSP70 by RNAi [57], suggesting a role for this chaperone in buffering pedunsaponin A-mediated damage. As already reported above for *A. fulica*, the immune system of *P. canaliculata* was also targeted by using a commercially available molluscicide based on the nematode *P. hermaphrodita*. *P. canaliculata* exhibited a significant resistance to this molluscicide. The recommended concentration of *P. hermaphrodita* determined an overall low mortality and reduced the synthesis of an antimicrobial peptide alternatively in two immune-related organs of the snails, i.e., the gills and the anterior kidney, in dependence

of the temperature of the treatment [58]. The ultrastructural observation and proteomic analysis of an organ associated with the oxidative stress resistance and potentially involved in the immune response, namely the aortic ampulla [59,60], revealed that *P. canaliculata* systemic reaction to the immune challenge, represented by the pathogenic nematode, involved a diffused oxidative stress response, as suggested by the increased expression in the ampulla of enzymes associated with ROS detoxification, such as Cu2+–Zn2+ superoxide dismutase, catalase-like isoform X1, glutathione peroxidase-like protein and peroxiredoxin. As the nematodes were not present in the ampulla of exposed snails, and no nematode proteins were isolated or sequenced among the ampulla proteome, the increase in antioxidant enzymes allowed the speculation that the organ was responding to a systemic increase in ROS. Consistently, the snail self-protection from ROS-mediated damage was also associated with the increased expression of HSP60, 70 and 90 [61].

#### **3. The Eco-Immunological Advantage of Managing Oxidative Stress Response**

While significant differences exist in the anatomical organization and physiology of the invasive species introduced above, their high tolerance versus oxidative stress represents a common trait in their immune response towards pathogens, pesticides and environmental or experimental physico-chemical stressors. The invasiveness of a species relies on different physiological features [62,63], not least the capacity to overcome or tolerate the new potential pathogens. Invertebrate innate-only immune response has been progressively described as anticipatory and highly specific, especially in light of the numerous hypervariable molecules discovered in invertebrate taxa [64–68] and the increasing evidence of circulating microbiotas [69–71] that some invertebrate species are able to manage together with the intestinal microbiota. However, a specific and anticipatory immune response could also miss new pathogens that may be encountered during the colonization of new environments by the invasive species. In this respect, well-conserved and unspecific responses, such as the increased synthesis of ROS, could become essential for the adaptation to new environments. As an unspecific response based on short-lived and volatile molecules, ROS-mediated immune defence is self-limited by the capability of the host to manage the potential harm that these volatile molecules could determine; in the majority of the invasive species mentioned here, a marked resilience versus oxidative stress has been reported.

The mussel *M. galloprovincialis* can populate subtidal and intertidal environments. It displays tolerance to temperature fluctuations [72] and its metabolic plasticity allows the Mediterranean mussel to adapt and to react differently to environmental challenges on the basis of the colonized habitat [73]. In *D. polymorpha*, an exceptional tolerance to variable environmental conditions associated with strong anti-oxidant defences has been reported [74], maybe in consequence of a high genetic diversity [75]. *P. amurensis* can colonize subtidal and intertidal habitats and present a high tolerance to wide ranges of water salinity and temperature [76,77], conditions that are connected to changes in oxygen availability and require efficient oxidative stress response. Among invasive freshwater gastropods, both *A. fulica* and *P. canaliculata* can undergo estivation [78,79], a physiological adaptative response to unsuitable environmental conditions that relies on the ability to manage the significant increase in ROS during the arousal, when oxygen concentration quickly increases. In the African giant snail, mitochondrial or cytosolic superoxide dismutase levels did not change during estivation, and the same held true for other anti-oxidant enzymes such as glutathione peroxidase, glutathione reductase or catalase. While their amount and activity are stable during estivation, during the arousal from estivation the antioxidant enzymes increase their detoxifying activities with a time- and tissue-specific distribution. Consistently, no signs of protein damage or lipid peroxidation were detected in the heart tissues of African giant snails after four weeks of experimental estivation [78]. During estivation, *A. fulica* can accumulate urea, a nitrogen waste product less harmful than ammonia. Evidence collected in *A. fulica* has demonstrated an astonishing capacity to detoxify exogenous and injected ammonia via a significant increase in the urea synthesis

rate, suggesting that urea could have other metabolic roles beside the excretory one [80]. In *P. canaliculata*, the involvement of nitrogen compounds in the anti-oxidant response associated with the arousal from estivation has been documented in detail. In agreement with the observations from *A. fulica*, the *P. canaliculata* presented a complex pattern of anti-oxidant responses that involved enzymatic and non-enzymatic components which fluctuated in an organ-specific fashion, especially during the arousal [81]. The nitrogen compound uric acid is likely to play an important role in buffering the ROS concentration increase during the arousal [82]. A specific tissue, labelled as urate tissue, is distributed among numerous organs, i.e., the lung, the digestive system, the anterior kidney (also identified as pallial ureter) and the ampulla, which are considered relevant organs in managing ROS levels during the arousal [60,79] (Figure 2).

**Figure 2.** The invasive gastropods *A. fulica* and *P. canaliculata* can cope with ROS peaks in different situations. Left: when the water and oxygen availability are sufficient (green), the snails do not display a significant activity in detoxifying enzymes. When the water availability is reduced, e.g., as a consequence of drought, the snails can enter estivation, reducing their metabolism, as a consequence of reduced oxygen availability (red). Once the environmental conditions become more suitable for their survival, the snails face a peak of ROS that follows the increase in the oxygen availability and in the metabolic activity. This peak is associated with an increased enzymatic activity and, at least in *P. canaliculata*, is sustained by nitrogen-containing compounds. Right: during the immune response against pathogenic nematodes (grey), a similar trend of ROS production is observed, although in this case it is also possible that immune-related components, e.g., haemocytes, can contribute to ROS synthesis and increase. The protective elements employed during the arousal can also be utilized in response to the immune challenge, allowing the snails to express a powerful ROS-mediated immune response, avoiding self-damage. Created with BioRender.com (accessed 2 May 2023).

Recently, the response of *P. canaliculata* circulating haemocytes has also been investigated, and differences have emerged in the mediators involved in the anti-oxidant response versus experimental hibernation or estivation [83], confirming the high plasticity and adaptability of the oxidative stress response in this invasive snail. The proteomic analysis of ampullae collected from snails challenged with the pathogenic nematode *P. hermaphrodita* evidenced a metabolic response that paralleled the adaptation to estivation, with a significant increase in anti-oxidant defences and an increase in aerobic metabolism, witnessed by the increase in mitochondrial enzymes related to ATP synthesis [61].

The ability to cope with the environmental conditions that require an increased capability of managing oxidative stress response represents a significant advantage that could help the adaptivity and invasiveness of some species. Nonetheless, that capacity is associated with the metabolic costs that the maintenance of a prompt anti-oxidant defence system requires. In the eco-immunological perspective of efficient energy expenditure and management [84,85], the advantage of controlling the self-damaging consequences of oxidative stress response could be more relevant if extended to other physiological functions such as the immune response against pathogens. While the physiological immune functions are deeply interconnected with metabolism, neural functions and development [86], and their energetic demand is balanced together with that of the other physiological systems, during the response against pathogens the energy demand of the immune system suddenly increases and must be managed [84]. In this context, the possibility to take advantage of a pre-set and dynamic anti-oxidant defence system such as those described above for invasive molluscs may allow the organisms to express a potent and efficacious ROS-mediated immune response without incurring the self-damage that ROS may determine for the host. In organisms adapted to sustain the oxidant stress response derived from changes in water temperature or salinity, or the arousal from estivation or hibernation, the self-limitations that must be applied to the ROS-mediated immune response [87] could be less marked. This would prove particularly functional for *A. fulica* and *P. canaliculata* in the presence of large pathogens, such as the nematode *P. hermaphrodita*. From this perspective, the metabolic costs associated with the adaptation to specific environmental conditions could be counterbalanced by the advantages also reflecting on the immune functions, thus giving the species endowed with this capacity a special advantage in the face of both environmental and immunological challenges [1].

#### **4. Conclusions**

The updated determination of alien species' potential of invasiveness and the consequent polices for alien species control are becoming more urgent in view of the increasing consequences of ongoing climate change. For instance, in 2012, the EU Parliament classified *Pomacea* as a pest and invading genus and requested a comprehensive risk assessment on *Pomacea* species [88] that included the potential establishment of these snails in EU territory. Similar studies highlighted regions of India considered as more prone to invasion by the African giant snail, *A. fulica* [89]. The different scenarios regarding the areas potentially invaded and prospected in those risk assessments almost ten years ago, which considered specific patterns of air, water temperature and rainfall fluctuations, might now need some adjustments or updates. At the same time, climate change has also promoted an unanticipated reduction of sea water pH. This acidification has been demonstrated to modify the freeze tolerance of subtidal species, such as for instance *M. galloprovincialis*, and it will likely modify the area of distribution of subtidal and intertidal *Mytilus* species [90].

Current evidence suggests that most invasive molluscs present a relevant capacity to manage ROS increase, either derived from environmental cues or from immune stimuli. From an eco-immunological perspective, the possibility of taking advantage of the same metabolic ability in diverse functional contexts allows a better use of resources and energy trade-offs. This shared capacity may likely represent a trait of potential invasiveness which, associated with more species-specific characteristics, could justify the success of species such as *M. galloprovincialis*, *D. polymorpha, A. fulica* and *P. canaliculata* to invade and adapt to new environments and niches. From this perspective, the resistance to experimental oxidative stress of emerging alien species could be further investigated in order to gain more complete information on their potential of invasiveness and to formulate consistent policies for their control.

**Author Contributions:** Conceptualization, D.M.; investigation, D.M., N.F. and S.S.; resources, D.M.; writing—original draft preparation, D.M.; writing—review and editing, D.M., N.F. and S.S.; visualization, N.F.; supervision, D.M.; project administration, D.M.; funding acquisition, D.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the National Recovery and Resilience Plan (NRRP), Mission 4, Component 2 Investment 1.4—Call for tender No. 3138 of 16 December 2021, rectified by Decree n.3175 of 18 December 2021 of the Italian Ministry of University and Research funded by the European Union—NextGenerationEU, grant number Project code CN\_00000033, Concession Decree No. 1034 of 17 June 2022 adopted by the Italian Ministry of University and Research, CUP E93C22001090001, Project title "National Biodiversity Future Center—NBFC" and by the Department of Life Sciences, University of Modena and Reggio Emilia, Bando FAR2020. The APC was waived by the Editor.

**Institutional Review Board Statement:** Ethical review and approval were waived for this study, as the use of bivalves and gastropods as experimental animals is neither detailed in the Directive 2010/63/EU of the European Parliament and of the Council (https://eur-lex.europa.eu/legalcontent/EN/TXT/HTML/?uri=CELEX:32010L0063&from=EN, accessed on 2 June 2023) nor in the Italian legislative decree n. 26/2014 (https://www.gazzettaufficiale.it/eli/id/2014/03/14/14G00036 /sg, accessed on 2 June 2023). Experiments have been performed and presented in accordance with the ARRIVE guidelines (https://arriveguidelines.org/arrive-guidelines, accessed on 2 June 2023).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** No new data were created or analysed in this study. Data sharing is not applicable to this article.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

**Disclaimer/Publisher's Note:** The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.


3.2.4. Pentavalent Dopants

For the pentavalent dopants V5+ and Mo5+, no charge compensation is required for the substitution at the Nb5+ host site, but it is required when the substitution is at the Li+ host site, as shown in Tables 8 and 9.

**Table 8.** Types of defects considered due to M = V5+ incorporation in LiNbO3.

The solution energies for the pentavalent (V5+) and (Mo5+) dopants with different charge compensation mechanisms were evaluated and plotted as a function of the reaction scheme. Based on the lowest energy value, it seems that the incorporation of pentavalent (V5+) and (Mo5+) ions at an Nb site is energetically more favourable than at an Li site, according to scheme (iv) as shown in Figures 6 and 7 at temperatures 0 K and 293 K. This can be attributed to the similarity between the charge of the V5+ and Mo5+ ions and the Nb5+ host, which can contribute to a small deformation in the lattice and consequently a lower solution energy. Experimental results by Kong et al. [17] and Tian et al. [16] show that substitution occurs at the Nb5+ site.

**Figure 6.** Bar chart of solution energies vs. solution schemes for pentavalent dopant (V5+) at the Li and Nb sites, considering the first neighbours in relation to the c axis.

**Figure 7.** Bar chart of solution energies vs. solution schemes for pentavalent dopant (Mo5+) at the Li and Nb sites, considering the first neighbours in relation to the c axis.

### 3.2.5. Hexavalent Dopants

For the hexavalent dopant Mo6+, as with the pentavalent ions, there is no self-compensation mechanism and charge compensation schemes are possible when replacing Li and Nb in the LiNbO3 matrix as shown in Table 10.

**Table 10.** Types of defects considered due to Mo6+ incorporation in LiNbO3.

The solution energies for the hexavalent (Mo6+) dopants with different charge-compensation mechanisms were evaluated and plotted as a function of the reaction scheme. Based on the lowest energy value, it seems that the incorporation of hexavalent (Mo6+) ions at an Nb site is energetically more favourable than at an Li site, according to scheme (iv) as shown in Figure 8 at temperatures 0 K and 293 K. This can be attributed to the similarity between the ionic radii of Mo6+ ions and the Nb5+ host site (0.32–0.71 Å) [40]. The ionic radii of Mo6+, taking into account the coordination number, vary between 0.42 and 0.67 Å [40], and the small difference between the Mo6+ dopant ions and Nb5+ ions can contribute to a small deformation in the lattice and consequently a lower solution energy. This result reveals that global trends of dopant solution energies are controlled by the combination of dopant ion size [40] and its electrostatic interactions, demonstrating that there is a relation between the energetically preferred site and the types of defect mechanisms involved in the doping process. Experimental results from Kong et al. [17] and Zhu et al. [41] show that substitution occurs at the Nb5+ site.

**Figure 8.** Bar chart of solution energies vs. solution schemes for hexavalent dopant (Mo6+) at the Li and Nb sites, considering the first neighbours in relation to the c axis.

In all cases, the energy involved in doping was obtained by calculating the solution energy, which includes all terms of the thermodynamic cycle involved in the solution process. For example, the solution energy, Esol, corresponding to the incorporation of V2+ at the Li+ site (second equation in Table 3) is given by:

$$\mathrm{E\_{Sol}} = \mathrm{E\_{Def}(5M\_{Li} + V\_{\mathrm{Nb}}^{\prime\prime\prime\prime})} + 2.5\mathrm{E\_{Latt}(Li\_2O)} + 0.5\mathrm{E\_{Latt}(Nb\_2O\_5)} - 5\mathrm{E\_{Latt}(MO)}\tag{2}$$

where the Elatt and EDef terms are lattice energies and defect energy.

All energies were normalised by the number of dopants, i.e., the solution energy is divided by the number of dopants involved. For example, for scheme (ii) of Table 3, the energy must be divided by five, since five lithium sites are occupied. This is done because the number of dopants varies for each mechanism. Lattice energies, Elatt, required to calculate the solution energies are given in Table 11.

**Table 11.** Lattice energies used in the solution energy calculations (eV).


**NEURAL STEM CELLS - NEW PERSPECTIVES**

#### **Neural Stem Cells - New Perspectives**

http://dx.doi.org/10.5772/56573 Edited by Luca Bonfanti

#### **Contributors**

Kaneyasu Nishimura, Luca Colucci-D\'Amato, Maria Teresa Gentile, Luca Bonfanti, Emilia Madarász, Caetana Carvalho, Bruno P Carreira, Ines Araujo, Ana Isabel Santos, Angelique Bordey, Manavendra Pathania, Shan Bian, Emmanuel Moyse, Young Gyu Chai, Nando Dulal Das, Verdon Taylor, Stefano Pluchino, Matteo Donega, Elena Giusto, Chiara Cossetti, Teri Belecky-Adams, Luciano Conti, Simona Casarosa, Jacopo Zasso, Joshua Goldberg

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First published in Croatia, 2013 by INTECH d.o.o. eBook (PDF) Published by IN TECH d.o.o. Place and year of publication of eBook (PDF): Rijeka, 2019. IntechOpen is the global imprint of IN TECH d.o.o. Printed in Croatia

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*3.1. Data*

The SD data were obtained from the long-term series of the daily snow depth dataset in China (1979–2020), released by the National Tibetan Plateau data center (http://data. tpdc.ac.cn, accessed on 11 October 2021). The data were derived from the inversion of daily passive microwave brightness temperature (SMMR, SSM/I, and SSMI/S), with a spatial resolution of 0.25◦ [51]. Based on the daily SD data, the monthly and annual maximum SD data were obtained using the maximum value composite (MVC) method, which represented the maximum value in the process of snowmelt and accumulation [52]. The snow cover data were adopted from the MODIS Daily Cloudless 500 m Snow Area Product Dataset over China during the period from 2000 to 2019, released by the National Cryosphere Desert Data Center (http://www.ncdc.ac.cn, accessed on 28 October 2021), used for LSSP calculation [53]. A DEM with a 30 m spatial resolution released by the Chinese Academy of Sciences Geospatial Data Cloud (http://www.gscloud.cn, accessed on 5 July 2018) was used to calculate the slope and aspect across the SGP. The surface net solar radiation data were released by the European Centre for Medium-Range Weather Forecasts (ECMWF) (https://www.ecmwf.int/, accessed on 17 April 2019), with a spatial resolution of 0.25◦ and a temporal resolution of 3 h. The meteorological data, including precipitation, maximum temperature, and minimum temperature, were from the National Meteorological Information Center of China Meteorological Administration (http://data.cma.cn/, accessed on 21 August 2021). Snow depth observation data came from the National Meteorological Information Center of the China Meteorological Administration and are used to test the accuracy of the downscaling. The time spans of the above data (except for the DEM) were unified to the same period from 2002 to 2018 for simultaneity.


*2.5. The Effect of GCB, Terr, and Their Combination on the Colorectal Cell Lines' Growth and Proliferation*

The combination of Terr and GCB was found to show a synergistic effect in normoxia and an antagonistic effect in hypoxia. This was evident when checking the cell growth regulators and cell proliferation regulators in both normoxia and hypoxia conditions. We examined and quantified the fold changes of several genes responsible for cellular growth (AKT1, TGF-B1, HIF1-α, and PRKDC) and cellular proliferation (PCNA and RAD18) using the RT-qPCR technique.

In HCT-116, the AKT1 gene was over-expressed when the cells were treated with terrein in normoxia by 45.8-fold and were under-expressed for other treatments in the same condition. The contrary was evident in hypoxia. TGF-1β showed no change in expression for cells treated with terrein but was under-expressed when the cells were treated with other treatment conditions under normoxia. HIF1-α showed similar results to the ones seen by AKT1 under normoxic conditions. PRKDC showed no change in expression for all treatments except the combination treatment (GCB + Terr) under normoxic conditions. At hypoxia, TGF-1β, HIF1-α, and PRKDC were under-expressed in all treatments (Figures 8A,B and 9A,B).

**Figure 8.** Fold change of genes by Terr, GCB, and their combination under normoxic and hypoxic conditions. A fold change value below 0.5 indicates under-expression, above 2.0 indicates overexpression, and between 0.5 and 2.0 indicates no change in expression. HCT-116 under normoxia and hypoxia (**A**,**B**), HT-29 under normoxia and hypoxia (**C**,**D**), and SW620 under normoxia and hypoxia (**E**,**F**). The data are presented as mean fold change ± SD.

**Figure 9.** *Cont*.

**Figure 9.** Fold change of genes by Terr, GCB, and their combination under normoxic and hypoxic conditions. A fold change value below 0.5 indicates under-expression, above 2.0 indicates overexpression, and between 0.5 and 2.0 indicates no change in expression. HCT-116 at 24 h under normoxia and hypoxia (**A**,**B**), HT-29 at 24 h under normoxia and hypoxia (**C**,**D**), and SW620 at 24 h under normoxia and hypoxia (**E**,**F**). The data are presented as mean fold change ± SD.

On the other hand, the cellular proliferation gene, PCNA, was over-expressed when the cells were treated with terrein in normoxic conditions by 40-fold. However, it was under-expressed for the rest of the treatments. At hypoxia, the combination treatment (GCB + Terr), resulted in no change in the expression of PCNA. Yet, the remaining treatments resulted in the under-expression of the gene. The RAD18 gene was under-expressed in all treatments in both oxygen conditions except for terrein in normoxia where the gene did not change in expression (Figure 8A,B).

In HT-29, AKT1 was only over-expressed 2.62-fold after treatment with GCB. TGF-1β was under-expressed in all treatments under both oxygenation conditions. HIF1-α was under-expressed in all treatments and oxygen conditions, except after treatment with (GCB + Terr) where it showed no change in expression. PRKDC showed no change in expression in normoxia in response to all treatment conditions (Figure 8C,D). PCNA was under-expressed due to hypoxic conditions and no change in expression was observed under normoxia (Figure 9C,D).

In SW620, AKT1 showed no change in gene expression for all treatments under normoxia and hypoxia. However, TGF-1β and HIF1-α were under-expressed in all treatments under normoxia and hypoxia. The PRKDC gene was only expressed under hypoxia conditions for all treatments (Figure 8C,D). PCNA and RAD18 were under-expressed in this cell line for all treatment groups under both oxygenation conditions (Figure 9E,F).


**1. Introduction**

Diabetes mellitus is a heterogeneous primary disorder of carbohydrate metabolism which exists everywhere in the world and interests approximately 371 million people worldwide. The prevalence of diabetes mellitus is increasing with ageing of the population and lifestyle changes associated with rapid urbanization and westernization. The disease is found in all parts of the world and is rapidly increasing in its coverage [1, 2]. WHO projects that diabetes will be the 7th leading cause of death in 2030 [2, 3]. It is a metabolic disorder initially charac‐ terized by a loss of glucose homeostasis with disturbances of carbohydrate, fat and protein metabolism resulting from defects in insulin secretion, insulin action, or both [4]. Without enough insulin, the cells of the body cannot absorb sufficient glucose from the blood; hence blood glucose levels increase, which is termed as hyperglycemia. If the glucose level in the blood remains high over a long period of time, this can result in long-term damage to organs, such as the kidneys, liver, eyes, nerves, heart and blood vessels. Complications in some of these organs can lead to death [5]

The roles of certain organs such as the pancreas, liver and kidney in diabetes mellitus are very important.The pancreas plays a primary role in the metabolism of glucose by secreting the hormones insulin and glucagon (Figure 1). The islets of Langerhans secrete insulin and glucagon directly into the blood. Insulin is a protein that is essential for proper regulation of glucose and for maintenance of proper blood glucose levels [6]. Glucagon is a hormone that opposes the action of insulin. It is secreted when blood glucose level falls. It increases blood glucose concentration partly by breaking down stored glycogen in the liver by a pathway

© 2014 Arise et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

known as glycogenolysis. Gluconeogenesis is the production of glucose in the liver from noncarbohydrate precursors such as glycogenic amino acids [7].

The conventional treatment for diabetes mellitus is oral hypoglycemic agents/insulin therapy [12]. However these have been shown to have prominent side effects and they do not modify the course of diabetic complications [13, 14]. The need to develop new antidiabetic drugs has led to studies that have attempted to screen some indigenous plants for antidiabetic activity [15, 16]. Traditional preparations of plant sources are widely used almost everywhere in the worldto treatthisdisease.Thereforeplantmaterials are consideredto be the alternative sources for finding out new leads for antihyperglycemic agents. The plant drugs are frequently consideredtobe less toxicwhencomparedto syntheticdrugs [17].More than1,123plant species havebeenusedtotreatdiabetesandmorethan200purecompoundshavebeenshowntopossess characteristics of lowering blood glucose activity [18]. *Acacia ataxacantha* or *Flame Thorn* is an African tree species with conspicuous red pods and numerous hooked prickles. It is wide‐ spread in sub-Saharan Africa from Senegal in the west to Sudan in the east, Namibia, Botswa‐ na, Zimbabwe, and in the Transvaal and KwaZulu-Natal. In arid regions it prefers low-lying sites near streams, water courses and in valleys, but in higher rainfall areas it is a common memberofthevegetation,oftenfavouringforestmargins.Theflakingbarkis lightgrey, splitting longitudinally and transversely, and revealing a buff under-colour. The persistent prickles are profuse on young twigs, but can also be found on older wood. Unlike most Acacias,the prickles

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are not in pairs, but scattered along young twigs ('ataxacantha' i.e orderless prickles).

**Source :** Davidson and Jeppe [19]

**Figure 2.** *Acacia ataxacantha*

WHO classification of diabetes introduced in 1980 and revised in 1985 was based on clinical characteristics. The two most common types of diabetes were insulin-dependent diabetes mellitus (IDDM) or (type I) and non-insulin-dependent diabetes mellitus (NIDDM) or (type II). WHO classification also recognized malnutrition-related diabetes mellitus and gestational diabetes. Malnutrition-related diabetes was omitted from the new classification because its etiology is uncertain, and it is unclear whether it is a separate type of diabetes [8, 9, and 10].

International Diabetes Federation [11] reported that one in 10 adults will have diabetes by 2030, posing a huge challenge to health care systems around the world. According to the report, the number of people living with diabetes worldwide will increase to 552 million by 2030 from 366 million in 2011 unless action is taken. Over 80 percent of its related deaths occur in lowand middle-income countries. It has been predicted that the number of cases may jump by 90 percent even in Africa, where infectious diseases have previously been the top killer [11].

**Figure 1.** The role of pancreas in the body

The conventional treatment for diabetes mellitus is oral hypoglycemic agents/insulin therapy [12]. However these have been shown to have prominent side effects and they do not modify the course of diabetic complications [13, 14]. The need to develop new antidiabetic drugs has led to studies that have attempted to screen some indigenous plants for antidiabetic activity [15, 16]. Traditional preparations of plant sources are widely used almost everywhere in the worldto treatthisdisease.Thereforeplantmaterials are consideredto be the alternative sources for finding out new leads for antihyperglycemic agents. The plant drugs are frequently consideredtobe less toxicwhencomparedto syntheticdrugs [17].More than1,123plant species havebeenusedtotreatdiabetesandmorethan200purecompoundshavebeenshowntopossess characteristics of lowering blood glucose activity [18]. *Acacia ataxacantha* or *Flame Thorn* is an African tree species with conspicuous red pods and numerous hooked prickles. It is wide‐ spread in sub-Saharan Africa from Senegal in the west to Sudan in the east, Namibia, Botswa‐ na, Zimbabwe, and in the Transvaal and KwaZulu-Natal. In arid regions it prefers low-lying sites near streams, water courses and in valleys, but in higher rainfall areas it is a common memberofthevegetation,oftenfavouringforestmargins.Theflakingbarkis lightgrey, splitting longitudinally and transversely, and revealing a buff under-colour. The persistent prickles are profuse on young twigs, but can also be found on older wood. Unlike most Acacias,the prickles are not in pairs, but scattered along young twigs ('ataxacantha' i.e orderless prickles).

**Source :** Davidson and Jeppe [19]

**Figure 2.** *Acacia ataxacantha*

known as glycogenolysis. Gluconeogenesis is the production of glucose in the liver from non-

WHO classification of diabetes introduced in 1980 and revised in 1985 was based on clinical characteristics. The two most common types of diabetes were insulin-dependent diabetes mellitus (IDDM) or (type I) and non-insulin-dependent diabetes mellitus (NIDDM) or (type II). WHO classification also recognized malnutrition-related diabetes mellitus and gestational diabetes. Malnutrition-related diabetes was omitted from the new classification because its etiology is uncertain, and it is unclear whether it is a separate type of diabetes [8, 9, and 10].

International Diabetes Federation [11] reported that one in 10 adults will have diabetes by 2030, posing a huge challenge to health care systems around the world. According to the report, the number of people living with diabetes worldwide will increase to 552 million by 2030 from 366 million in 2011 unless action is taken. Over 80 percent of its related deaths occur in lowand middle-income countries. It has been predicted that the number of cases may jump by 90 percent even in Africa, where infectious diseases have previously been the top killer [11].

carbohydrate precursors such as glycogenic amino acids [7].

4 Antioxidant-Antidiabetic Agents and Human Health

**Source:** Worthley [6]

**Figure 1.** The role of pancreas in the body

Hence, the aim of this study is to investigate the anti-diabetic potential and safety evaluation of ethanolic extract of *Acacia ataxacantha* bark in streptozotocin-induced diabetic rats. The results were compared with standard anti-diabetic drug, metformin.

Group 4: Diabetic and treated with 250 mg/kg b.w of ethanolic extract of *Acacia ataxacantha*

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Group 5: Diabetic and treated with 500 mg/kg b.w of ethanolic extract of *Acacia ataxacantha* bark

At the end of the experimental period, venous blood was collected from the experimental animals and serum was prepared by centrifuging the blood samples at 3000 rpm for 5 minutes [23] and serum collected by pippeting. The animals were thereafter quickly dissected and the liver and pancreas removed. The pancreas and liver were suspended in ice-cold 0.25 M sucrose solution (1:5 w*/*v) and homogenized. The homogenates were kept frozen overnight to ensure

Hepatic glucose and glycogen were estimated using the method of Barham and Trinder [25]

The method described by Reitman and Frankel [27] was used for assaying the activity of

Total cholesterol, triglyceride, high density lipoprotein cholesterol, low density lipoprotein cholesterol were assayed using the method of Zlakis *et al.* [28], Foster and Dunn [29], Burstein

The procedure described by Doumas *et al.* [32] and Sherlock [33] was used for the determina‐

The method of Tietz *et al*. [34] was used to determine the levels of serum urea and creatinine.

**2.10. Determination of superoxide dismutase activity and malondialdehyde concentration** The pancreatic superoxide dismutase activity was determined by the method of Misra and Fridovich [35] while malondialdehyde concentration was assayed using the method described

All data are expressed as the mean of sixth replicates ± standard error of mean (S.E.M). Statistical evaluation of data was performed by SPSS version 16 using one way analysis of

Group 6: Diabetic and treated with standard drug (Metformin)

bark.

**2.5. Sample preparation**

maximum release of enzymes [24].

**2.8. Lipids profile analysis**

by Varshney and Kale [36].

**2.11. Statistical analysis**

**2.6. Estimation of hepatic glucose and glycogen**

and Passoneau and Lauderdale [26] respectively.

alanine aminotransferase and aspartate aminotransferase.

tion of albumin and bilirubin concentrations respectively.

**2.7. Determination of liver enzyme activities**

*et al.*[30] and Friedwald *et al.* [31] respectively.

**2.9. Determination of liver function indices**