*3.2.2. Cytokines and chemokines*

bites. The agent reaches the interior of macrophages when they phagocyte apoptotic bodies of previously infected neutrophils, where it can survive and multiply. This parasite escape

The main effector mechanism involved in protective immune response against *Leishmania* spp*.* is the activation of macrophages by IFN-γ and tumor necrosis factor alpha (TNF-α) stimulation, with consequent stimulation of nitric oxide synthesis, which is required for effective destruction of the pathogen and for controlling the spread of infection in dogs [81–83].

The major subpopulations of lymphocytes are CD4+ T cells (Th1, producing IFN-γ and TNFα; Th2, secreting IL-4, IL-5, and IL-13; and Th17, producing IL-17 and IL-22) and CD8+ T cells. Antigen-presenting cells submit *Leishmania* spp. antigens to CD4+ T cells via MHC class II, and because the agent is an intracellular parasite, there may be presentation via MHC class I with

The role of CD4+ T cells in the response to visceral leishmaniasis (VL) has not been fully elucidated. Research carried out so far points to a mixed response (Th1/Th2) during infection [84, 85]. It is reported, however, that control of infection depends on Th1 cells that activate macrophages, promoting elimination of intracellular parasites [86], whereas Th2 cells direct the immune system toward humoral response and negatively regulate cellular immunity,

CD8+ cells constitute a significant population in cellular immunity against canine visceral leishmaniasis (CVL), outnumbering CD4+ cells in the dermis [87]. They play an important role in resistance to infection. Guerra et al. [88] associated phenotypic changes with tissue parasitism in the spleen and skin of infected dogs. They noticed that the high frequency of CD8+ circulating lymphocytes is directly related to low splenic parasitism and that there is a negative correlation between CD8+ T cells with skin parasite density, indicating that this cell type relates

The regulatory role of FOXP3+ CD4+ T cells in canine VL has not been fully elucidated; however, reduction of Treg cell percentage in peripheral blood of infected dogs has been observed [89]. Silva et al. [90] reported increased production of IL-10 by splenic Treg cells of dogs with LV, along with decrease in the total number of T cells when compared to healthy dogs. The findings suggest that Treg cells are a major source of IL-10 in the spleen and participate in the modulation of immune response, while a small percentage of these cells in

T-cell exhaustion (CD4+ and CD8+ cells) in peripheral blood of dogs with LV, followed by reduction of the expression of cytokines (such as IFN-γ), was recently demonstrated. This phenomenon, called cell exhaustion, is mainly mediated by high expression of programmed death protein ("programmed cell death 1," PD-1) and may be related to the strong immuno-

infected dogs may be related to persistent immune activation [90].

mechanism is called "Trojan Horse" [80].

28 Canine Medicine - Recent Topics and Advanced Research

activation of CD8+ T cells as well [69].

promoting Th1 cell anergy [87].

to resistance against LVC [88].

**3.2. Acquired response**

*3.2.1. Lymphocytes*

Cytokine patterns for CVL have not been well established. Studies are inconclusive, so the pattern of immune response associated with resistance or susceptibility in infected animals is yet to be established.

One of the first studies on cytokine profiling in CVL was performed by Pinelli et al. [71], who observed high levels of IL-2 and tumor necrosis factor alpha (TNF-α) in asymptomatic dogs, compared with symptomatic ones, which suggests a role of these cytokines in resistance to *L. infantum* in dogs naturally or experimentally infected. Since then, much research has been done in order to elucidate the cytokine profiles found in various tissue compartments of infected dogs. They revealed contrasting cytokine profiles among different tissues, indicating that the immune response in LVC occurs in an organ-specific manner [71].

Profile of cytokines in peripheral blood mononuclear cells (PBMC) culture from asymptomatic dogs experimentally infected with *L. infantum* shows a predominantly Th1 response, mediated by expression of IL-2, IFN-γ, and IL-18 and very low IL-4 expression [95]. De Lima [96] dosed IL-6 and TNF-α in serum of symptomatic dogs naturally infected with *L. infantum* and uninfected control dogs. TNF-α was found in similar levels in dogs of both groups, yet IL-6 showed statistically higher levels in dogs with active VL than healthy dogs, thus demonstrating to be a good indicator for the disease [96].

After evaluating expression of cytokines in spleen cells from dogs naturally infected with *L. infantum*, Lage et al. suggested that CVL is characterized by a mixed response: with production of cytokines types I and II; involvement of IFN-y and IL-10 and a positive correlation between IL-10 levels and progression of parasitic load or clinical manifestations of the disease; and correlation between IFN-γ and increased parasitic intensity of the spleen [84]. In whole blood, the increase of IL-10 has been associated with detection of parasite DNA [97]. Do Nascimento et al., in accordance, state that in CVL there is increase in expression of pro-inflammatory cytokines IFN-γ and TNF-α in dogs with low splenic parasitism, while dogs with higher parasitic load show an increase in IL-10 [98].

Souza [99] states that asymptomatic dogs have low dermal parasitism and exhibit a mixed pattern of immune response, with simultaneous increase of type I (IFN-γ and TNF-α) and type II (IL-5 and IL-13) cytokines, but predominance of type I response. According to the author, increased and simultaneous expression of IFN-γ and TNF-α in the skin of infected dogs enables the speculation that these mediators are closely involved with protection mechanisms during CVL, since these cytokines increased in the skin of animals with the asymptomatic clinical form. Increased expression of IL-5 and IL-13 in the skin of healthy dogs and negative correlation of the latter with clinical disease progression were also observed. Furthermore, high simultaneous expression of IFN-γ and IL-13 was found in asymptomatic dogs, indicating the role of IL-13 in establishing milder clinical forms [99].

Regarding cytokine profile in the bone marrow, Quinnell et al. [94] reported that expression of mRNA for IL-10, IL-4, and IL-18 was not elevated in infected dogs. However, some infected dogs had detectable expression of mRNA for IL-4 significantly correlated with more severe clinical signs. Moreover, expression of mRNA for IL-13 was not detected either in control or in infected dogs, and unlike in human infection, immunosuppressive activity of IL-10 was not observed in CVL [94].

Dogs infected with *L. infantum* exhibit significant decrease in expression of mRNAs for IL-10, IL-17, TNF-α, IFN-γ, and iNOS in liver tissue. Deficiency in IL-17 mRNA expression was evident in the symptomatic dogs compared to the asymptomatic. Reduction in cytokine expression results in decreased iNOS expression and therefore higher parasite load. The increase in IL-17 expression in the liver of asymptomatic dogs and its correlation with elevated expression of iNOS indicates a protective role of that cytokine in canine infection by *L. infantum* [67]. However, Michelin et al. [100] reported increased TNF-α, IL-4, and IL-10 levels in the liver of infected dogs compared to healthy dogs. In addition, the association between TNF-α levels and an increase in parasitic load in the spleen suggests its importance in the evolution of the infection process [100].

Another subject lacking clarification is the participation of chemokines and their receptors in resistance or susceptibility to LVC. Knowledge surrounding the role of these modulators in response to *L. infantum* infection is critical, considering that the interaction between cytokines and chemokines may regulate the immune response against the parasite, activating and recruiting immune cells to areas of infection.

Menezes-Souza et al. [101] analyzed the expression of CCL2, CCl4, CCL5, CCL13, CCL17, CCL21, CCL24, and CXCL8 chemokines in the skin of 35 dogs naturally infected by *L. infantum*, comparing cutaneous parasitism and clinical manifestations of the disease. Increase of the parasitic load correlated with increased expression of CCL2, CCL4, CCL5, CCL21, and CXCL8. On the other hand, CCL24 expression negatively correlated with parasitism [101].

After connecting clinical findings in naturally infected dogs with liver and spleen parasitism and expression levels for cytokines, chemokines, and their receptors, Albuquerque [67] showed that symptomatic dogs exhibit low expression of these modulators, alongside lower inflammatory response, and higher parasite load—primarily in the liver—than asymptomatic animals. CXCL10 was the only chemokine found at a much higher concentration in both the liver and the spleen of symptomatic animals. It also positively correlated with clinical score. The author indicates that expression profiles of hepatic and splenic chemokines and their receptors are essential for induction of correct cell inflammatory profile, as it has potential to contain the infection and the disease. Impaired cell migration facilitates replication of the parasite and development of CVL symptoms [67].

Understanding of the immune response in canine visceral leishmaniasis may reveal the factors involved with the onset and severity of clinical signs and the damage to host tissues. Additionally, it takes place as an indispensable tool for development of an effective vaccine.


**Knowledge Mapping of the Literature on Fiber-Reinforced Geopolymers: A Scientometric Review**

**Hassan Ali Alkadhim 1, Muhammad Nasir Amin 1,\*, Waqas Ahmad 2, Kaffayatullah Khan 1, Mohammed Najeeb Al-Hashem 1, Sara Houda 3, Marc Azab 3 and Zaher Abdel Baki 3**

**Abstract:** This study examined the bibliographic data on fiber-reinforced geopolymers (FRGPs) using scientometrics to determine their important features. Manual review articles are inadequate in their capability to connect various segments of literature in an ordered and systematic manner. Scientific mapping, co-citation, and co-occurrence are the difficult aspects of current research. The Scopus database was utilized to find and obtain the data needed to achieve the study's aims. The VOSviewer application was employed to assess the literature records from 751 publications, including citation, bibliographic, keyword, and abstract details. Significant publishing outlets, keywords, prolific researchers in terms of citations and articles published, top-cited documents, and locations actively participating in FRGP investigations were identified during the data review. The possible uses of FRGP were also highlighted. The scientometric analysis revealed that the most frequently used keywords in FRGP research are inorganic polymers, geopolymers, reinforcement, geopolymer, and compressive strength. Additionally, 27 authors have published more than 10 articles on FRGP, and 29 articles have received more than 100 citations up to June 2022. Due to the graphical illustration and quantitative contribution of scholars and countries, this study can support scholars in building joint ventures and communicating innovative ideas and practices.

**Keywords:** fibers; geopolymers; fiber-reinforced geopolymers; bibliographic analysis

#### **1. Introduction**

Geopolymer (GP) is a type of inorganic silico-aluminum composite with a 3D network made of a SiO4 and AlO4 tetrahedral unit structure [1–3]. It is manufactured by the interaction of active low-calcium silico-alumina ingredients with alkaline activators [4,5]. Active solid aluminosilicates and activators comprising alkali silicates and metals are required for the production of GPs [6–8]. The alkaline solution functions as an activator of alkali, binder, and dispersant [9]. In comparison to cementitious materials, GPs offer the benefits of high initial-age strength, rapid hardening, and a large variety of raw ingredients [10,11]. GPs use less energy and emit fewer pollutants during manufacture, and they are regarded as the material having the greatest potential to substitute cement [12–14]. The concept of GPs was proposed in 1978 to explain inorganic aluminosilicate polymers made using natural ingredients [15–17]. The intention was to utilize alkali metal silicate solutions to encourage the formation of polymeric aluminum silicate materials from geological minerals under severe alkaline environments [18,19]. Consequently, various solid silicate raw materials such as fly ash, slag, silica fume, and other wastes were utilized to effectively produce GPs [20,21].

Conventional cementitious materials have reduced durability such as resistance to elevated temperatures and deterioration [22,23]. GP composites effectively solve this deficiency [24–26]. However, GPs are comparable to ceramics in that their tensile and

**Citation:** Alkadhim, H.A.; Amin, M.N.; Ahmad, W.; Khan, K.; Al-Hashem, M.N.; Houda, S.; Azab, M.; Baki, Z.A. Knowledge Mapping of the Literature on Fiber-Reinforced Geopolymers: A Scientometric Review. *Polymers* **2022**, *14*, 5008. https://doi.org/10.3390/ polym14225008

Academic Editors: Wensheng Wang, Yongchun Cheng, Heping (Fred) Chen and Guojin Tan

Received: 3 October 2022 Accepted: 10 November 2022 Published: 18 November 2022

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

**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/).

flexural strengths are inadequate, and they are very susceptible to microcracks [27,28]. By integrating fibers, the brittleness of GPs may be addressed by increasing the toughness of composites [29–31]. The addition of fibers to the GP can inhibit the emergence of fractures while simultaneously enhancing its ductility, toughness, and tensile strength [32–34]. In recent years, several researchers have investigated the durability of GPs, which focused mostly on their resistance to abrasion, weathering, freeze–thaw, sulfate, water absorption, chloride ions, and various dry and wet impacts [35,36]. The mechanical performance and durability of composites are enhanced by altering the concentration of the alkaline solution, silicon to aluminum ratio, curing conditions, and the addition of fibers [37–39]. The inclusion of fibers increases the material's fracture performance and flexural strength and enhances its toughening process [29,40]. GPs with fiber reinforcement are more durable than cementitious composites of the same grade [41].

Currently, natural fibers, inorganic fibers, synthetic fibers, and steel fibers are the most frequent types of fibers utilized in GP composites [42–44]. Numerous research studies have been conducted on synthetic-fiber-reinforced GPs, such as polypropylene (PP), polyvinyl alcohol (PVA), polyethylene (PE), etc., but their manufacturing method contaminates the atmosphere and has difficulty satisfying the needs of sustainable development [45]. The majority of natural fibers is cellulose or plant fibers. Natural fibers have low cost, are lightweight, have strong adhesion, have easy production methods, and are biodegradable and are attracting the attention of academics [46].

As scientists continue to study fiber-reinforced geopolymers (FRGPs), because of the growing worries regarding the initiation and development of cracks, there is an issue regarding knowledge limitations that might prevent the establishment of new research and academic relationships. Therefore, it is essential to develop and use a method that enables scholars to acquire important information from the highly trustworthy sources available. This issue could be resolved with the aid of a scientometric process. Therefore, the purpose of this study is to conduct a scientometric evaluation of the literature data on FRGP investigations that has been made accessible up until June 2022. A scientometric analysis is used to carry out a quantitative examination of vast bibliographic records using advanced tools. This is because the many aspects of the literature cannot be appropriately and completely linked in traditional review studies. Complex elements of advanced studies include scientific visualization, co-citations, and co-occurrence [47,48]. Scientometric evaluations highlight the regions actively engaged in a research issue as well as the outlets with the greatest publications, frequently used keywords, and highly cited researchers and papers. To achieve the objectives of the current study, data from 751 pertinent papers were found using the Scopus search engine. This data included abstracts, keywords, citations, and bibliographic details. Additionally, the restrictions related to the applications of FRGP in the building industry were explored, along with possible remedies to these limits. Due to the graphical interpretation and quantitative records of countries and scientists, this study will assist academics in developing collaborative developments and exchanging fresh concepts and techniques.

#### **2. Review Strategy**

This work identified the different facets of the literature using a scientometric assessment of bibliometric data. In scientometric studies, systematic visualization, a technique developed by experts for analyzing bibliographic records, is applied [49,50]. Data retrieval was done using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) approach. The PRISMA checklist is attached as Table S1 in supplementary materials. Since there are extensive articles published on the topic under investigation, it was vital to use a trustworthy database. For this reason, the very dependable databases Scopus and Web of Science were appropriate [51,52]. The Scopus database, which academics strongly suggest [53,54], was used to compile bibliometric data on FRGP research. As of June 2022, 949 results for the term "fiber-reinforced geopolymers" were returned by a Scopus search. There were several filter settings used to reduce unnecessary papers. The whole PRISMA technique for data extraction, assessment, and the various restrictions and filters is shown in Figure 1. A similar strategy was used in some earlier investigations across other subject areas [55–57]. Finally, 751 records were used for further analysis using the appropriate tools. The literature data were compiled in Comma Separated Values (CSV) format, and VOSviewer software, version 1.6.18, was used to create the scientific representation and quantitative valuation of the information acquired. VOSviewer is an open-source visualization tool and freely accessible [58–60]. Therefore, by using VOSviewer, the intentions of the current study were achieved. The generated data (CSV files) were imported to the VOSviewer to allow for analysis. The systematic research looked at the publication outlets, the highly popular keywords, the highly referenced authors and publications, and countries' participation. Tables were created to provide quantitative data, while graphs were given to show the different traits, their interactions, and co-occurrence.

**Figure 1.** Flowchart of PRISMA technique for data retrieval, filters applied, and analysis.

#### **3. Results and Discussion**

#### *3.1. Progress on the Research of FRGP*

For research development and subject area evaluation, the Scopus analyzer was utilized. As illustrated in Figure 2, engineering and materials science were revealed to be the two top disciplines that produced the most articles, each field producing around 39% of the total papers in the FRGP research. Furthermore, documents related to the study field were searched for on Scopus (Figure 3). According to this study, almost 73%, 18%, 6%, and 3% of all the data comprised journal articles, conference papers, conference reviews, and journal reviews, respectively. Figure 4 shows the annual progress of papers published on FRGP studies from 1991 to June 2022. The initial publication of the FRGP research was discovered to be from 1991. With an average of nearly three articles published yearly up to 2010, there has been a very slight increase in the progress of publications in the area of FRGP investigations. Thereafter, the rate of publications grew gradually, averaging nearly 21 articles per year between 2011 and 2016, with 37 publications in 2015. A significant increase in publications occurred between 2017 and 2021, with a yearly average of about

94 papers and 136 papers in 2021. With 111 articles published so far this year (June 2022), the publications in the field of the subject under study are increasing every year.

**Figure 2.** Relevant subject areas containing publications on FRGP studies.

**Figure 3.** Kinds of documents available on FRGP studies.

**Figure 4.** Yearly progress of publications on FRGP studies up to June 2022.

#### *3.2. Knowledge Visualization of Publishing Outlets*

Based on bibliographic records, VOSviewer was employed to assess publishing outlets (conferences/journals). A minimum limit of 10 articles was established for an outlet, and 17 of the 206 outlets complied with this requirement. Sources with at least 10 articles on FRGP till June 2022 are included in Table 1, along with the citations obtained during that period. The average citations for each outlet were calculated by dividing the citations with documents. With 95, 57, and 31 documents, respectively, *Construction and Building Materials* (*CBM*), *IOP Conference Series: Materials Science and Engineering*, and *Materials* were found to be the leading three publication outlets in terms of total publications. Moreover, *CBM*, which obtained 3188 citations, *Composites Part B: Engineering*, which obtained 1615 citations, and *Ceramics International*, which received 690 citations, were the top three journals based on citations gained till June 2022. When the comparison of each outlet was made using average citations, the leading outlets were noted to be *Composites Part B: Engineering*, with nearly 77, *Cement and Concrete Composites*, with about 37, and *Journal of Materials in Civil Engineering*, with nearly 35 average citations. In particular, this study would serve as the foundation for the next scientometric analyses of FRGP research. Additionally, past standard review studies were unable to offer a comprehensive summary of the data published. An illustration of sources with at least 10 articles published is shown in Figure 5. The frame size in Figure 5a is connected to the source's contribution to the topic under investigation based on document count; a greater frame size denotes a stronger influence. For instance, *CBM* has a broader frame than the others, suggesting that it is a key outlet in the subject area. On the map, three clusters were created, each with its unique color (green, red, and blue). Clusters were created using the research outlet's breadth or how frequently they are mentioned together in relevant articles [61]. The co-citation rates of the sources were categorized by VOSviewer in publications. Ten articles, for instance, were included in the red cluster and were co-cited several times in similar articles. Moreover, in a cluster, linkages between close outlets were stronger than those between widely scattered frames. Compared to *Materials Today: Proceedings* or *MATEC Web of Conferences*, *CBM* showed a greater association with *Composites Part B: Engineering*. Varying colors corresponded to different density concentrations for an outlet, as noticed in Figure 5b. Red, yellow, green, and blue were in order of declining density concentration, with red having the maximum intensity. The red/yellow shades in *CBM*, *Materials*, and other well-known outlets signified a stronger commitment to FRGP investigations. Additionally, the text of some outlets was found to be smaller, faded, and unclear because of the low-density concentration, implying their lower contribution to the research of FRGP.

**Figure 5.** *Cont*.

**Figure 5.** Systematic map of publication outlets: (**a**) network map; (**b**) density map.

#### *3.3. Knowledge Visualization of Keywords*

In research, keywords are vital because they differentiate and draw attention to the main domain of the study [62]. The least number of repeats for a term was set at 20, and 88 of the 4261 keywords met this requirement. The top 30 keywords that were used the most frequently in the literature are shown in Table 2. The five very frequent terms in the FRGP research included inorganic polymers, geopolymers, reinforcement, geopolymer, and compressive strength. FRGP has mainly been researched to increase mechanical performance and durability, mainly to decrease brittle behavior by bridging fractures, according to the keyword evaluation. Figure 6 displays a systematic map of terms with relationships, co-occurrences, and densities according to their frequency of occurrence. In Figure 6a, the size of a keyword node reveals its rate of recurrence, and its location reveals where it co-occurs in articles. The leading keywords also have wider nodes on the map than the rest, suggesting that they are important keywords for careful evaluation in the research of FRGP. The graph highlights clusters in a way that shows how frequently they appear together across different publications. The color-coded classification of keywords is based on their co-occurrence in articles. Six clusters of varying colors are shown in Figure 6a. Figure 6b illustrates how different hues correspond to various levels of keyword density. Indicating a higher number of occurrences, inorganic polymers, geopolymers, reinforcement, and other noteworthy keywords are shaded in red/yellow. This discovery will help ambitious scholars choose keywords that will make it simpler to find publications on a specific topic.

**Table 2.** List of 30 highly employed keywords in publications of FRGP studies.

#### *3.4. Knowledge Visualization of Researchers*

Citations serve as evidence of a scholar's importance in a specific area of research [63]. The minimal publication requirement for a scientist was set at 10, and 27 of the 1583 writers reached this constraint. According to bibliometric data, Table 3 lists the authors of FRGP research with the most publications and citations. By dividing the overall citations by the publications, the average number of citations for a writer was calculated. When all factors, including the quantity of papers, the average citations, and the overall citations, were taken into account, it was difficult to assess a researcher's success. Instead, each element's evaluation for the scientist was evaluated separately. Shaikh F.U.A. was found to be the most productive scholar, based on the data analysis, with 26 papers, followed by Korniejenko K., with 25, and Ganesan N., with 18 publications. In the research of FRGP, Shaikh F.U.A. was first in the research area based on total citations with 1486, Alomayri T. was second with 714, and Jia D. was third with 638 total citations. However, Nematollahi B. may be placed at the top with about 62 average citations, Sanjayan J. may be in second place with about 58, and Shaikh F.U.A. may be in third place with approximately 57 average citations. The association between the most well-known writers and authors with at least 10 publications is seen in Figure 7. Figure 7a displays the scientific visualization of the researcher's co-authorship with at least 10 published articles in the investigation of FRGP. Additionally, Figure 7b shows the greatest number of citation-based related authors. A few FRGP scholars were connected by citations, as it was discovered that 8 of the 27 authors made up the largest group of connected authors based on citations.

(**a**)

**Figure 6.** Systematic map of keywords in the research of FRGP: (**a**) visualization map; (**b**) density.

**Table 3.** Authors together with their publications and citations in FRGP research.

#### *3.5. Knowledge Visualization of Documents*

An article's importance in a particular academic field is shown by the number of citations it has obtained. In their respective academic domains, articles having the highest citations are interpreted as revolutionary. A minimum of a 50 citation limit was set for an article, and 84 of 751 articles met this requirement. The top five documents in the field of FRGP, together with their citation details, are shown in Table 4. The article "Geopolymers—Inorganic Polymeric New Materials", by Davidovits J. [64], had 2553 citations. Additionally, Yan L. [65] and Lyon R.E. [66] were in the top three, with 310 and 291 citations, respectively, for their publications. However, as of June 2022, only 29 papers had received more than 100 citations. The systematic map of papers and their ties to the subject area based on citations is also demonstrated in Figure 8. A map of connected articles with a minimum of 50 citations up to June 2022 is shown in Figure 8a. According to the data analysis, 82 out of 84 articles were linked by citations. As a result, the majority of important papers in the current study field is connected together by citations. The map of density for the linked articles based on citations is shown in Figure 8b. Clearly, papers with more citations showed larger density concentrations.

**Figure 7.** Systematic map indicating author's collaborations: (**a**) authors with at least 10 articles; (**b**) connected authors based on citations.

**Figure 8.** Systematic mapping of papers: (**a**) connected articles based on citations; (**b**) density of connected articles.

#### *3.6. Knowledge Mapping of Countries*

In comparison to other states, some have contributed more documents to the subject topic and plan to keep on doing so. Readers can view sections of the scientific graph that are specifically for the FRGP study. The least articles limit for a country was set at 10, and 20 countries complied with this condition. According to Table 5, the represented countries have published at least 10 articles on the FRGP study. With 139, 129, and 116 publications each, Australia, China, and India published the most research. The top three countries in terms of citations were also discovered to be Australia (4153 citations), China (3439 citations), and the United States (1864 citations). Figure 9 indicates the scientific framework and the density concentration of the countries connected by citations. According to the quantity of papers released, a country's influence on a subject is reflected in the frame size shown in Figure 9a. The regions with the top levels of engagement had a larger density concentration, as seen in Figure 9b. Young researchers will be able to develop scientific alliances, start joint businesses, and discuss novel ideas and techniques with the aid of the graphical interpretation and statistical data of the participating states. Researchers from

nations with an interest in FRGP investigations can collaborate with experts in the research area and gain from their experience.

**Table 5.** Detail of regions actively devoted to FRGP investigations.

(**a**)

**Figure 9.** Systematic map of participating countries: (**a**) scientific visualization; (**b**) density.

#### **4. Discussion and Potential Applications**

Using literature data, this study carried out a systematic mapping and quantitative assessment of the FRGP research. Past conventional reviews lacked the ability to correctly and completely link various areas of the literature. This research identified the journals and conferences that presented the most articles, the commonly used keywords, the authors and publications that received the most citations, and the countries actively involved in FRGP investigations. The FRGP has mainly been researched to increase mechanical performance and durability, mostly to control brittle behavior by bridging cracks, according to keyword analysis. Additionally, bibliographic data were examined to sort out highly dedicated and prolific writers and countries based on publications and citations. New scientists will benefit from the graphical representation and statistical analysis of active nations and researchers as they establish joint ventures, form scientific alliances, and exchange new approaches and notions. Researchers from different countries motivated to further expand their studies on the application of FRGP can collaborate with specialists in the research field and benefit from their experience. This study explored and discussed the potential applications of FRGP based on an assessment of the literature data and a review of the highly pertinent documents.

As shown in Figure 10, the application of the material ranged from low-tech/low-cost to high-tech/high-cost, mostly dependent on the type of fiber and binder utilized to form the composites. It is essential to realize that correct conclusions on the effectiveness of each composite should be described separately for various uses. Below are some instances of the uses of FRGP composites.

**Figure 10.** Fiber-reinforced geopolymer's possible applications [28].

Due to their accessibility and affordability, steel fibers have been frequently employed in cementitious materials for structural purposes. Steel fibers are incorporated to minimize shrinkage and enhance flexural performance, post-cracking performance, and energy absorption capability of GPs [69]. The highly alkaline nature of GPs maintains the passive condition of the steel reinforcement, hence making it a robust composite for several infrastructure applications [70,71]. Due to the high mechanical strength, flexibility, and hydrophilicity of PE and PVA fibers, niche applications have been examined for PE and PVA-FRGP, such as the progress of strain-hardening GP composites, which require a material with ultra-high impact resistance and ductility [28,72]. A similar use for PP-FRGP has been examined to make the composite more cost-efficient and environmentally friendly [73,74]. In addition, PP and PVA-FRGP have been explored for extruding-based 3D printing processes to construct formwork-less structures with complicated geometries and minimal water curing requirements [67,75,76]. The inflammability of GPs, coupled with the exceptional elevated-temperature resilience of inorganic and carbon fibers, can be utilized in the fabrication of materials where thermal resistance is necessary [66,77]. In addition to their great strength and low weight, carbon fibers have the possibility to be used in the production of lightweight, durable, and robust huge constructions. In addition, carbon-nanotubes were utilized not only to improve the fracture energy of the GPs but also to offer electrical conductivity and piezoresistive responses to examine micro-crack development [78,79]. Inorganic fibers such as silicon carbide and basalt are cost-efficient substitutes for carbon fibers in the production of elevated-temperature GP composites [80–82]. Natural fibers are often inexpensive and flexible, and they may be employed as reinforcement in GPs at high concentrations. Several production procedures have been developed and used to address the poor compaction of GPs reinforced with natural fibers. In the presence of 8.3% short cotton fiber, for instance, roller compaction was utilized to drive the GP binder into the fiber system, resulting in a material with a tensile strength of around

32 MPa [83]. However, there is a need for in-depth investigations, methods, and guidelines for large-scale practical applications of FRGP.

#### **5. Conclusions**

This study conducted a scientometric review of the available literature data on fiberreinforced geopolymers (FRGP) research to assess several criteria. The Scopus search engine was explored for 751 relevant records and evaluated by employing VOSviewer software. The main findings of this study are as follows:

**Supplementary Materials:** The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/polym14225008/s1, Table S1. PRISMA checklist.

**Author Contributions:** H.A.A.: funding acquisition, visualization, validation, writing—reviewing and editing. M.N.A.: conceptualization, validation, investigation, project administration, funding acquisition, writing—reviewing and editing. W.A.: conceptualization, methodology, resources, supervision, software, validation, investigation, writing—original draft, reviewing, and editing. K.K.: data curation, visualization, writing—reviewing and editing. M.N.A.-H.: investigation, writing—reviewing and editing. S.H.: data curation, funding acquisition, formal analysis, writing—reviewing and editing. M.A.: resources, validation, investigation, writing—reviewing and editing. Z.A.B.: formal analysis, visualization, writing—reviewing and editing. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the Deanship of Scientific Research, Vice Presidency for Graduate Studies and Scientific Research, King Faisal University, Saudi Arabia (Project No. GRANT1697), through its KFU Research Summer Initiative.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data used in this research have been properly cited and reported in the main text.

**Acknowledgments:** The authors acknowledge the Deanship of Scientific Research, Vice Presidency for Graduate Studies and Scientific Research, King Faisal University, Saudi Arabia (Project No. GRANT1697), through its KFU Research Summer Initiative. The authors extend their appreciation for the financial support that made this study possible.

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

#### **References**

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*4.1. Introduction of the Coast–Ship Bistatic HFSWR*

A coast–ship HFSWR experiment was conducted in July 2019 using two Compact Over-horizon Radar for Marine Surveillance HFSWR systems (CORMS). One radar was located on the coast near the city of Weihai (Shandong Province, China; Figure 16), and the other was deployed on the M/V *Shun Chang 28*. The experimental area comprised the open-water area off the coast near Weihai. The experiment was divided into two periods. The first period, from 09:18:00 to 10:07:00 local time, is indicated by the green line in Figure 16. During this period, measured experimental data using the CTSR bistatic HFSWR system were obtained. The second period, from 10:50:00 to 14:40:00 local time, is indicated by the red dashed line in Figure 16. During this period, measured experimental data using the STCR bistatic HFSWR system were acquired. The velocity and heading of the shipborne platform during the experiment are shown in Figure 17. Here, the heading is given clockwise relative to north.

**Figure 16.** Location of coast-based radar station and navigation route of shipborne platform.

**Figure 17.** Velocity and heading of shipborne platform.

The CORMS HFSWR system used in the experiment had a solid-state transmitter with maximum peak power of 500 W. The output power of the transmitter could be adjusted continuously. A linear frequency-modulated interrupting continuous wave signal was used, and a double-whip transmitter antenna at the height of 11 m transmitted an omnidirectional pattern. The HF radar receiver was fully digitalized with eight channels, although only five channels were used in the experiment. Each element of the receiving array was a small magnetic cylindrical antenna (length: 0.5 m, diameter: 0.4 m), suitable for shipborne platforms. The radar frequency was 4.7 MHz and the bandwidth was 60 KHz.

In the coast–ship bistatic HFSWR experiment, synchronous transmitter–receiver monostatic HFSWR data were also obtained using a system collocated with the transmitter station. The transmitters and receivers of both radar systems, as well as the two radars, were synchronized using GPS. Both the shore-based receiving array and the shipborne receiving array were composed of five elements but with different element spacing. Owing to the limitation that the length of the shipborne platform was only 88 m, the available array aperture of the shipborne receiving array was only 62 m and its antenna spacing was 15.5 m. Conversely, the antenna spacing of the shore-based receiving array was 29 m, and the array aperture was 116 m, i.e., >100 m. Besides, motion attitude information of the shipborne platform was recorded synchronously using the shipborne inertial navigation system. The measured radar echo spectrum data of the coast–ship bistatic HFSWR system obtained in the experiment are discussed in the following section.


**Author details**

Dayane Juliate Barros1 , Marília de Souza Bento2 , Murilo de Souza Ferreira3 , Janaina Braga do Carmo4 , Rogério Hartung Toppa4 , Vania Neu5 , Maria Bernadete de Amâncio Varesche6 , Paula Benevides de Morais1 and Acacio Aparecido Navarrete1,4,7\*

1 Graduate Program in Biodiversity and Biotechnology – BIONORTE, Federal University of Tocantins (UFT), Palmas, Tocantins, Brazil

2 Graduate Program in Biotechnology and Environmental Monitoring (PPGBMA), Federal University of São Carlos (UFSCar), Sorocaba, São Paulo, Brazil

3 Graduate Program in Hydraulic and Sanitation Engineering (PPG-SHS) of the São Carlos School of Engineering, University of São Paulo (USP), São Carlos, São Paulo, Brazil

4 Department of Environmental Sciences, Federal University of São Carlos (UFSCar), Sorocaba, São Paulo, Brazil

5 Social Environmental and Water Resources Institute, Federal Rural University of Amazonia (UFRA), Belém, Pará, Brazil

6 Department of Hydraulics and Sanitation, São Carlos School of Engineering, University of São Paulo (USP), São Carlos, São Paulo, Brazil

7 Graduate Program in Agronomy (PPGA), Federal University of Mato Grosso do Sul (UFMS), Chapadão do Sul, Mato Grosso do Sul, Brazil

\*Address all correspondence to<EMAIL_ADDRESS>
© 2019 The Author(s). Licensee IntechOpen. This chapter is 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.


*Article* **Dystonia: Sparse Synapses for D2 Receptors in Striatum of a DYT1 Knock-out Mouse Model**

**Vincenza D'Angelo 1 , Emanuela Paldino 2 , Silvia Cardarelli 3 , Roberto Sorge 1 , Francesca Romana Fusco 2 , Stefano Biagioni 3 , Nicola Biagio Mercuri 1,2, Mauro Giorgi 3,\* and Giuseppe Sancesario 1,\***

Received: 27 December 2019; Accepted: 1 February 2020; Published: 6 February 2020

**Abstract:** Dystonia pathophysiology has been partly linked to downregulation and dysfunction of dopamine D2 receptors in striatum. We aimed to investigate the possible morpho-structural correlates of D2 receptor downregulation in the striatum of a DYT1 Tor1a mouse model. Adult control Tor1a+/+ and mutant Tor1a+/− mice were used. The brains were perfused and free-floating sections of basal ganglia were incubated with polyclonal anti-D2 antibody, followed by secondary immune-fluorescent antibody. Confocal microscopy was used to detect immune-fluorescent signals. The same primary antibody was used to evaluate D2 receptor expression by western blot. The D2 receptor immune-fluorescence appeared circumscribed in small disks (~0.3–0.5 µm diameter), likely representing D2 synapse aggregates, densely distributed in the striatum of Tor1a+/+ mice. In the Tor1a+/− mice the D2 aggregates were significantly smaller (µm2 2.4 ± SE 0.16, compared to µm2 6.73 ± SE 3.41 in Tor1a+/+) and sparse, with ~30% less number per microscopic field, value correspondent to the amount of reduced D2 expression in western blotting analysis. In DYT1 mutant mice the sparse and small D2 synapses in the striatum may be insufficient to "gate" the amount of presynaptic dopamine release diffusing in peri-synaptic space, and this consequently may result in a timing and spatially larger nonselective sphere of influence of dopamine action.

**Keywords:** dystonia; striatum; D2 receptors; synapses; dopamine volume transmission

#### **1. Introduction**

Dystonia is a disorder of movement characterized by disturbed agonist-antagonist muscle activation [1], with particular difficulty switching between sequential muscles involved in a complex task, either voluntary or involuntary [2]. Symptomatic dystonia can be observed in various neurological disorders, including cerebrovascular diseases, Parkinson's disease, and Wilson's disease [3]. Unlike symptomatic dystonia, no pathologic correlate is still known for many dystonic disorders categorized as idiopathic dystonia, and further divided into generalized, focal, and segmental dystonia [4].

In the past, various dystonic forms were often interpreted in psychological or psychiatric terms until the late 19th century [3,5], when the descriptions of familial forms of generalized primary torsion dystonia suggested that it is an organic disease [6,7]. The identification of genetic mutations in some families in the late 20th century established an organic framework for idiopathic dystonia (see 3).

The most common and severe form of genetic dystonia is caused by a 3 bp deletion (GAG) in the coding region of the TOR1A (DYT1) gene, which results in a defective protein called torsinA [8]. (see 3).

Although hereditary, DYT1 dystonia has a childhood or adolescent clinical onset [4]. However, significant neurodegeneration could not be documented at the histological level in any brain areas of patients with DYT1 dystonia [9,10], suggesting that dystonia pathophysiology is determined by functional rather than structural abnormalities. Several neurophysiological and neuroimaging studies, as well as new genetic insights, have been so far performed in DYT1 dystonia, contributing to define functional abnormalities in the basal ganglia [11–15]. Clinical neuroimaging studies have revealed decreased caudate-putamen dopamine D2 receptor availability in DYT1 patients compared to controls [16,17]. Moreover, reduced striatal D2 receptor binding and protein level have also been reported in several different DYT1 experimental models [18–20]. Aside from D2 receptor downregulation, multiple lines of evidence demonstrated reduced coupling between the D2 receptor and its cognate G proteins, as well as severely altered D2 receptor electrophysiological plasticity in the striatum but not in the *substantia nigra* [18,21–25]. Comparative studies on the functions of D1, D2, an A2A receptors, as well as of different neurotransmitters (dopamine, GABA, glutamate, acetylcholine) have been performed by Pisani et al. in mouse models of dystonia, demonstrating a selective downregulation and dysfunction of D2 receptors [18,21,23]. In addition, a recent paper has clarified the mechanisms of D2 receptor downregulation in the striatum, mediated by increased lysosomal degradation, associated in turn with lower levels of striatal RGS9-2 and spinophiling, opening a new approach to the therapy [26]. Therefore, it is generally assumed that abnormal striatal synaptic plasticity, and D2 receptor-dependent striatal outflow abnormalities have a leading role in determining basal ganglia pathophysiology in DYT1 dystonia [27,28]. The developmental profile of the aberrant D2 receptor function has been studied in DYT1 mutant mice, recording in cholinergic neurons an abnormal excitatory response to the D2 receptor agonist quinpirole since postnatal day 14, which persisted at three and nine months in hMT mice [22]. significant neurodegeneration could not be documented at the histological level in any brain areas of patients with DYT1 dystonia [9,10], suggesting that dystonia pathophysiology is determined by functional rather than structural abnormalities. Several neurophysiological and neuroimaging studies, as well as new genetic insights, have been so far performed in DYT1 dystonia, contributing to define functional abnormalities in the basal ganglia [11–15]. Clinical neuroimaging studies have revealed decreased caudate-putamen dopamine D2 receptor availability in DYT1 patients compared to controls [16,17]. Moreover, reduced striatal D2 receptor binding and protein level have also been reported in several different DYT1 experimental models [18–20]. Aside from D2 receptor downregulation, multiple lines of evidence demonstrated reduced coupling between the D2 receptor and its cognate G proteins, as well as severely altered D2 receptor electrophysiological plasticity in the striatum but not in the *substantia nigra* [18,21–25]. Comparative studies on the functions of D1, D2, an A2A receptors, as well as of different neurotransmitters (dopamine, GABA, glutamate, acetylcholine) have been performed by Pisani et al. in mouse models of dystonia, demonstrating a selective downregulation and dysfunction of D2 receptors [18,21,23]. In addition, a recent paper has clarified the mechanisms of D2 receptor downregulation in the striatum, mediated by increased lysosomal degradation, associated in turn with lower levels of striatal RGS9-2 and spinophiling, opening a new approach to the therapy [26]. Therefore, it is generally assumed that abnormal striatal synaptic plasticity, and D2 receptor-dependent striatal outflow abnormalities have a leading role in determining basal ganglia pathophysiology in DYT1 dystonia [27,28]. The developmental profile of the aberrant D2 receptor function has been studied in DYT1 mutant mice, recording in cholinergic neurons an abnormal excitatory response to the D2 receptor agonist quinpirole since postnatal day 14, which persisted at three and nine months in hMT mice [22]. We aimed to investigate possible morpho-structural correlates of D2 receptor downregulation

*Int. J. Mol. Sci.* **2019**, *20*, x FOR PEER REVIEW 2 of 12

in some families in the late 20th century established an organic framework for idiopathic dystonia

Although hereditary, DYT1 dystonia has a childhood or adolescent clinical onset [4]. However,

We aimed to investigate possible morpho-structural correlates of D2 receptor downregulation in striatum of an adult DYT1 knock-out mouse model. in striatum of an adult DYT1 knock-out mouse model.

#### **2. Results 2. Results**  We first quantified the levels of D2 receptors on proteins extracted from the striatum. In line

We first quantified the levels of D2 receptors on proteins extracted from the striatum. In line with a previous study [26] western blotting analysis revealed a significant ~ 30% reduction (*p* < 0.05) of D2 receptor levels in the striatum of mutant Tor1a+/− compared to control Tor1a+/+ mice (Figure 1). with a previous study [26] western blotting analysis revealed a significant ~ 30% reduction (*p* < 0.05) of D2 receptor levels in the striatum of mutant Tor1a+/− compared to control Tor1a+/+ mice (Figure 1).

 **Figure 1.** (**a**) Comparative immunoblots of D2 receptors from the striatum of control Tor1a+/+, and mutant Tor1a+/− mice. β-actin content was detected as internal reference standard. (**b**) Densitometric analysis of relative optical density (OD) on D2 receptors immune-stained bands. Results were expressed as the mean ± SEM of the values obtained for each separate hemisphere from four mice per group. \* *p* < 0.05.

Light microscopy immune-histochemistry demonstrated an intense D2 receptor brown peroxidase reaction product reactivity in the striatum (Figure 2A). In control Tor1a+/+, the striatum displayed D2 positive neuronal perikarya, peripherally outlined by an intense reaction product, and surrounded by a diffuse lighter neuropil staining. These data document a large distribution of D2 receptors on neuronal bodies, and neuropil of striatal neurons. In Tor1a+/− the D2 peroxidase reaction product appeared less intense around neuronal bodies, as well as in the neuropil of the striatum (Figure 2B), confirming the western blot analysis. However, the diffuse brown reaction product detectable by the D2 light microscopy immune-histochemistry can give just a rough idea of the D2 densitometric changes around neuronal bodies and neuropil, but does not allow a precise definition of the morpho-structural characteristics of the D2 receptor aggregates in control and mutant mice.

**Figure 2.** Representative microphotographs of D2 receptor immune-histochemistry in control Tor1a+/+ (**A**), and mutant Tor1a+/− knock-out (**B**) mice. The brown reaction product is clustered around neuronal bodies and diffused in the neuropil. Scale bar in **B** = 100 µm.

Immune-fluorescence images were acquired with a LSM700 Zeiss confocal laser scanning microscope (Zeiss, Germany): 5× and 20× objectives were used to define areas of interest in the dorsolateral striatum; distribution of D2 receptors was first acquired using 63× oil immersion lens (1.4 numerical aperture), and thereafter with an additional digital zoom factor (1×–1.5×–2×). Images of D2 immune-fluorescence acquired with a 63× oil immersion lens at first look appeared as a shiny galaxy in a starkly sky, with clusters of extremely small grains covering diffusely the neuronal compartments of the striatum, without apparent difference between perikarya and neuropil, whereas grains were rare and almost absent on the cell nuclei and in striatal axonal bundles (Figure 3). The density of D2 positive fluorescent grains was clearly different between the striatum of Tor1a+/+ and Tor1a+/− mice. In Tor1a+/+ the D2 positive grain were contiguous and even superimposed each other, whereas in the striatum of Tor1a+/− mice the D2 positive grains were close but separated from each other (Figure 3).

*Int. J. Mol. Sci.* **2019**, *20*, x FOR PEER REVIEW 4 of 12

grains were rare and almost absent on the cell nuclei and in striatal axonal bundles (Figure 3). The density of D2 positive fluorescent grains was clearly different between the striatum of Tor1a+/+ and Tor1a+/− mice. In Tor1a+/+ the D2 positive grain were contiguous and even superimposed each other, whereas in the striatum of Tor1a+/− mice the D2 positive grains were close but separated from each

**Figure 3.** Representative immune-fluorescence microphotographs of confocal laser scanning microscopy (63×), double-labeled for D2 receptors and for nuclei in the striatum of control Tor1a+/+ (**A**), and of mutant Tor1a+/−knock-out (**B**) mice. D2 receptor immune-labeling is visualized in red-Cy3 fluorescence, while nuclei are visualized by DAPI fluorescence in blue. Bar in **B** = 10 µm. **Figure 3.** Representative immune-fluorescence microphotographs of confocal laser scanning microscopy (63×), double-labeled for D2 receptors and for nuclei in the striatum of control Tor1a+/+ (**A**), and of mutant Tor1a+/− knock-out (**B**) mice. D2 receptor immune-labeling is visualized in red-Cy3 fluorescence, while nuclei are visualized by DAPI fluorescence in blue. Bar in **B** = 10 µm.

A better understanding of D2 receptors subcellular distribution came out in images acquired using a 63× oil immersion lens (1.4 numerical aperture) with an additional digital zoom factor (1×–1.5×–2×). The immune-fluorescent signal appeared extremely specific without background staining so that the D2 receptor localization appeared circumscribed in very small disks, roundish or elliptical in shape, of size variable with a diameter between ~0.3–0.5 µm in the striatum of Tor1a+/+ mice (Figure 4A). A better understanding of D2 receptors subcellular distribution came out in images acquired using a 63× oil immersion lens (1.4 numerical aperture) with an additional digital zoom factor (1×– 1.5×–2×). The immune-fluorescent signal appeared extremely specific without background staining so that the D2 receptor localization appeared circumscribed in very small disks, roundish or elliptical in shape, of size variable with a diameter between ∼0.3–0.5 µm in the striatum of Tor1a+/+ mice (Figure 4A).

**Figure 4.** Representative immune-fluorescence microphotographs of high magnification (63× plus zoom factor 1×–1.5×–2×) confocal laser scanning microscopy, double-labeled for D2 receptors and for nuclei in the striatum of control Tor1a+/+ (**A**), and of mutant Tor1a+/− knock-out (**B**) mice. D2 receptor immune-labeling is visualized in red-Cy3 fluorescence, while nuclei are visualized by DAPI fluorescence in blue. Bar in **B** = 2 µm. **Figure 4.** Representative immune-fluorescence microphotographs of high magnification (63× plus zoom factor 1×–1.5×–2×) confocal laser scanning microscopy, double-labeled for D2 receptors and for nuclei in the striatum of control Tor1a+/+ (**A**), and of mutant Tor1a+/− knock-out (**B**) mice. D2 receptor immune-labeling is visualized in red-Cy3 fluorescence, while nuclei are visualized by DAPI fluorescence in blue. Bar in **B** = 2 µm.

In the striatum of Tor1a+/− mice, the D2 positive disks appeared sparse (Figure 4B), and the number and size of D2 positive disks and their total area per microscopic field was significantly lower in the striatum of Tor1a+/− than in corresponding areas of Tor1a+/+ mice (Figure 5A–C). Moreover, the mean distance between D2 positive disks was <1 µm for most of the disks in the striatum of Tor1a+/+ but > 2 µm for most of the disks in corresponding areas of Tor1a+/− mice (as can be easily seen in Figure 4).

**Figure 5.** Comparative densitometric analysis of the D2 immune-fluorescence in the striatum of control Tor1a+/+ and mutant Tor1a+/− knock-out transgenic mice. (**A**) Total D2 positive area per microscopic field through 63× oil immersion objective. df = 1, F = 31.202; *p* < 0.0001. (**B**) Number of D2 positive disks per microscopic field through 63× oil immersion objective (1.4 numerical aperture) with an additional digital zoom factor (1×–1.5×–2×). df = 1, F = 13.406; *p* < 0.001. (**C**) Size of D2 disk (µm2 ) through 63× oil immersion objective (1.4 numerical aperture) with an additional digital zoom factor (1×–1.5×–2×). df = 1, F = 6.543; *p* < 0.04. Results were expressed as the mean + SE of average values detected within the randomly selected microscopic areas of the dorsolateral caudate-putamen of both hemispheres from four mice in each group. Comparison among groups was performed with the ANOVA one-way.

However, quantitative analysis of the relative immune-fluorescence intensity per single D2 positive disk in the striatum was slightly less intense in Tor1a+/− than in Tor1a+/+ mice (Figure 6, Table 1), suggesting that the density of D2 receptors in large and small aggregates is substantially similar in control and mutant animals.

**Figure 6.** Table 1—Comparative fluorescence intensity of D2 receptor spots evaluated by Java image processing and ImageJ in microphotographs from confocal laser scanning microscopy. D2 positive spots were randomly selected and marked in red in control Tor1a+/+ (**A**), and mutant Tor1a+/− knock-out (**B**) mice. Bar in **B** = 2 µm. Table 1—Results were expressed as the mean + SE of the area and the average grey values (0–255) within the selected spots of the dorsolateral caudate-putamen of both hemispheres from three mice in each group. Marked decrease of the area (see analysis in Figure 5), but not of the grey level in D2 positive spots was observed in mutant Tor1a+/− knock-out mice, compared with controls.

**Table 1.** Control Mutant.

#### **3. Discussion**

The main finding of our work is that the reduced expression of D2 receptors in the Tor1a+/−striatum is associated with marked reduction in the number and size, but not in fluorescence intensity of the D2 positive residual aggregates. These results have however, some limitations in providing a morpho-structural correlate of the downregulation of D2 receptors in dystonia.

In our study, high-resolution confocal microscopy can give well-defined immune-fluorescent images of every D2 receptors' aggregate in the microscopic field, but without configuring at the same time the mosaic of the surrounding cell structures, unlike electron microscopy immunocytochemistry. Indeed, immune-fluorescent D2 receptors appear arranged in homogeneous roundish shapes, variable in size with a diameter between ~0.3–0.5 µm, likely approaching dimensions of dopaminergic synapses on small dendritic spines (with shaft diameter ~0.04–1 µm and length ~0.1–2 µm) of striatal medium spiny neurons [29]. The D2 receptor positive disks likely represent the subcellular structures formed by D2 molecular aggregates, but we can just hypothesize the subcellular sites where the D2 aggregates are inserted in Tor1a+/+ and in Tor1a+/− mice.

Instead, at the electron microscopy level, in the striatum the D2 receptors were extensively localized to 38% of dendrites and 48% of spines of medium spiny neurons, and quite frequently on axon terminals [30]. Moreover, synapses formed by D2 immune-reactive terminals were not always easy to be identified in the striatum due to a lack of pronounced pre- or postsynaptic densities, suggesting a significant D2 extra-synaptic localization [30]. Indeed, in the striatum 60%–70% of dopaminergic axon terminals, supposed to be dopamine release sites, do not make synaptic junctional complex [31]. On the other hand, while photomicrographs obtained by transmission electron microscopy can allow detailed morphological characteristics of synapses, they can just give a rough estimate of synaptic sizes, shapes [32], and of spatial distribution frequency, due to the high magnification and limited electron microscopic fields.

By confocal microscopy in our study, we can obtain a definite spatial arrangement of the D2 signals with an estimate of sizes and shapes of the D2 receptor aggregates, and of their distribution density through a relatively large microscopic field. D2 receptor aggregates have generally a disk-shape and a size in the range of dimension of dendritic spines, and so D2 receptor aggregates have morphological similarity with synapses which can be categorized as macular synapses among the three main types of synaptic junctions (macular, perforated, and horseshoe-shaped synapses) [33]. Therefore, we would try to approximately interpret the reduction in number and size of the D2 aggregates in the striatum of Tor1a+/− mice as a global reduction of D2 synapses, but without differentiating between the synaptic and extra-synaptic sites. Reduction in synapses' number and size is a well-known mechanism reducing efficiency of neuronal connectivity. However, the residual D2 synapses in Tor1a+/− mice display immune-fluorescence intensity and macular shape similar to those seen in Tor1a+/+ mice, suggesting that the synaptic arrangement and intensity of D2 receptors can be preserved in mutant animals.

A reduced topographic density of D2 synapses would be particularly relevant for dopamine transmission in the striatum of Tor1a+/− mice. Indeed, dopaminergic transmission is predominantly characterized as nonsynaptic or peri-synaptic volume transmission, since most dopaminergic receptors are extra-synaptic, i.e., rather distant from directly opposing dopaminergic terminal varicosities [29]. Once released by tyrosine hydroxylase (TH) positive terminals, dopamine is normally not limited in a synaptic cleft but diffuses far away from release sites, traveling through extracellular spaces usually along an effective radius of 7 µm in three dimensions, until dopamine molecules bind to synaptic and extra-synaptic D1 and D2 receptors or are cleared up by presynaptic dopamine transporters [29,34,35].

In Tor1a+/− mice, sparse D2 synapses in the striatum have to face a normal density of dopamine axon terminals, since the TH-positive axonal networks in mutant animals do not show morphologically obvious abnormalities in the striatum [36]. Moreover, there was no significant difference in striatal dopamine content between mutant and control mice, while its metabolite homovanillic acid (HVA) was higher suggesting that dopamine turnover was significantly increased in the mutant mice [36,37], according to evidence for increased dopamine turnover observed in the striatum of DYT1 patients [38]. Therefore, in DYT1 mutant mice the dopamine peri-synaptic landscape appears specifically changed, since D2 synapses are sparse in the striatum, but the sites of presynaptic dopamine release appear to be preserved [36,37]. In this scenario, dopamine molecules diffusing away from release sites can have a minor probability of binding in contiguous D2 synapses [39], so that they may have a large radius of diffusion, until they are bound by D1 and D2 receptors and/or are cleared up by presynaptic dopamine transporters. On the basis of the peri-synaptic dopamine volume efflux model, we hypothesize that in DYT1 mutant mice the sparse D2 synapses may be insufficient to "gate" a relatively abundant dopamine efflux, and this consequently may result in a timing and spatially larger nonselective sphere of influence of dopamine action.

The model of sparse D2 synapses can help interpret some aspects of DYT1 physiopathology in basal ganglia. At first glance, it seems inexplicable that the baseline and amphetamine-stimulated dopamine release detected by microdialysis is lower in mutant DYT1 mice compared with controls, while at the same time the caudate-putamen tissue dopamine levels were normal [36]. We can interpret the reduced dopamine release in mutant animals to be compensatory to D2 sparse synapses. Moreover, sparse D2 synapses can explain why DYT1 dystonia individuals do not respond to medications that alter dopamine transmission [36], since D2 synapses are sparse, there would be already a relative ceiling action of endogenous dopamine quantal release from dopamine preserved synaptic terminals. Finally, sparse D2 synapses can clearly differentiate the morpho-functional correlates of basal ganglia physiopathology in dystonia and Parkinson's disease, the one characterized as post-synaptic pathology with D2 sparse synapses insufficient to gate dopamine efflux from preserved dopaminergic terminals, the other as a presynaptic pathology with loss of striatal dopamine release terminals but conserved D1 and D2 receptors in experimental parkinsonian monkey [40]. Moreover, in tissues from parkinsonian patients who died without receiving L-dopa in the last weeks of life, the binding of a radioactive ligand to Dl or D2 was elevated 20%–60% above the neurological control tissue [41]. Accordingly, dopamine receptor agonists are useful medications even regarded as first choice to delay the starting of L-dopa therapy in Parkinson's disease [42], but ineffective in DYT1 dystonia [36].

Although the sparse D2 synapses may have a relevant role in DYT1 physio-pathology, it cannot be inferred that the observed D2 alteration is primary and specific, since the D2 receptors distribution in the striatum was not compared with other receptors in our study. In a previous work, we have demonstrated that in DYT1 mice carrying human mutant torsinA (hMT), the enkephalinergic neurons express a higher cellular content of the neuropeptide enkephalin and of PDE10A, a key enzyme in the catabolism of the second messenger nucleotides [43]. Therefore, we can infer that the downregulation of D2 receptors, predominantly expressed by the medium spiny enkephalinergic neurons, seems to be a primary process not secondary to reduced cellular capacity of protein synthesis in DYT1 dystonia.

However, it is not known whether the downregulation of the D2 receptors is selective and specific to the physiopathology of dystonia, or it is part of a larger process involving altered expressions of other receptor types, and in particular of the adenosine 2A (A2A) receptor subtype. It is worth noting that in the enkephalin positive striatal medium spiny neurons there is evidence for colocalization of the D2 receptors with the A2A receptors, which together form intramembrane cell complexes called heteromers, functionally interacting in a reciprocal antagonistic manner [44]. Likely, sparse D2 synapses may also result in an impaired D2/A2A interaction in DYT1 dystonia.

Further studies should investigate the synaptic and extra-synaptic distribution of the sparse D2 aggregates and of D2/A2A heteromers, evaluating their relationship with dopamine volume transmission in the physiopathology of basal ganglia microcircuits in DYT1 mutant mice. Moreover, clarifying the developmental morpho-structural characteristics of the D2 receptor aggregates and of D2/A2A etheromers in the DYT1 mouse model would be helpful to develop preventative treatment before clinical manifestation of disease.

#### **4. Materials and Methods**

#### *4.1. Animals and Methods*

C57BL/6 Tor1a+/− knock-out transgenic mice, that mimic the loss of function of the *DYT1* dystonia *TOR1A* mutation [45], were bred at the Santa Lucia Foundation Animal Facility; mice were sacrificed at 5–6-months-old. DNA was isolated and amplified from 1- to 2-mm tail fragments with the Extract-N-Amp Tissue polymerase chain reaction (PCR) kit (XNAT2 kit; Sigma-Aldrich, Milan, Italy, and genotyping performed as reported by Bonsi et al. [26]. All the efforts were made to minimize the number of animals utilized and their suffering. Treatment and handling of mice were carried out in compliance with both the EC and Italian guidelines (2010/63EU, D.lgs. 26/2014; 86/609/EEC; D.Lvo 116/1992), according to experimental protocols approved by the Animal Ethics Committee of the University of Rome "Tor Vergata" (D.M. 153/2001-A and 43/2002-A), and by the Santa Lucia Foundation Animal Care and Use Committee (D.M. 9/2006-A), and authorized by the Italian Ministry of Health (authorization # 223/2017-PR).

For biochemical studies, the animals (*n* = 5 Tor1a+/+, *n* = 5 Tor1a+/− knock-out) were killed by cervical dislocation, and their brains were removed rapidly and placed on an ice-cold plate. Thick brain sections were cut with an Oxford vibratome, and the caudate-putamen was dissected out rapidly from both hemispheres, and promptly frozen in liquid nitrogen and stored at –80 ◦C [46].

For morphological studies, the animals were deeply anesthetized with tiletamine/zolazepam (80 mg/Kg) and xylazine (10 mg/Kg), and perfused trans-cardially with 1% heparin in a 50 mL 0.1 M sodium phosphate buffer, and with 250 mL 4% paraformaldehyde in a 0.1 M sodium phosphate buffer (pH 7.4). The brains were removed immediately and post-fixed in the same fixative solution overnight at 4 ◦C. Coronal brain sections 40-µm-thick were cut with an Oxford vibratome across the entire rostro-caudal extent of the basal ganglia, and collected and stored at 4 ◦C in a 0.1 M phosphate buffer that contained 0.02% sodium azide, as previously described [43].

#### *4.2. Quantitative Analysis of D2 Protein*

The quantitative analysis of D2 receptor expressions in the striatum was assessed by western blotting. Tissues were lysated in 10 mM Tris–HCl pH 8, 150 mM NaCl, 1% *v*/*v* Triton X-100, 0.1% *w*/*v* sodium deoxycholate, 0.1% *w*/*v* sodium dodecyl sulfate, 1 mM EDTA, 5 mM β-mercaptoethanol, 1 mM PMSF, and protease inhibitor cocktail (Sigma-Aldrich). Thirty µg of proteins were loaded on a 9% SDS polyacrylamide gel and subjected to electrophoresis under reducing condition. The proteins were then transferred to a nitrocellulose membrane (Bio-Rad). The blots were incubated overnight at 4 ◦C with a rabbit polyclonal anti-D2 receptor antibody (1:1000, AB5084P; Millipore); or mouse anti-β-actin (1:10,000; Sigma-Aldrich) as a reference standard. Immune-reactive bands were revealed by horseradish peroxidase-conjugated secondary antibodies (1:10,000, Jackson Immunoresearch), incubated in a lumi-light-enhanced chemiluminescence substrate (Bio-Rad) and exposed to chemidoc (Bio-Rad). Densitometric analysis of scanned blots was performed using the NIH ImageJ version l.29 program (NIH, Bethesda, MD, USA).

#### *4.3. Immune-Histochemistry*

Coronal brain sections including the sensorimotor cerebral cortex, and the caudate-putamen were processed for identification of D2 positive receptors. Briefly, free floating sections were washed three times with Tris-buffered saline, pH 7.4, and endogenous peroxidase activity was inactivated by incubation for 5 min in Tris-buffered saline that contained 2% H2O2. Sections were rinsed with a Tris-buffered saline that contained 0.1% Triton X-100, and incubated with 2% normal goat serum, followed by overnight incubation at 4 ◦C with the primary polyclonal antibody (1:500) rabbit anti-D2 receptors (AB5084P, Millipore). The primary antibody was detected using a biotinylated secondary antibody (Vectastain ABC kit; Vector Laboratories, Burlingame, CA, USA), and an avidin horseradish peroxidase/diaminobenzidine/H2O2 chromogen system (Sigma-Aldrich, Milan, Italy). The specificity of the immunocytochemical reaction was confirmed by the absence of staining after omission of the primary antibody. After the diaminobenzidine reaction, the sections were rinsed with Tris-buffered saline and mounted on gelatine-coated slides, dehydrated, and cover-slipped with Permount for light microscopy examination. The sections were observed and photographed with a light microscope (Olympus BX51, Tokyo, Japan) equipped with an automatic micro camera (LeicaDC 300 F, Q550 IW Soft, Wetzlar, Germany).

#### *4.4. Immune-Fluorescence Techniques and Confocal Microscopy*

To evaluate the D2 receptor immune-fluorescence in striatal neurons, slices processing and confocal image acquisition were performed according to Bonsi et al. [26]. Mice were deeply anesthetized and perfused with cold 4% paraformaldehyde in a 0.12 M phosphate buffer (pH 7.4). The brain was post-fixed for at least 3 h at 4 ◦C and equilibrated with 30% sucrose overnight.  Slices were dehydrated with serial alcohol dilutions (50%–70%–50%) and then incubated 1 h at room temperature in a 10% donkey serum solution in PBS 0.25%-Triton X-100 (PBS-Tx). The primary rabbit anti-D2 antibody (AB5084P, Millipore) was utilized (1:500, three days at 4 ◦C). The following secondary antibodies were used (1:200, room temperature, 2 h): Alexa 488 and Alexa 647 (Invitrogen), and cyanine 3 (cy3)-conjugated secondary antibodies (Jackson ImmunoResearch, Cambridge House, UK). Cell nuclei were detected with a blue-fluorescent DNA stain by 40 ,6-diamidino-2-phenylindole (DAPI) (D9542, Sigma-Aldrich, Milan, Italy). After washout, slices were mounted on plus polarized glass slides with a Vectashield mounting medium (Super Frost Plus; Thermo Fisher Scientific) and coverslipped.

Images were acquired with a LSM800 Zeiss confocal laser scanning microscope (Zeiss, Germany), with a 5×, 20× objective, or 63× oil immersion lens (1.4 numerical aperture) with an additional digital zoom factor (1×–1.5×–2×) under no saturation conditions. Single-section images (1024 × 1024) or *z*-stack projections in the *z*-dimension (*z*-spacing, 1 µm) were collected. *Z*-stack images were acquired to analyze the whole neuronal soma, which spans multiple confocal planes. The confocal pinhole was kept at 1, the gain and the offset were adjusted to prevent saturation of the brightest signal and sequential scanning for each channel was performed. The confocal settings, including laser power, photomultiplier gain, and offset, were kept constant for each marker. For quantitative immune-fluorescence analysis, images were collected from at least 3–4 slices processed simultaneously from each striatum (*n* ≥ 4 mice/genotype) and exported for analysis with the ImageJ software (NIH). Software background subtraction was utilized to reduce noise.

The intensity of D2 fluorescence per D2 positive spots was evaluated by Java image processing and ImageJ. Briefly, the D2 positive spots in immune-fluorescence microphotographs were randomly selected using a drawing selection circle closely around, and the red color image of D2 fluorescence intensity was analyzed by converting each pixel in greyscale (0–255). The minimum and maximum grey values within the selection were detected, and the mean grey value (the sum of the grey values of all the pixels in the selection area divided by the number of pixels) per spot was calculated.

#### *4.5. Statistical Analysis*

Data analysis was performed using the Statistical Package for the Social Sciences Windows, version 15.0 (SPSS). Descriptive statistics consisted of the mean ± SE for parameters with Gaussian distributions (after confirmation with histograms and the Kolgomorov–Smirnov test). The homogeneity of the variance was evaluated by Levene's test. Comparison among groups was performed with the ANOVA one-way. *P*-value < 0.05 was considered statistically significant.

#### **5. Conclusions**

The reduced expression of D2 receptors in the striatum of Tor1a+/− dystonic mice is associated with marked reduction in number and size of the D2 positive synapses. D2 sparse and small synapses may be insufficient to "gate" a relatively abundant dopamine release diffusing extrasynaptically through the interstitial space among striatal neurons. This may result in a longer duration and larger sphere of influence of dopamine transmission, making its action non-selective in striatal micro-circuitries. Our study suggests a relevant role for dopamine volume transmission in DYT1 dystonia physiopathology.

**Author Contributions:** V.D., M.G., and G.S. conceived and designed the study. V.D., E.P., and S.C. performed the experiments and acquired the data. R.S. performed data analysis. G.S. evaluated the results and wrote the manuscript. V.D., F.R.F., S.B., and N.B.M. contributed to critical reviewing and editing the manuscript. All authors approved the manuscript. All authors have read and agreed to the published version of the manuscript.

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

**Acknowledgments:** We wish to thank Paola Bonsi and Antonio Pisani for kindly providing the control Tor1a+/+ and the mutant Tor1a+/− mice. We are also grateful to Paola Bonsi, and to Antonio Pisani for valuable technical suggestions and critical reading of the manuscript. We kindly acknowledge James Linch for style reviewing.

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

#### **References**

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