*3.3. Water Vapor Permeability*

The results of the water vapor permeability test, as expressed by the water vapor diffusion resistance coefficient (μ), show a μ decrease for all hydrophobic treatments on limestone and mortar specimens, reducing the breathability of the substrates (Table 7). The reduction in water vapor permeability is an inevitable consequence of the water repellence properties of polymer film; however, the lowest possible decrease is pursued [43].

This reduction is more significant in limestone specimens, whereas it is almost negligible in mortar specimens. In fact, an increase of μ of 227% (HSila/Sil), 129% \*(HSil) and 74% (HNST) is observed on treated limestone specimens, when compared to untreated ones, whereas a minimal μ increase (<4%) is observed on the treated mortar specimens.

After artificial aging, only the HNST treatment applied on limestone maintains reasonably higher μ values (increase of 7%) when compared to untreated specimens, whereas HSil and HSila/Sil treatments still induce a drastic μ increase (46% and 188%, respectively). In the case of mortar specimens, the higher increase of μ was observed with HSila/Sil treatment (9%), and only a slight μ decrease in the case of HSil and HNST treatments (<2%). In general, the hydrophobic treatment that illustrates the most suitable behavior to water vapor permeability was HNST, with a moderate reduction of the μ on both substrates, even after artificial aging.

#### **4. Discussion**

The variation of the moisture transport properties of the substrates treated with the hydrophobic products is presented in Figure 4. In general, it can be observed that the hydrophobic products induce a decrease of the capillary water absorption coefficient (C), this decrease being more relevant with HNST treatment on limestone specimens. However, after artificial aging, HSila/Sil and HSil treatments show a higher durability, with a higher decrease of the C, if compared to specimens treated with HNST. Concerning mortar specimens, all treatments maintain a similar water absorption coefficient (considerably lower than untreated specimens), even after artificial aging.

**Figure 4.** Percentage variation of (**a**) capillary water absorption coefficient (C), (**b**) drying index (DI) and (**c**) diffusion resistance of the water vapor coefficient (μ) of treated specimens, before and after artificial aging, when compared to untreated specimens.

The different moisture transport properties of mortar and limestone can be attributed to the higher open porosity (30%) of the mortar when compared to the studied limestone (11%). Additionally, mortar specimens generally have a higher volume of coarse pores (>100 μm) when compared to this type of compact Moleanos limestone [40].

Regarding the difference in the effectiveness and durability of the hydrophobic products, it is worth noting that (monomer) silane molecules are considerably smaller (10 to 15 Å) when

compared to (oligomeric) siloxane molecules (25 to 75 Å) [44]. Thus, the longer Si–O chains of the siloxanes compared to the silane ones have a lower penetration depth in the compact limestone. Additionally, organo-modified siloxanes have an extremely high reactivity, which can hinder their in-depth penetration [2]. The silane has a potentially deeper penetration in the treated surface, with a higher reduction of the hydrophilicity and, thus, improved hydrophobic effectiveness. Furthermore, concerning the silane, it is generally assumed that the larger the molecule of the alkyl group linked to the silicon atom (which constitutes the structure of this compound), the higher the water repellency of the silane [8,45]. On the other hand, the long siloxane chains are more affected by environmental agents and weathering, undergoing degradation processes which can reduce their effectiveness as hydrophobic products [14].

The optimal performance obtained with the nanostructured product based on SiO2 and TiO2 (HNST) on the limestone can probably be attributed to the chain arrangement of the TiO2-SiO2 nanoparticles, due to the creation of the Si–O–Ti bond [23]. In fact, the copolymerization of the TiO2 and silane within the silica network can give rise to the formation of homogeneous organic–inorganic hybrid xerogel [24]. Additionally, the nanosize (<0.1 μm) of the silicon titanium oxide particles can match the dimension of pore network of the limestone. On the other hand, the low durability of HNST treatment can be attribute to the photocatalytic oxidation (of the organic radicals) and thermal degradation of the SiO2-TiO2 composite, which can weaken the adhesion and thus, durability of the coating [38].

Concerning the drying index, the hydrophobic products induced an increase of the DI on limestone specimens, with higher variation in the case of the HSil and HSila/Sil treatments. After aging, HSila/Sil significantly increase the DI of the treated limestone specimens, whereas HSil and HNST maintain values similar to unaged specimens. Greater variations are observed in mortar specimens; in fact, the HNST treatment induces an increase of the DI, and a drastic decrease is observed after artificial aging, indicating the probable degradation of the HNST treatment. On the other hand, the HSila/Sil and HSil treatments decrease the DI on mortar specimens, with a lower decrease of the latter after artificial aging compared with the HNST treatment. As reported in other works [46], the silane/siloxanes products can modify the pore netwok of the treated material by increasing the volume of capillary pores, probably also due to an air entraining effect of liquid siloxane. This feature can justify the improved resistance to freeze-thaw cycles, and thus, durability of HSila/Sil and HSil treatments compared to HNST.

Furthermore, the difference in the moisture transport properties of treated limestone and mortar can be attributed also to the higher roughness of the mortar, compared to flatter surface of the limestone, which accounts for better adhesion of both fissured and crack free SiO2-TiO2 films to the substrate [37].

All treatments induce an increase of the μ in limestone specimens. More specifically, specimens treated with HSila/Sil and HSil show the highest μ increase, which significantly decreases after artificial aging in the case of the HNST treatment. The HNST treatment shows a lower μ after artificial aging compared with unaged specimens. Considering the mortar specimens, it can be concluded that μ variation is extremely low with all treatments, both before and after artificial aging. The HSila/Sil is the only treatment which induced a slight increase of the μ after artificial aging.

Ultimately, it would be expectable that a substrate with higher DI would have also a higher μ (i.e., lower WVP). For treated limestone specimens this trend is confirmed; however, specimens treated with HSila/Sil have a higher μ/DI ratio (even after artificial aging) compared to the other treatments. A different behavior is observed with treated mortar specimens: Aged and unaged specimens with HNST treatment show the trend mentioned above, whereas unaged HSil treatment and aged HSila/Sil treatment show a DI decrease and a μ increase.

#### **5. Conclusions**

In this paper, the effectiveness and durability of three commercially available hydrophobic products (a silicon and titanium dioxides-based nanostructured dispersion—HNST; a silane/oligomeric siloxane—HSila/Sil; and a siloxane—HSil) when applied to a Moleanos limestone and on a cement-based mortar, were analyzed. The alteration of the moisture transport properties (water absorption by capillarity and under low pressure, drying kinetics and water vapor permeability) of the treated substrates, prior to and after artificial aging tests, was evaluated.

Results show that the effectiveness and durability of the water-repellent treatment is influenced both by the type of hydrophobic product and by the treated substrate.

Although the products were applied at different concentrations, following the recommendations of the producers, all treatments induce a significant decrease of the values of the capillary water absorption and water absorption under low pressure on the mortar specimens. A lower decrease was observed in the limestone specimens. This difference is attributed to the higher open porosity of the mortar specimens compared to limestone specimens, thus allowing a deeper penetration of the hydrophobic products, which increases the water-repellency of the treated mortar. After artificial aging, all hydrophobic treatments show a significant durability to the type and duration of the artificial aging cycles considered in this work, maintaining a reasonably low water absorption in both mortar and limestone specimens, when compared to untreated specimens. The HNST treatment shows a slightly greater loss of efficacy in terms of water capillary absorption and water vapor permeability after artificial aging, mostly when applied on limestone. Therefore, it can be considered less durable.

The treatments induce also a variation of the drying index, which increases with all treatments on limestone specimens (even after artificial drying) and a general decrease on mortar specimens, except for the HNST treatment before aging, which slightly increases the DI. On the other hand, the HNST treatment shows a lower drying index after artificial aging.

HSila/Sil and HSil treatments significantly reduce the water vapor permeability of limestone specimens, whereas the HNST treatment induces a smaller decrease, with values similar to those of untreated specimens after artificial aging. The WVP of the treated mortar specimens was not significantly affected by the hydrophobic treatments, even after aging tests.

These observations confirm that the hydrophobic products are generally more effective and durable on mortar specimens, rather than on low-porosity limestone, as in the case of the studied Moleanos limestone.

When pondering the variation of all the moisture transport properties, the hydrophobic product based on siloxane (HSil) has the best performance on cement-based mortar; in fact, the molecular structure of siloxanes matches to the higher porosity of this substrate. On the other hand, although it has a lower durability compared to the other treatments, HNST has the best performance when applied on Moleanos limestone, with a significant decrease of the capillary water absorption and a low variation of the drying index and of the WVP. This behavior can result from the combination of the low porosity and micro-sized pores of the stone with the surface deposition of a nanostructured layer. Additionally, the presence of TiO2 confers antibacterial activity against the microorganism growth and pollutant absorption.

The HSila/Sil treatment significantly decreases its water-repellent properties. However, it hinders the drying process and the breathability of the substrates, even after artificial aging. Thus, based on the results of this study, its use is not recommended in either limestone or mortar.

Further tests (e.g., optimization of the protocol; artificial aging cycles with UV light, pollutants and/or biological colonization; FTIR analysis of the hydrophobic products; morphological analysis by SEM-EDS; contact angle measurements of the treated substrates, among others) are ongoing to correlate the effect of the physical-chemical aging on the effectiveness of hydrophobic products, and ultimately, to verify the durability to more prolonged weathering action (from lab to real natural scale tests) of the hydrophobic products.

**Author Contributions:** Conceptualization, G.B.; Methodology, I.F.-C. and R.V.; Investigation, C.E.; Writing—Original draft preparation, G.B.; Writing—Review and editing, I.F.-C. and R.V.; Supervision, I.F.-C.; Project administration, I.F.-C. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Portuguese Foundation for Science and Technology (FCT), grant number PTDC/ECI-EGC/30681/2017 (WGB\_Shield – Shielding building' facades on cities revitalization. Triple resistance for water, graffiti and biocolonization of external thermal insulation systems).

**Acknowledgments:** The authors acknowledge the companies CIN, Saint-Gobain Weber and NanoPhos for the supply of the hydrophobic products.

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


**Author details**

Kondwani Kapinga1 \*, Jules Christian Zekeng2 , Lackson Chama3 , Nalukui Matakala2 , Stanford Siachoono3 , Obote Shakacite3 , Concilia Monde4 and Stephen Syampungani2,5

1 Dag Hammarskjöld, Institute for Peace and Conflict Studies (DHIPS), Copperbelt University, Kitwe, Zambia

2 ORTARChI Chair Environment and Development, Copperbelt University, Kitwe, Zambia

3 Department of Plant and Environmental Sciences, Copperbelt University, Kitwe, Zambia

4 Department of Zoology and Aquatic Sciences, Copperbelt University, Zambia

5 Department of Plant and Soil Sciences, Plant Sciences Complex, University of Pretoria, South Africa

\*Address all correspondence to<EMAIL_ADDRESS><EMAIL_ADDRESS>
© 2022 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.


**3. Primary ectopic (extracranial) meningiomas**

To begin this section with an aside, the designation of ectopic meningiomas as "primary" necessarily implies that there may be other categories of meningiomas as "secondary", "tertiary" and so on. The terminology "primary ectopic meningioma" (PEM for brevity sake) then designates those meningiomas that are outside of the CNS that develop presumably from separate meningeal precursor cells; either ectopic arachnoid cells or perhaps cells that are multipotent (e.g. neural crest) as we have briefly noted. In some sense, this terminology is redundant since if the meningioma (or any lesion) is designated as "ectopic" that lesion is not indigenous to its normal anatomic location and would therefore be "primary". So called "secondary extracranial meningiomas" are defined as those lesions that are an extension of an intracranial meningioma. The author has not seen further designations of meningiomas beyond secondary and it is difficult to conjure a scenario where the designation of "tertiary extracranial meningioma" would be applicable. In addition, in perusing this literature, the terminology is a bit confusing because different names have been applied descriptively to lesions that probably ultimately fall under an umbrella category (see above description of Type I cutaneous meningioma). Some have described extracranial meningothelial proliferations in four categories: 1) extracranial extension of intracranial meningioma, 2) extracranial meningioma, 3) the aforementioned primary cutaneous meningioma, and 4) metastatic meningioma [6]. We already see that category 1 is what has been referenced as a Type III cutaneous meningioma and 3 presumably encompasses the spectrum of cutaneous meningiomas that include Type III. In this author's simplistic way of thinking and since this is a review, primary ectopic meningioma is not an unreasonable designation because the terms denote a meningioma that is outside the CNS as a "stand alone" or primary tumor in that location. If the designation were only "ectopic meningioma" then extracranial extensions of intracranial meningiomas could technically be under that rubric. Additionally, and similarly, the term "meningothelial" seems to imply tissue that is by all standards meningeal but not a tumor (more later). Meningothelial tissue or proliferations would seem by definition to be primary and ectopic since intracranial meningothelial proliferations are really not a diagnostic category of CNS lesions except for the possibility of meningeal tissue as part of another tumor.

That aside, it is interesting when trying to quell cases of PEM from the searchable medical literature, the variety of names given to these lesions as hinted at, and the ensuing difficulty that arises in attempting to exactly define the numbers and types of PEM reported. As Gibson and Prayson aptly alert the reader in their excellent chapter on this subject [7], a variety of designations have been used when reporting these lesions including ectopic, extracranial, extraneuraxial, extradural, cutaneous, intraosseous, calvarial etc. In addition, case reports may designate one of these categories to describe the lesion, but indeed the lesion may actually represent a secondary extracranial meningioma, or the patient may have had a previous remote intracranial meningioma.

Combined with a previous review of 178 reported cases from the English language medical literature by Lang et al. [8], Gibson cited an additional 100 cases of PEM at the time of publication of their chapter (2009). In this author's brief survey of the entire medical literature not restricted to the English language from 1/1/2008 to the present, approximately 184 cases of PEM are noted after using search terms that Gibson identified as descriptors for PEMs such as "ectopic",

"extracranial", "extraneuraxial", "extradural", "cutaneous", "calvarial", "intraosseous" and "meningioma". By far, the most cases are reported from the head/neck anatomic location with intraosseous PEM by far exceeding any other location in the head and neck or elsewhere outside of the head and neck (**Table 1**). The most common site/location for PEM outside the head and neck was interestingly pulmonary followed by spinal/mediastinal/thoracic (**Table 2**).

It should not be terribly surprising that from a teleological perspective and given the developmental biology of meninges that the most commonly cited location of PEM is in the head and neck location and more specifically in the calvarial/skull bones. The intimate association of mesenchymal and/or neural crest cells in the development of the calvarial bones and meninges, particularly the dura which is directly apposed to the bone, makes this causative possibility very plausible. Likewise could be said for the greater number of cases of PEM found in the spinal/mediastinal/thoracic location. In children, certain tumors particularly neuroblastoma and its variants, can be found in this paraxial location along the sympathetic chain where neuroblasts are migrating during development.

PEM are quite uncommon as might be expected and the multiple reviews all cite basically the same incidence of approximately 1–2% of all meningiomas. Several

#### **Table 1.**

*Approximate location and number of primary ectopic meningiomas in the head and neck from 2008 to present.*

#### **Table 2.**

*Approximate location and number of primary ectopic meningiomas outside the head and neck from 2008 to present.*

*Meninges Outside the Meninges: Ectopic Meningiomas and Meningothlelial Proliferations DOI: http://dx.doi.org/10.5772/intechopen.100206*

excellent reviews have been published identifying the clinical and pathological characteristics of PEM in general and in specific locations including intraosseous (calvarial) [9], ear and temporal bone [10], sinonasal tract [11], and pulmonary [12–15]. In addition, PEMs have been reported in skin, kidney, retroperitoneum, mediastinum, extremities, adrenal gland, ovary (mature cystic teratoma) [7], In general, for PEM, in contrast to CNS meningiomas, there is only a slight female predominance in incidence with the exception of perhaps those presenting in the ear and temporal bone where at least in the series by Rushing et al., the M:F ratio (1,2) more closely approximated the M:F ratio for CNS meningiomas in adults [16]. PEM arising in the ear and temporal bone are particularly troublesome for the pathologist as they can have a broad differential diagnosis in a location that is not the genesis of a comparatively large number of specimens that might traverse a general pathologist's microscope. This differential diagnosis includes entities such as paraganglioma, schwannoma, melanoma, middle ear adenoma or carcinoid, and carcinoma. The middle ear was the most common location for these PEMs with a relatively high association with cholesteatoma (9/36 cases). A smattering of histological subtypes were identified in these PEM and this is the case for PEM in general. The most common histological subtype identified by far is meningothelial (range 47% to over 90% depending on site) followed at a significant distance by atypical or psammomatous [16]. As an aside, in searching our pathology archives for cases of meningioma, we found approximately 730 cases of meningioma over 20 years, with less than 5 PEMs found that were either intraosseous (calvarial) or middle ear (mastoid) (**Figure 1**) and no cases of extracranial soft tissue, visceral, or pulmonary PEMs.

#### **Figure 1.**

*Meningothelial meningioma of the mastoid presenting with tinnitus in a 46-year-old woman. No connection to the CNS was seen by imaging or intraoperatively. A: Low magnification view of meningioma composed of spindled and epitheliod cells in a whirling pattern with areas of more concentrated fibrous tissue. Scattered psammoma bodies were seen throughout the biopsy pieces (arrow, HE 40X). B: High magnification view of the relatively bland appearing nuclei with numerous pseudoinclusions, a hallmark nuclear feature of meningiomas (arrow, HE 600X). C: Diffuse staining of the meningeal lesional cells with epithelial membrane antigen (EMA 100X).*

A few reviews have been published specifically addressing primary pulmonary meningiomas (PPM), that is, meningiomas that occur in the lung parenchyma as cited. Three of the four reviews included part of the time span included in the review here [12, 14, 15]. Our review provides perhaps more emphasis on the pathological phenotypes and concurrence with other tumors than other reviews.

PPMs are interesting entities for several reasons. One, their pathogenesis might not be as teleologically obvious compared to PEMs that occur say in the head and neck. Two, pulmonary meningothelial proliferations/nodules are not a terribly uncommon finding incidentally in lung resections and will be the subject of the latter part of this review as part of the spectrum of extracranial/extraneuraxial lesions of meningothelial lineage. Three, there are multiple case reports of concurrent CNS and pulmonary meningiomas [17] as well as reports of concurrent PEMs in lung and bone [18], metastases from CNS meningiomas to the lung [19] in particular, and metastases from other tumors into CNS meningiomas [20] and even metastases from PEMs to the lung [21]. Four, PPM can be mimickers of a primary lung cancer, now the second most common cancer in both men and women in the United States and therefore should be considered (albeit somewhat down the differential diagnostic list) by every pathologist who looks at lung biopsies and resections [22]. Five, pulmonary meningothelial proliferations, either sporadic or diffuse, may even be more troublesome for the pathologist. These interesting categories in the spectrum of meningiomas cannot, unfortunately, be further elucidated in this brief review.

**Table 3** represents a review of the medical literature (English and non-English) searched in PubMed using the search terms "pulmonary" or "lung" and "meningioma" contained in the title/abstract from January 1, 2008 to August 16, 2021 (roughly corresponding to the last cases of PEM reported by Gibson et al. as noted). This search yielded 407 results of which 34 case reports were included reporting PPM [12–15, 18, 22–50]. One patient had two case reports approximately 10 years apart and one article published in a Spanish journal could not be obtained. The clinical characteristics (age, gender, location, size, recurrence rate) are in keeping with other reviews of PPM [12–15]. The immunohistochemical profiles were also in line with other reviews with most PPMs demonstrating staining with epithelial membrane antigen (EMA), progesterone receptor (PR), and vimentin. This review demonstrated perhaps higher numbers of both WHO grades II and III tumors (if presuming the remainder of the undesignated cases were WHO grade I which is a reasonably safe assumption).

An interesting aspect in the review of these cases and not overly emphasized in other reviews is the seemingly high number of patients with a history of other malignancies; almost one-third of the patients (10/34) with one patient having multiple (3) other tumors (osteosarcoma, fibrous dysplasia, and giant cell tumor of bone). This patient had a somatic mutation in the giant cell tumor of H3.3A and a germline mutation of BRCA2. Of course, one must be careful in drawing conclusions from a small dataset and in this case the patients with other malignancies were all in the age range where other malignancies are not uncommon (mean age 65 years, range 52–80). The malignancies in this group included buccal (1) (presumably squamous cell carcinoma), breast (2), gastric adenocarcinoma (1), papillary thyroid carcinoma (1), thymoma (1)/renal cell carcinoma (1)(same patient), rectal adenocarcinoma (1), concurrent lung adenocarcinoma (1) and teratoma (1) (immature and mature teratoma of the ovary and retroperitoneum, respectively). With the exception of the teratomas and thymoma, the other tumors are not uncommonly seen in this age group. As an aside, the PPM in the patient with teratomas does raise the remote possibility of a meningothelial/ meningioma metastases from one of the teratomas particularly since one teratoma was designated as immature.

*Meninges Outside the Meninges: Ectopic Meningiomas and Meningothlelial Proliferations DOI: http://dx.doi.org/10.5772/intechopen.100206*

**Table 3.**

*Review of clinical and pathological characteristics of primary pulmonary meningioma 2008–2021.*

A 21-year-old female with Diamond-Blackfan anemia was included in this cohort who presented with a chordoid histology PPM [39]. Diamond-Blackfan anemia (DBA) is an inherited bone marrow failure syndrome that has approximately 20 known genetic aberrations in genes encoding small/large subunit associated ribosomal proteins (RPS and RPL genes) [51]. A cursory review shows no definitive RPS or RPL genes to be associated with any of the non-neurofibromatosis, type 2 (NF2) associated genes known to be mutated in meningiomas [52–54]. While DBA is associated with higher rates of other cancers including osteosarcoma, vaginal squamous cell carcinomas, esophageal cancer, colon adenocarcinoma, and myeloid leukemias, increased risk for meningiomas have not been seen in patients with DBA to date [51]. However, subsequent to the publication of this young patient with DBA and chordoid meningioma, she was found to have a mutation of the RSP19 gene [51].

As noted, one patient in this cohort had papillary thyroid carcinoma (PTC). An interesting epidemiological study by Sughrue et al. [55] showed that compared to the expected prevalence for a similar population, patients with meningioma had a statistically higher incidence of papillary thyroid cancer and acute leukemia. From a pathology perspective, it is also interesting that both tumors share at least one characteristic histopathological feature; psammoma bodies. Psammoma bodies can be seen characteristically in several tumors including PTC, meningiomas, serous cystadenocarcinoma of the ovary, and melanotic schwannoma (one of the tumors with high prevalence in Carney syndrome). The pathogenesis of psammoma bodies is still controversial with theories involving vascular thrombosis of papillae, followed by calcification and endothelial necrosis. More recently, osteopontin (OPN), a calcium-binding glycophosphotein has been implicated in the formation of psammoma bodies being expressed in CD68 positive macrophages along with other factors that include alkaline phosphatase, osteocalcin, metalloproteinases, bone sialoprotein, and others [56]. OPN expression has been shown to be increased in meningiomas compared to normal meninges, correlates with histological grade, and is a predictor of recurrence in WHO grade I tumors [57, 58].

The pathogenesis of PPM and PEM particularly those outside of the head and neck, of course, remains speculative as do many aspects of normal meninges development as noted earlier. Hypotheses regarding the origin of PEMs have included ectopic arachnoid cells that are present in cranial and peripheral nerve sheaths and in cranial sutures, misplacement of arachnoid cells during development, pluripotent mesenchymal cells, perineurial cells of peripheral nerves, and entrapped meninges at sites of trauma [7, 16]. Meningiomas, arachnoid cap cells (cells making up the outer layer of the arachnoid mater and villi), and spindle cells within perineurium express EMA that is detected immunohistochemically. While nearly all meningiomas and perineuriomas express EMA, only about one-third of meningiomas express glucose transporter-1 (GLUT-1) while most perineuriomas express GLUT-1. Similarly, SSTR2 and PR are expressed in a high percentage of meningiomas compared to perineuriomas while the reverse is true for claudin-1 [59]. Indirect support for pluripotent mesenchymal cells as the cell of origin for PEMs derives from the fact that meningiomas can exhibit histologically other tissue types including bone, cartilage, muscle, vascular, and other tissues. With regards to PPM specifically, two theories have arisen regarding their pathogenesis. The first is similar to what has been proposed for PEMs elsewhere; pluripotent mesenchymal cells that reside in the subpleural region or from "precursor" lesions known as minute pulmonary meningothelial nodules (MPMN, more on these later) [12]. Both PPM and MPMN share some similar phenotypes particularly the expression of similar immunohistochemical markers (EMA, PR, vimentin) but it is certainly unclear if MPMNs are truly precursors of PPM. Indeed, there seems to be some

discrepancy in incidence of MPMN and meningiomas in autopsy series leading some to conclude that MPMN cannot be a precursor lesion [12]. Some have argued that MPMN are not precursor lesions based on genotyping studies demonstrating higher frequencies of loss of heterozygosity (LOH) and LOH affecting different loci in meningiomas compared to MPMN [60]. It is interesting that a progression of increasing frequency of LOH was noted from solitary MPMN to meningotheliomatosis to meningioma. Weissferdt et al. demonstrated that MPMN do share some genotypic alterations with PPM and CNS meningioma particularly deletion of the NF2 gene and gains of chromosome 22q [61]. The distinction between the two has mostly been by somewhat arbitrary size criteria (≤ 3 mm for MPMN). PPMs also tend to form a "mass" displacing lung.

From the pathology perspective, PEMs present specific and diverse diagnostic challenges. This is largely due to the variable histomorphologies that have been described not only in CNS meningiomas but also in PEMs and the various locations that PEMs can occur which raise different differential diagnoses. The differential diagnoses for PEM in the middle ear and temporal bone has already been discussed. For all PEMs in the head and neck location, Rushing et al. found paraganglioma, schwannoma, and metastatic carcinomas to be the most frequent misdiagnoses for ear and temporal bone PEM; Carcinoma, melanoma, olfactory neuroblastoma, and aggressive psammomatoid ossifying fibroma for sinonasal tract PEM, and dermatofibroma, melanoma, fibrosarcoma, leimyosarcoma, and synovial sarcoma for soft tissue and skin PEM [16]. The differential diagnosis for PPMs brings a variety of entities that can occur in the lung and mediastinum. These include sarcomatoid mesothelioma, solitary fibrous tumor, spindle cell thymoma, spindle cell carcinomas, inflammatory myofibroblastic tumor, synovial sarcoma, epithelioid hemangioendothelioma, and of course metastases [12]. As noted, meningiomas can present with a wide range of histological features including meningothelial, fibrous, microcystic, transitional (spindle cell component) psammomatous, angiomatous, secretory, metaplastic (lipidized cells), lymphoplasmacytic rich (all WHO grade I), clear cell (mimicking clear cell renal cell carcinoma), chordoid, atypical (WHO grade II), and rhabdoid (cells with eccentric nuclei and abundant eosinophilic cytoplasm), papillary, and anaplastic (WHO grade III). There are several excellent reviews of the histopathology of meningiomas along with the criteria for atypical and anaplastic variants [52, 54, 62]. And as mentioned before, other tissue types can be seen occasionally within meningiomas (i.e. bone, cartilage/chondroid, muscle). The variety of tissue types and potential to produce other tissues speaks highly for a pluripotent cell of origin (neural crest, mesenchymal) and are reminiscent of some other tumors that have such potentially variable histological appearances (e.g. yolk sac/endodermal sinus tumor which is derived from a pluripotent cell). Suffice it to say that given these variations, the differential diagnoses become expanded and criteria for narrowing the diagnoses are critical. Most of the identified histological subtypes have been identified in PEMs. In the review by Rushing et al. of extracranial head and neck PEMs, meningothelial, psammomatous, clear cell, atypical, and anaplastic were noted [16]. In sinonasal tract meningiomas, meningothelial is the most common subtype and transitional, metaplastic, and psammomatous types have been identified [11]. In PPMs, as noted in this review and others, meningothelial, transitional, fibrous, chordoid, rhabdoid, psammomatous, atypical, and anaplastic have been reported. Transitional histology is reported as the most common histological subtype in PPMs [12]. As seen in our review and noted in many publications, the immunohistochemical trio of EMA, vimentin, and progesterone receptor is present in the majority of cases of meningioma including PPMs. A smattering of cases will show staining with CD34, S100, and CD68 but are almost always negative for cytokeratins. In cases where these stains and morphology still leave doubt,

other markers should be included in the work-up panel. A cytokeratin stain (both high and low molecular weight or pankeratin) is very important in the exclusion of entities such as metastases from carcinoma, mesothelioma, and thymoma. Neural markers (chromogranin, synaptophysin) are usually negative in meningiomas and positive in paragangliomas for instance. CD56 has been shown to mark some meningiomas (4 positive cases in our series although most cases in the series did not perform or report this marker) but is also strongly positive in most paragangliomas. Since melanoma can be a great mimicker of many tumors, markers such as HMB-45, melan-A, MART1, and MITF are helpful in this distinction since these are almost always negative in meningiomas. S100 may be useful also in the distinction of melanocytic tumors and peripheral nerve sheath tumors (schwannoma) with usual strong diffuse staining of these entities. However, as seen, 6/14 cases that reported results of S100 staining in PPMs were positive in our series. While most did not report the extent or intensity of staining for S100, in general, it is probably not to the degree seen in melanocytic or peripheral nerve sheath tumors. Vascular markers, CD31 and CD34, can be helpful since most meningiomas are negative for these markers. These vascular markers should be positive in hemangioendothelioma. CD34 is usually positive in solitary fibrous tumor along with bcl-2 and is almost always negative for EMA. Bcl-2 is also useful for distinguishing synovial sarcoma. Muscle markers (smooth muscle actin, muscle specific actin, calponin and others) can helpful in the distinction of smooth muscle neoplasms and inflammatory myofibroblastic tumors along with ALK-1.

Another marker that demonstrates very high positive and negative predictive values for meningiomas is the monoclonal antibody to SSTR2a (somatostatin receptor 2a) [63]. This receptor is highly expressed in meningiomas as are other somatostatin receptors. Somatostatin ligand analogs such as DOTATOC and DOTATATE have been linked to 68Ga and used in PET imaging of meningiomas with great success. 68Ga-DOTATATE uptake correlates with SSTR2 expression and has high sensitivity for detecting active tumor in untreated and recurrent meningiomas [64]. In tissue specimens, the monoclonal antibody to SSTR2a has higher sensitivity than EMA or PR and did not stain with high intensity other lesions in the differential diagnoses including peripheral nerve sheath tumors (malignant and benign) and other carcinomas, mesenchymal tumors, or melanomas [63]. A high percentage of meningiomas also express p40 which is one isoform of p63 (p53 homolog gene) and the intensity of expression correlates with histological grade and recurrence [65].

All meningiomas are not sporadic. Multiple familial syndromes associated with specific genetic aberrations have increased risk for development of meningiomas. Neurofibromatosis 2 (NF2 gene, chromosome 22q12) has the highest lifetime risk for development of meningioma at 50%. Approximately 40% of sporadic meningiomas are driven by other genetic mutations other than NF2 [54]. **Table 4** highlights the known familial syndromes associated with varying degrees of increased risk of meningioma. As somewhat of an aside, patients with Werner syndrome (Progeria, premature aging syndrome) as noted have an increased risk of meningioma. In the excellent review by Lauper et al. [66], they noted 27 meningiomas in their cohort of 189 confirmed Werner syndrome patients. Eight of these tumors occurred in patients with multiple tumors and 5/9 patients with meningiomas also had thyroid neoplasia (1 PTC, 1 thyroid carcinoma NOS, 3 adenomas). Four of 5 patients had only meningioma and thyroid neoplasia as manifestation of their multiple tumors. Gardner/Turcot syndrome (APC-associated polyposis), PTEN hamartoma syndrome (Cowden, Bannayan-Riley-Ruvalcaba, Lhermitte-Duclos syndromes), MEN1 (Wermer syndrome), and Werner syndrome are familial inherited syndromes also highly associated with thyroid neoplasia [67]. Carney complex also has a high

**Familial syndrome Gene Chromosome location Risk %** Neurofibromatosis 2 NF2 22q12 >50 Multiple spinal meningiomas SMARCE1 17q21.2 BAP1 tumor predisposition BAP1 3p21.1 2 Familial schwannomatosis SMARCB1 22q11.23 Gorlin syndrome PTCH1 9q22.3 5 Cowden disease PTEN 10q23.31 8 Familial multiple meningiomas SUFU 10q24.32 Li-Fraumeni TP53/CHEK2 17p13.1/22q12.1 Rubinstein-Taybi CREBBP 16p13.3 Gardner syndrome/Turcot syndrome APC 5q21–22 Multiple endocrine neoplasia I MEN 11q13 Werner syndrome WRN (RECQL) 8p11.1–21.2 14 Von Hippel–Lindau VHL 3p25.3 Ataxia telangiectasia ATM 11q22

*Meninges Outside the Meninges: Ectopic Meningiomas and Meningothlelial Proliferations DOI: http://dx.doi.org/10.5772/intechopen.100206*

#### **Table 4.**

*Familial syndromes associated with increased risk of meningioma.*

association of thyroid neoplasia and has as a pathognomonic lesion the melanotic schwannoma, a tumor characterized by schwannian differentiation with the feature of psammoma bodies. Carney complex is known to result from mutations in PRKAR1A (chromosome 17q22–24) and CNC2 (2p16) while WHO grades II and III meningiomas have been shown to be associated with gains and amplification of 17q [53, 54, 67]. In addition, interestingly, Gardner/Turcot syndrome, Carney complex, and Werner syndrome all have various bone tumors as part of the manifestation/ diagnostic criteria for their respective syndromes. In the review by Lauper et al. [66], 17/189 Werner syndrome patients had osteosarcoma with 7/19 osteosarcomas occurring in patients with multiple tumors and one of these patients had meningioma/thyroid adenoma/osteosarcoma as the manifestation of multiple neoplasia. Another association is the presence of neoplasia associated with derivatives of the neural crest (retinal epithelial hypertrophy, benign and malignant peripheral nerve sheath tumors, benign and malignant melanocytic lesions). Gardner/Turcot, Carney complex, PTEN hamartoma, Werner syndromes all have melanocytic lesions as part of their neoplasia spectrum.

These are very interesting associations and can and will lead us to further elucidation of unifying mechanisms of molecular and cellular origins of tumors and in this case meningiomas. It is worth noting here that there are very few reports of PEMs associated with a familial inherited syndrome. What was presented above was data for associations with primary CNS meningiomas. Very few reports of PEMs in patients with a familial syndrome can be found. One case report describes an ectopic meningioma of the right parapharyngeal space in a 14-year-old girl with NF2 [68]. A second report describes an intranodal meningothelial proliferation in a 55-year-old woman with confirmed Cowden syndrome [69]. There seems likely a true difference in familial genetic syndromes and association with PEMs since one would think that specific case reports would address this as part of the unique spectrum of individual presentations of PEM.


**6. Landslides prevention and control**

Initially, the materials with a huge volume of water in the convex part of the slope begin to slide and roll over the concave part and finally on a fairly regular part (settlement areas). The extraordinary rapid movement of sediment and water destroys and buries people and houses, and finally due to the non-cohesive nature of slope materials, the slide terminates as dry flow. The steps involving the development of landslides (**Figure 5**) can be summarized

• Due to torrential rain, rainwater starts to infiltrate into the slope materials increasing pore

• On top of hills, crown cracks developed and those cracks allow more and more water to

• Landslides initiate when threshold for shear resistance is exceeded by pore water pressure.

• The resulting pore water pressure exceeds the shear resistance of the slope materials.

as follows:

water pressure.

infiltrate into the materials.

140 Engineering and Mathematical Topics in Rainfall

**Figure 5.** Stages of development of landslides.

The choice of landslides prevention and control measures depend upon the geological condition, physiography, available technology and above all the socioeconomic condition of the people. Important landslides prevention and control methods are [11] as follows:

Avoid method: If slope is extremely hazardous and landslides control measures are expensive, complete avoidance of the area is the only sustainable solution.

Water control method: Usually, rainwater infiltrates into the slope materials and makes them exposed to landslides. Proper management of rainwater through drainage from the slope instead of infiltration into the soil can significantly reduce landslide vulnerability. This can be realized by afforestation and building suitable drainage structures on slopes.

Excavation method: A slope turns out to be vulnerable when the natural angle of the slope becomes higher than the angle of internal friction of slope materials. The main objective of excavation method is to stabilize the slope by modification of the slope angle so that the internal friction angle of slope materials is higher than the slope angle.

Retaining structure: Structures that can be used for slope stability are buttresses, retaining walls, shear keys, guide walls, and so on.

The best prevention method for Chittagong area might be complete avoidance of hazardous slopes. A second option might be excavation method which suggests reforming the slope so that the slope angle becomes less than the angle of friction of slope materials. Thirdly, retaining structures like retaining wall, guide walls or buttresses can be constructed to protect the slopes from further landslides. However, if we could harness the modern communication and information technology to provide an in-advance warning prior to an incidence by developing real-time landslides early-warning system, then that should be one of the best options at least to save lives.


**Author details**

Mohd Muslim and Musheer Ahmad\* Faculty of Engineering and Technology, Department of Applied Chemistry, ZHCET, Aligarh Muslim University, Aligarh, UP, India

\*Address all correspondence to<EMAIL_ADDRESS>
© 2022 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.


LDH Ternary Nanocomposites: g-C3N4 Intercalated ZnO\Mg-Al for Superior Photocatalytic Activity towards Dye Degradation

*Kandasamy Bhuvaneswari,Thangavelu Pazhanivel, Govindasamy Palanisamy and Ganapathi Bharathi*


**2. Globalization**

Globalization is linking people and things at a faster pace than ever before. With global markets, supply chains have become more intricate, uncertain, and unpredictable. Therefore, globalization presents challenging problems to assuring the integrity of components used to build trustworthy information systems and networks. Critical information systems should be composed of parts that are trusted to do only that which is expected or specified and to do so reliably and dependably. Global supply chains are vulnerable to questions of unknown product or service provenance, which subsequently leads to questionable trustworthiness of the supplied items and the suppliers in the supply chain.

Both globalization and outsourcing are creating longer supply chains. Outsourcing creates a greater dependency on outsiders – procuring ever-more-complex and more critical products from external strategic suppliers instead of developing products in-house (Bolgar, 2010). Outsourcing projects can provide a number of benefits, including cost savings, increased productivity, improved schedule performance, and higher quality of work (Kliem, 2004). However, extended supply chains greatly increase the complexity of the supply network and decrease the visibility of risks. Nevertheless, globalization provides an opportunity to increase the security of mission critical information systems. The global marketplace can be leveraged to propagate better information assurance techniques and security practices in designing and building trusted information systems.


*1.4. Endothelial Dysfunction and Changes in Vascular Structure*

Endothelial dysfunction plays an important role in the pathogenesis of CKD and albuminuria [50]. Insulin resistance, low levels of adiponectin, high plasma leptin, increased levels of plasma glucose, and FFAs induce an inflammation profile that causes endothelial dysfunction, which causes increased protein loss from the kidneys.

Nitric oxide (NO) is produced from the endothelium, promotes vasodilation, reduces inflammation, and platelet aggregation. Phosphoinositide 3-kinase activation is causing phosphorylation of endothelial NO synthase (e-NOs) that produce NO [51]. Obesity is associated with reduced NO bioavailability. In the presence of insulin resistance this pathway is down-regulated, while hyperinsulinemia increases endothelin-1 levels resulting in imbalance between vasodilator and vasoconstrictor endothelium factors causing hypertension [52,53]. Vascular cell adhesion molecule-1 (VCAM-1), inter-cellular adhesion

molecule-1 (ICAM-1), and E-selectin increase monocyte adhesion to the vascular wall, inducing atherosclerosis. These vascular factors promote a cycle that is causing renal damage and hypertension [33].


**18. Autologous neurosensory retinal transplant (ART)**

In 2016, Grewal and Mahmoud introduced the use of an autologous full-thickness retinal free flap for closure of refractory myopic MHs. They applied endolaser and diathermy in a circular pattern around a 2-disc diameter area of the retina and using a bimanual approach with vertical scissors and forceps obtained a retinal free flap. Instillation of perfluoro-noctane heavy liquid over the flap was followed by a direct PFC-silicone oil exchange [98–102].

#### **19. Human amniotic membrane (hAM) transplantation**

Rizzo [103] describes the use of a human amniotic membrane (hAM) for MH treatment. A 2 mm disk of hAM grasped by forceps under fluid or perfluorocarbon was transplanted into the subretinal space. It is speculated that the hAM stimulates retinal pigment epithelium (RPE) cells division.

Kuriyan [104] described the use of commercially available human amniograft from Bio-Tissue (Tissue Tech, Miami) using sub- and pre-retinal placement. A dermal punch may be used to trim the tissue for hole placement. The chorion side is supposed to face the retinal pigment epithelium. Once in place inside or on top of the MH, the fluid-air exchange was performed.

### **20. Autologous platelet concentrate**

Gaudric [105] explored the effects of autologous platelet concentrate (APC) in macular hole closure by injecting some APC in the hole at the end of a

vitrectomy and obtained an improvement in anatomical closure rate. In a study comparing ILM peeling versus ILM peeling plus platelet-rich plasma (PRP) a significant improvement in anatomic and functional results was reported in eyes that had an application of PRP [106]. Several growth factors and cytokines are released by platelets and platelet-rich plasma has been used in various medical conditions [107]. Autologous PRP has been used in more complex holes such as myopic and refractory MH's with encouraging anatomical and functional results [108]. PRP derived from a patient's peripheral blood requires special tools, which may not be always available. One study found low rates of closure in refractory MHs with autologous blood [109] but a further study found high rates of closure when combining the inverted ILM flap technique with autologous blood for large MHs [110].

#### **21. Subretinal blebs**

The concept underlying this technique is that it is thought to increase retinal compliance by releasing the adhesions of photoreceptors to the retinal pigment epithelium (RPE).

The ILM is peeled in the usual manner at the sites of the injection. A small-gauge subretinal cannula (usually 38-41gauge) is used to inject a small volume of fluid under the retina, and usually few blebs are created by injecting BSS into the subretinal space. A confluent perifoveal serous detachment is induced, using a Tano diamond-dusted scraper or a Flex Loop to help massage the sub-retinal fluid until the retina surrounding the macular hole is detached. A fluid-air exchange is performed, and gas or silicone oil is used. This procedure has been shown to be a viable option for the closure of recurrent or persistent holes [20, 111, 112].

Multiple entry sites into the retina may be avoided as suggested by Felfeli [113] using a silicone extrusion cannula to inject fluid through the macular hole. Once the central retina has been detached by refluxing fluid into the macular hole the margins of the hole are once more re-opposed by carefully massaging them and this is followed by a fluid-air exchange and then gas tamponade. In a series of 39 complex cases, this technique resulted in a 95% closure rate. This procedure is especially useful where chorioretinal scarring is present at the macula resulting in extra adhesion of the retinal layers.

### **22. Retinal relaxing incisions**

Charles [114] reported using retinal relaxing incisions in six eyes to release tangential traction and increase retinal elasticity. This procedure does result in damage to the neuroretina and potentially the underlying RPE and visual improvement were limited to 3 cases.

Reiss [115] described 7 patients treated with five radial full-thickness incisions and gas tamponade. Using this procedure, the investigators reported 100% anatomic success in patients with refractory MH's.

The need to perform a deep incision in the retina with no damage to the underlying retinal pigment epithelium and choroid makes this procedure more technically challenging than others and with potentially greater risk.


**8. Radical resection for a locally advanced basal cell carcinoma involving floor of the orbit and zygomatic bone**

This demonstrated case has long standing basal cell carcinoma of the left lower eyelid with extension into the orbit (**Figure 22**). The CT scan showed soft tissue mass with enhancement involving left lateral eyelid skin and subcutaneous tissue with extension to the pre-septal and post-septal left orbit. The lesion invades anterior portion of the floor of the orbit and zygomatic bone (**Figure 23**). The plan of surgical resection includes access through upper eyelid preservation with upper eyelid incision marked 2 mm beyond the eyelash margin, while on the lower lid the involved skin and soft tissue is generously resected en-bloc with the specimen (**Figure 24**). The upper lid dissection is done between anterior and posterior lamella of the eyelid, while the lower lid incision is full thickness at least 1 cm away from the visible tumor margin. The skin incision is deepened, leaving a generous amount of soft tissue on the lateral and inferior wall of the orbit to secure adequate margins around the tumor. The skin flap is elevated laterally, directly over the zygoma and the anterior bony wall of the maxilla. The upper lid dissection and periosteal elevation is similar to the steps described for above first case. The bone cuts to encompass the tumor are depicted on a skull (**Figures 25** and **26**). A power saw is used to make bone cuts for the proposed inferior orbital wall and zygoma resection. The inferior

**Figure 22.** *Basal cell carcinoma of the left lateral lower eyelid with orbital invasion.*

**Figure 23.**

*CT scan showing orbital invasion by periocular basal cell carcinoma. Erosion of the orbital rim and floor is noticed.*

rim of the orbit is divided medial to the infraorbital foramen to keep the lower lateral quadrant with the specimen with adequate bone margins (**Figure 25**). This bone cut extends through the floor of the orbit up to the optic foramen posteriorly. Bone cuts are made on the lateral wall of the orbit at fronto-zygomatic suture and zygomatico-temporal suture (**Figure 26**). This superior bone cut is extended through to the superior orbital fissure (**Figure 25**). Small osteotomes are used to

#### **Figure 24.**

*The plan of surgical resection through a upper lid sparing, while generous amount of skin and soft tissues are kept on lower lateral region.*

**Figure 25.** *The inferior rim of the orbit is divided medial to the infraorbital foramen.*

### **Figure 26.**

*Bone cuts are made on the lateral wall of the orbit at fronto-zygomatic suture (blue circle) and zygomaticotemporal suture (black circle).*

#### **Figure 27.**

*Anterior view of the total orbital exenteration and lateral orbital wall resection specimen.*

#### **Figure 28.**

*Lateral view of the total orbital exenteration and lateral wall removal specimen.*

complete the bone cuts and to mobilize the bony attachments of the surgical specimen. Once all bone cuts are completed, the surgical specimen remains attached only at the cone of the orbit posteriorly through the attachments of the extraocular muscles and the optic nerve. The orbital contents are retracted laterally and the posterior attachments of the extraocular muscles and the optic nerve are transacted with curved enucleation curved scissors to deliver the specimen (**Figures 27** and **28**). Rest of the steps are similar to as described for the above case for achieving hemostasis. Specimen and orbital defect is examined after hemostasis is achieved and reconstruction of the defect done.


**3. Buchu and the reproductive system**

The benefits of Buchu may be applied to treat ailments of the reproductive system. As previously sited, Buchu is used as a urinary tract antiseptic and as an anti-inflammatory agent [2], thereby reducing the inflammation seen in UTI's and treating infections of the urethra and prostate. Buchu has many direct and indirect effects on the reproductive system via its ability to act as a diuretic, an anti-oxidant, anti-microbial and an anti-inflammatory agent. The ability of Buchu to do this will be looked at in more detail below, focussing on its actions in Benign Prostatic Hypertrophy/Hyperplasia (BPH), prostatitis, in UTI's and on male fertility.

### **3.1. Benign prostatic hypertrophy/hyperplasia**

BPH is the non-malignant enlargement of the prostate, and is the most common benign tumour in men over the age of 60 [17]. BPH has many predisposing factors, which include age, ethnicity, medical and family history and lifestyle factors such as cigarette smoking [17]. Men who have suffered from chronic diseases such as hypertension, coronary artery disease and diabetes mellitus are at risk of developing BPH [17].

The prostate is an exocrine gland [18] composed of fibromuscular tissue in a network of glands embedded in stroma [19]. Androgens are essential to growth of the prostate during early development, purberty and aging [20]. Androgens do not essentially cause BPH, but have been found to play a role in hypertrophy of the prostate along with other factors such as aging, diet, an increase in inflammatory mediators, hormones and oxidative stress [19].

BPH is characterised by nodular overgrowth of the epithelium and fibromuscular tissue [18] of the prostate, with the differentiation of fibroblasts into myofibroblasts being the hallmark of tissue remodelling [18] in the disease. Increased deposition of collagen in the ultrastruscture of the penis and the loss of smooth muscle causes urethral obstruction [21], causing a list of clinical symptoms related to urinary tract obstruction.

The pathophysiology of BPH is multifaceted, with local and endocrine factors being involved [17]. One of the main instigators is age-related changes to sex steroid hormone levels which leads to alterations in the metabolism of androgens locally. This disrupts the growth factor signalling pathways which interact with the stroma/epithelium, promoting tissue remodelling, leading to an increase in the size of the prostate [17]. The remodelled epithelium has an altered production of cytokines and chemoattractants, which encourages inflammation of the prostate and the production of ROS and RNS. With the increased proliferation of stromal and fibro‐ muscular cells comes an increased demand of nutrition and oxygen to be delivered to the prostate [17]. This leads to local tissue hypoxia, promoting neovascularisation to cope with the increased demand for blood flow, and the production of ROS [17]. The increase in ROS leads to further fibroblast to myofibroblast differentiation, perpetuating the cell proliferation cycle and resulting in a further increase in prostate volume.

Buchu, is hepatotoxic [2]. It has been found to reduce the levels of glutathione, a substance used by the liver in several detoxification steps [2], which would allow the accumulation of toxins within the liver. The depletion of glutathione and the excess amounts of pulegone found at high dosages lead to hepatocellular necrosis. Care should thus be taken when Buchu is consumed to ensure that the maximal therapeutic benefits are acquired, without any of the

The benefits of Buchu may be applied to treat ailments of the reproductive system. As previously sited, Buchu is used as a urinary tract antiseptic and as an anti-inflammatory agent [2], thereby reducing the inflammation seen in UTI's and treating infections of the urethra and prostate. Buchu has many direct and indirect effects on the reproductive system via its ability to act as a diuretic, an anti-oxidant, anti-microbial and an anti-inflammatory agent. The ability of Buchu to do this will be looked at in more detail below, focussing on its actions in Benign

BPH is the non-malignant enlargement of the prostate, and is the most common benign tumour in men over the age of 60 [17]. BPH has many predisposing factors, which include age, ethnicity, medical and family history and lifestyle factors such as cigarette smoking [17]. Men who have suffered from chronic diseases such as hypertension, coronary artery disease and

The prostate is an exocrine gland [18] composed of fibromuscular tissue in a network of glands embedded in stroma [19]. Androgens are essential to growth of the prostate during early development, purberty and aging [20]. Androgens do not essentially cause BPH, but have been found to play a role in hypertrophy of the prostate along with other factors such as aging, diet,

BPH is characterised by nodular overgrowth of the epithelium and fibromuscular tissue [18] of the prostate, with the differentiation of fibroblasts into myofibroblasts being the hallmark of tissue remodelling [18] in the disease. Increased deposition of collagen in the ultrastruscture of the penis and the loss of smooth muscle causes urethral obstruction [21], causing a list of

The pathophysiology of BPH is multifaceted, with local and endocrine factors being involved [17]. One of the main instigators is age-related changes to sex steroid hormone levels which leads to alterations in the metabolism of androgens locally. This disrupts the growth factor signalling pathways which interact with the stroma/epithelium, promoting tissue remodelling, leading to an increase in the size of the prostate [17]. The remodelled epithelium has an altered production of cytokines and chemoattractants, which encourages inflammation of the prostate and the production of ROS and RNS. With the increased proliferation of stromal and fibro‐

an increase in inflammatory mediators, hormones and oxidative stress [19].

Prostatic Hypertrophy/Hyperplasia (BPH), prostatitis, in UTI's and on male fertility.

unwanted toxic effects.

300 Antioxidant-Antidiabetic Agents and Human Health

**3. Buchu and the reproductive system**

**3.1. Benign prostatic hypertrophy/hyperplasia**

diabetes mellitus are at risk of developing BPH [17].

clinical symptoms related to urinary tract obstruction.

The roles of other steroidal hormones [20] have been proposed in the pathophysiology of BPH. The reason for this stemmed from the finding that prostatic cells continue to grow, even in the face of declining androgen levels [23]. This led to the rationale that other factors being secreted by the testes may stimulate growth or even sensitize cells of the prostate to the actions of androgens [23]. As men age, the increase in their body weight increases the amount of adipose tissue within the body, leading to an increase in circulating oestrogen levels [24]. This is evident in the levels of free estradiol found circulating in the bloodstream, which remains constant due to the age-related increases in body weight [25, 26]. Adipose tissue causes increased secretion of the enzyme aromatase, which stimulates the conversion of androgen to oestrogen. Increased circulating oestrogen levels stimulate the increased proliferation of cells seen in BPH as oestrogens have been found to increase the number of prostatic epithelial and stromal cells [27, 29]. Oestrogen also has an indirect effect on the increase in prostate volume through its role in mediating the alterations in other circulating serum hormones [20].

The changes seen in androgen levels with age disrupt the interaction of growth factors with prostate cells [22]. The growth of prostate cells is enhanced by the production of growth factors, which are supplied to the prostate via circulation [17] or locally through autocrine production by stromal cells [18]. The locally produced growth factors control cell differentiation and proliferation, and matrix protein production through a network that is interactive, providing for a negative feedback control mechanism that controls normal cell growth [18]. Any disrup‐ tion in this network leads to abnormal proliferation and stromal hyperplasia [18], which manifects as BPH. Stromal cell proliferation is enhanced by fibroblast growth factors (FGF 1 and 9), insulin-like growth factors (IGF I and II) and transforming growth factor β (TGFβ1) [18]. Expression of these growth factors is upregulated in BPH and are a mechanism which may be altered to reduce accumulation of prostatic cells.

The prostate is located at the neck of the bladder, enveloping the urethra [17]. This location plays a role in the obstructive symptoms seen in patients with BPH, which also correlate with the size of the prostate [17]. There are two components of prostatic enlargement that play a role in obstruction of the bladder outlet which lead to lower urinary tract symptoms, namely static and dynamic [17].

The static component is related to enlargement of the prostate which is attributed to the nodular proliferation classically seen in benign prostatic hypertrophy and hyperplasia [17]. This component accounts for the symptoms related to obstruction of the lower urinary tract seen in cases of BPH. The dynamic component related to the tone of prostatic smooth muscle [17]. Smooth muscle accounts for a rather large percentage of prostatic volume in BPH [17]. These provide binding sites for α1-adrenoreceptors, making the prostate susceptible to changes in adrenoreceptor signalling. α-adrenergic signalling has a significant influence on survival of stromal cells of the prostate and on the activity of smooth muscle cells in the prostate and the rest of the body [17]. α-adrenergic signalling alters expression of TGFβ1 and extracellular matix turnover [18].

and growth of infectious agents, which ultimately leads to further stimulation of the inflam‐

Buchu – The Multi-Purpose Ethnomedicinally Important Specie and Its Benefits in the Reproductive System

Inflammation of the prostate increases the risk of urinary retention, which suggests that the

Prostatic inflammation is a possible target for the prevention and treatment of BPH. Phyto‐ therapy has become a popular treatment modality for BPH, and the anti-inflammatory properties of Buchu may be applied to help alleviate inflammation found in BPH. The presence of essential oils and limonene in buchu oil can inhibit the synthesis of leukotrienes by blocking

and maintenance of inflammatory processes, thereby limiting an infection and preventing its

strengthen connective tissue, reducing inflammation and any downstream effects that it may have. The reduction in inflammation can assist in reducing inflammation-induced damage seen in prostatic tissue, putting an end to chronic would healing that is activated to repair the cells damaged by inflammatory processes. It will also diminish inflammation-based tissue

Oxidative stress is a condition caused by the imbalance between free radicals and free radical scavengers [15]. Free radicals are molecules with one or more unpaired electron(s) [36] that are highly reactive, attacking nearby stable molecules to gain an electron [15]. The molecule from which the electron was acquired becomes damaged, and it itself may initiate events that lead to damage of surrounding cells [37]. Free radicals are present at physiological concentra‐ tions which help maintain homeostasis via their actions as signal transducers [38]. Free radicals come in two common forms, i.e. ROS and RNS [15]. ROS include the superoxide ion, hydrogen peroxide, the peroxyl and the hydroxyl radical [39]. Reactive nitrogen species include nitric oxide, nitrous oxide, peroxynitrite, peroxynitrous acid and the nitroxyl anion, and are considered to be a subsection of ROS [39]. Free radical scavengers are known as antioxidants, and these assist in keeping free radicals at physiologically homeostatic levels [15]. By doing this, they help to prevent any pathological effects that may be activated by the development of oxidative stress, where ROS exceed the number of antioxidants due to increased ROS,

The body produces ROS on a daily basis via metabolic processes that occur in a normal cell [40]. ROS are generated endogenously by mitochondria and through metabolic and inflam‐ matory processes, but are also introduced into the body via external sources [40]. Physiological levels of ROS are essential to the regulation of many biological processes [40]. Increased generation of ROS is related to tissue injury and DNA damage, which are processes found in cellular proliferation, aging, infections and mutations in mitochondrial DNA [40]. Increased levels of ROS have also been associated with neoplastic transformation, abnormal cell

remodelling and prevent further enlargement of the prostate.

. Buchu can also prevent the attachment of bacteria to internal surfaces, inhibiting

. The flavonoids found in buchu may also modulate of inflammation and

. This will reduce inflammation by preventing the initiation

http://dx.doi.org/10.5772/57233

303

inflammatory processes occurring in BPH lead to lower urinary tract symptoms [19].

matory response [19].

progression2

biofilm formation2

synthesis of the enzyme 5-LOX2

**3.3. Oxidative stress in BPH**

decreased levels of antioxidants, or both [15].

proliferation and growth [40].

BPH presents clinically with lower urinary tract symptoms such as frequent urination, nocturia (frequent urination at night), the feeling of a full bladder even after voiding, post urination dribbling, weak urine stream and difficulty initiating urination [30]. It is associated with serious morbidity and may lead to complications which include acute urinary retention, and a reduction in the quality of life [17].

### **3.2. Inflammation in BPH**

A substantial number of inflammatory cells and pro-inflammatory cytokines have been found to be involved in prostatic cell proliferation [19]. The prostate is generally populated by a small amount of T and B lymphocytes, mast cells and macrophages [19]. These are chronically activated in BPH and release pro-inflammatory cytokines such as Interleuken 2( IL-2), Interferon gamma (IFN-γ) and TGFβ, which stimulate growth of the fibromuscular stroma [19]. Cytokines released by neighbouring cells stimulate the release of cyclo-oxygenase 2 (COX-2) in prostatic epithelial cells, increasing the cell turnover within the prostate [31, 32].

The local hypoxia stimulated by high cell turnover in BPH brings about the release of ROS which act as inflammatory mediators, causing enlargement of the prostate via stimulating differentiation of fibroblasts to myofibroblasts [33]. Local hypoxia also stimulates the release of growth factors and cytokines by stromal cells, including IL-8, vascular endothelial growth factors, fibroblast growth factors (FGF-2 and 7) and TGFβ [19]. TGFβ regulates stromal cell proliferation and differentiation in BPH, and is a key factor in the control of prostatic growth by androgens [34].

The prostate gland's immune response is mainly a cell mediated one, with regulatory T cells found in the stromal tissue and cytotoxic T cells in the gland's epithelia [35]. The accumulation of activated lymphocytes seen in inflammation of the prostate contributes to the development of BPH by causing tissue destruction and subsequent tissue rebuilding [20]. The cytokines produced may drive growth factor production leading to angiogenesis and cell proliferation as a "wound healing" mechanism [20]. The damage induced by inflammation of the prostate is thus a chronic process of wound healing where the excessive proliferation of prostatic cells leads to a cyclic reactivation of inflammation within the prostate [20]. This hyperproliferation and wound healing leads to the formation of prostatic nodules seen in benign prostatic hypertrophy/hyperplasia [19]. Inflammation leads to prostatic enlargement by stimulating growth of the prostate directly or indirectly by reducing apoptosis [19].

Whilst the mechanism of how inflammation leads to the pathogenesis of BPH is well under‐ stood, the origin of the inflammation is not as clearly understood [19]. Numerous aetiologies have been brought forward, including bacterial infections, an autoimmune response, urine reflux combined with chemical inflammation, hormones, diet and any combination of these [19]. The origin thus seems to be multifactorial [19]. It has been proposed that constant injury to epithelial cells leads to the loss of its function as an effective barrier, facilitating the passage and growth of infectious agents, which ultimately leads to further stimulation of the inflam‐ matory response [19].

Inflammation of the prostate increases the risk of urinary retention, which suggests that the inflammatory processes occurring in BPH lead to lower urinary tract symptoms [19].

Prostatic inflammation is a possible target for the prevention and treatment of BPH. Phyto‐ therapy has become a popular treatment modality for BPH, and the anti-inflammatory properties of Buchu may be applied to help alleviate inflammation found in BPH. The presence of essential oils and limonene in buchu oil can inhibit the synthesis of leukotrienes by blocking synthesis of the enzyme 5-LOX2 . This will reduce inflammation by preventing the initiation and maintenance of inflammatory processes, thereby limiting an infection and preventing its progression2 . Buchu can also prevent the attachment of bacteria to internal surfaces, inhibiting biofilm formation2 . The flavonoids found in buchu may also modulate of inflammation and strengthen connective tissue, reducing inflammation and any downstream effects that it may have. The reduction in inflammation can assist in reducing inflammation-induced damage seen in prostatic tissue, putting an end to chronic would healing that is activated to repair the cells damaged by inflammatory processes. It will also diminish inflammation-based tissue remodelling and prevent further enlargement of the prostate.

#### **3.3. Oxidative stress in BPH**

rest of the body [17]. α-adrenergic signalling alters expression of TGFβ1 and extracellular matix

BPH presents clinically with lower urinary tract symptoms such as frequent urination, nocturia (frequent urination at night), the feeling of a full bladder even after voiding, post urination dribbling, weak urine stream and difficulty initiating urination [30]. It is associated with serious morbidity and may lead to complications which include acute urinary retention, and

A substantial number of inflammatory cells and pro-inflammatory cytokines have been found to be involved in prostatic cell proliferation [19]. The prostate is generally populated by a small amount of T and B lymphocytes, mast cells and macrophages [19]. These are chronically activated in BPH and release pro-inflammatory cytokines such as Interleuken 2( IL-2), Interferon gamma (IFN-γ) and TGFβ, which stimulate growth of the fibromuscular stroma [19]. Cytokines released by neighbouring cells stimulate the release of cyclo-oxygenase 2 (COX-2) in prostatic epithelial cells, increasing the cell turnover within the prostate [31, 32].

The local hypoxia stimulated by high cell turnover in BPH brings about the release of ROS which act as inflammatory mediators, causing enlargement of the prostate via stimulating differentiation of fibroblasts to myofibroblasts [33]. Local hypoxia also stimulates the release of growth factors and cytokines by stromal cells, including IL-8, vascular endothelial growth factors, fibroblast growth factors (FGF-2 and 7) and TGFβ [19]. TGFβ regulates stromal cell proliferation and differentiation in BPH, and is a key factor in the control of prostatic growth

The prostate gland's immune response is mainly a cell mediated one, with regulatory T cells found in the stromal tissue and cytotoxic T cells in the gland's epithelia [35]. The accumulation of activated lymphocytes seen in inflammation of the prostate contributes to the development of BPH by causing tissue destruction and subsequent tissue rebuilding [20]. The cytokines produced may drive growth factor production leading to angiogenesis and cell proliferation as a "wound healing" mechanism [20]. The damage induced by inflammation of the prostate is thus a chronic process of wound healing where the excessive proliferation of prostatic cells leads to a cyclic reactivation of inflammation within the prostate [20]. This hyperproliferation and wound healing leads to the formation of prostatic nodules seen in benign prostatic hypertrophy/hyperplasia [19]. Inflammation leads to prostatic enlargement by stimulating

Whilst the mechanism of how inflammation leads to the pathogenesis of BPH is well under‐ stood, the origin of the inflammation is not as clearly understood [19]. Numerous aetiologies have been brought forward, including bacterial infections, an autoimmune response, urine reflux combined with chemical inflammation, hormones, diet and any combination of these [19]. The origin thus seems to be multifactorial [19]. It has been proposed that constant injury to epithelial cells leads to the loss of its function as an effective barrier, facilitating the passage

growth of the prostate directly or indirectly by reducing apoptosis [19].

turnover [18].

a reduction in the quality of life [17].

302 Antioxidant-Antidiabetic Agents and Human Health

**3.2. Inflammation in BPH**

by androgens [34].

Oxidative stress is a condition caused by the imbalance between free radicals and free radical scavengers [15]. Free radicals are molecules with one or more unpaired electron(s) [36] that are highly reactive, attacking nearby stable molecules to gain an electron [15]. The molecule from which the electron was acquired becomes damaged, and it itself may initiate events that lead to damage of surrounding cells [37]. Free radicals are present at physiological concentra‐ tions which help maintain homeostasis via their actions as signal transducers [38]. Free radicals come in two common forms, i.e. ROS and RNS [15]. ROS include the superoxide ion, hydrogen peroxide, the peroxyl and the hydroxyl radical [39]. Reactive nitrogen species include nitric oxide, nitrous oxide, peroxynitrite, peroxynitrous acid and the nitroxyl anion, and are considered to be a subsection of ROS [39]. Free radical scavengers are known as antioxidants, and these assist in keeping free radicals at physiologically homeostatic levels [15]. By doing this, they help to prevent any pathological effects that may be activated by the development of oxidative stress, where ROS exceed the number of antioxidants due to increased ROS, decreased levels of antioxidants, or both [15].

The body produces ROS on a daily basis via metabolic processes that occur in a normal cell [40]. ROS are generated endogenously by mitochondria and through metabolic and inflam‐ matory processes, but are also introduced into the body via external sources [40]. Physiological levels of ROS are essential to the regulation of many biological processes [40]. Increased generation of ROS is related to tissue injury and DNA damage, which are processes found in cellular proliferation, aging, infections and mutations in mitochondrial DNA [40]. Increased levels of ROS have also been associated with neoplastic transformation, abnormal cell proliferation and growth [40].

Androgens have been found to increase oxidative stress in the prostate gland [41]. Pathological levels of ROS bring about lipid peroxidation, apoptosis and DNA damage [15]. Lipid peroxi‐ dation is one of the main signs of oxidative damage, and has been found to stimulate alterations in membrane structure and has been linked to enzyme inactivation [41]. ROS cause this by abstracting a hydrogen atom from side chains of fatty acids in the cell membrane, initiating lipid peroxidation which has been found to enhance carcinigenesis [41].

**4. Urinary tract infections**

which is known to have bactericidal activity [45].

prostatitis) [46].

smooth muscle [2

in erectile dysfunction.

UTI's have a varied incidence that is dependent on age, sex and predisposing factors [45]. UTI's are an uncommon occurrence in healthy young men, but incidence does increase with an

Buchu – The Multi-Purpose Ethnomedicinally Important Specie and Its Benefits in the Reproductive System

http://dx.doi.org/10.5772/57233

305

The most common cause of UTI'sare bacteria, the most common being Escherichia coli, Klebsiella pneumonia, enterococci and Staphylococcus epidermidis, to name a few [45]. Bacteria causing UTI'sin men have been found to reach the urinary tract via an ascending route [45]. The bacterial flora found around the region of the anus is a reservoir for potential pathogens of the urinary tract [45]. Invasion of the urinary tract is lead by colonisation of the opening of the urethra, followed by adhesion and attachment to the epithelial lining of the urethra, with growth of bacteria on the urethral surface leading to ascension of bacteria into the urinary tract [45]. Invasion of the urethra by bacteria is difficult in men due to the distance between the opening of the urethra and the perianal region where the bacteria causing UTI'sare known to reside, the length of the urethra and the presence of prostatic fluid in the urethra,

Co-infection of the prostate in patients with UTI'sis unknown [45], but a mechanism has been suggested. Reflux of urine into ducts of the prostate lead to the retrograde transfer of bacteria from the urethra into the prostate, bringing about infection of the prostate gland (bacterial

Men with UTI'sare usually predisposed to them by functional or structural abnormalities [47, 49]. Lesions of the urinary tract which predispose men to UTI'sinclude bladder outlet obstruc‐ tion, bladder stones, stricture of the urethra, cancer of the bladder and kidney stones [50].

Signs and symptoms of UTI'sinclude frequency, pain on urination, flank pain and/or costo-

Lower UTI'sare related to erectile dysfunction and BPH [51]. This interaction may be affected by extraneous variables such as diabetes, aging and coronary artery diseases such as athero‐ sclerosis, but it was found to be independent of these variables when the co-morbidities were controlled for [51]; the association between lower UTI's(UTI) and erectile dysfunction remains. Men who are found to have more severe lower UTI's have more severe erectile dysfunction [51]. The mechanism that gives rise to this interaction is obstruction of the urethra which is propagated by collagen deposition in the penile ultrastructure and the subsequent loss of

Buchu has been found to have antimicrobial activity against bacteria that cause urinary tract

Flavonoids found in buchu may modulate inflammation and strengthen connective tissue. They may be used in the acute and long-term use for chronic or recurrent UTI, reducing the symptoms caused by UTI's [52]. The diuretic actions of Buchu will also assist in the reduction of inflammation by helping to flush bacteria out of the urinary tract through its ability to

infections, i.e. Staphylococcus aureus and Klebsiella pneumonia.

. The loss of smooth muscle leads to the reduction in erectile tissue, resulting

vertebral angle tenderness, which is tenderness in the area overlying the kidney [45].

increase in age due to predisposing factors and urological abnormalities [45].

Intracellular changes in the levels of ROS occur during proliferative, apoptotic and senescent processes, which can lead to the activation of signalling pathways [40]. As mentioned previ‐ ously, changes in tissue oxygenation during these processes causes and upregulation of cytokines and growth factors, which trigger prostatic cell proliferation, prostatic growth and inflammation. These processes themselves are a source of oxidative stress, which then leads to further proliferation and the initiation a chronic inflammation. The presence of ROS is thus an essential component in the pathogenesis of BPH when present at high levels within the prostate. This will help reduce ROS levels to physiologically homeostatic levels, allowing them to carry out their functions as signal transducers without activating any pathological mecha‐ nisms.

Endogenous antioxidant levels decrease with age [42], and this is compounded by other agerelated changes which ultimately lead to the manifestation of BPH. Oxidative stress can lead to cell mutations, which may lead to carcinogenesis and remodelling of the gland's internal structure [43]. BPH is an alternative, benign pathway of unregulated prostatic growth which is encouraged by inflammatory mediators and increased oxidative stress [41]. Management of oxidative stress is thus an imperative step in the management of BPH [41].

Management of oxidative stress is also an important factor in the prevention and treatment of BPH [41]. Buchu is an antioxidant-rich natural remedy that may be used to reduce oxidative stress. The polyphenolics contained within the plant scavenge free radicals, allowing them to act as anti-oxidants1 . The hydrogen donating ability of the phenolic groups1 will help reduce the bulid up of ROS. By donating hydrogen groups, these polyphenolics will stabilise free radicals, preventing them from attacking nearby cells to acquire it.

An increase in free radicals stimulates cyclooxygenase(COX) [44], a key enzyme in initiantion of the inflammatory process. Increase COX activity stimulates the production of an array of pro-inflammatory substances called prostaglandins. In patients with BPH and prostatitis there is an increased production of cyclooxygenase 2 (COX-2) and a reduction in the levels of prostaglandin E1 [44]. Free radical scavengers, particularly hydroxyl radical scavengers, suppress the over expression of COX and prostaglandin synthesis [44]. Ethanolis extracts of Buchu (Agathosma betulina) possess an inhibitory effect on COX-2 synthesis [44]. This inhibition will, as a consequence, suppress the inflammatory response and reduce hypertro‐ phy/hyperplasia and inflammation of the prostate, which is the goal of antioxidant treatment.

Reducing levels of ROS allows Buchu to interrupt the pathogenesis of BPH by reducing tissue injury, DNA damage, neoplastic transformation, and activation of growth factors that lead to abnormal cell proliferation and growth [40]. A diet rich in antioxidants such as vitamins A, C and E may also be prescribed to work in unison with Buchu to reduce oxidative stress.


**Abstract**

Fear conditioning is a behavioral paradigm of learning to predict aversive events. It is a form of associative learning that memorizes an undesirable stimulus (e.g., an electrical shock) and a neutral stimulus (e.g., a tone), resulting in a fear response (such as running away) to the originally neutral stimulus. The association of concurrent events is implemented by strengthening the synaptic connection between the neurons. In this paper, with an analogous methodology, we reproduce the classic fear conditioning experiment of rats using mobile robots and a neuromorphic system. In our design, the acceleration from a vibration platform substitutes the undesirable stimulus in rats. Meanwhile, the brightness of light (dark vs. light) is used for a neutral stimulus, which is analogous to the neutral sound in fear conditioning experiments in rats. The brightness of the light is processed with sparse coding in the Intel Loihi chip. The simulation and experimental results demonstrate that our neuromorphic robot successfully, for the first time, reproduces the fear conditioning experiment of rats with a mobile robot. The work exhibits a potential online learning paradigm with no labeled data required. The mobile robot directly memorizes the events by interacting with its surroundings, essentially different from data-driven methods.

**Keywords:** neuromorphic computing, associative memory learning, mobile robot, Hebbian learning, fear conditioning


**2.1 Catecholaminergic polymorphic ventricular tachycardia (CPVT)**

CPVT is an inherited cardiac disease with the prevalence of about 1:5000/10,000. This disease is characterized by premature ventricular contraction and/or polymorphic ventricular tachycardia (VT) induced by adrenergic stimulation in response to emotional stress or physical exercise in structurally normal heart. Over 150 mutations in ryanodine receptor type 2 (*RYR2* gene) are responsible for ~ 55% of CPVT type 1 cases (CPVT1), and mutation in calsequestrin 2 (*CASQ2* gene) CPVT accounts for 3–5% CPVT type 2 (CPVT2) cases [28, 29]. In addition, mutations in calmodulin (CALM1) genes and in triadin (TRDN) have been reported causing CPVT. *RYR2*, *CASQ2*, CALM1, and TRDN are involved in ECC, and mutation in any of these genes results in elevated intracellular Ca2+, which leads to abnormal Ca2+ handling and arrhythmias [28, 29]. In consistency with clinical phenotype, many hiPSC-CM model had demonstrated the exacerbation of electrophysiological and Ca2+ handling abnormalities upon adrenergic stimulation [26, 30–32]. Furthermore, Zhang and colleagues had modeled hiPSC-CMs harboring CPVT1-associated F2483I mutation in *RYR2* gene and demonstrated that CPVT1 hiPSC-CMs had longer and wandering Ca2+ sparks and smaller sarcoplasmic reticulum Ca2+ content [32]. Later on, the same group corrected this mutation using clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9) gene editing technique and showed that this mutation is causative rather than associative to the disease [33]. hiPSC-CM model for CPVT has also been used in studying the efficacy of various drugs. Previously we had directly compared the clinical results from CPVT1 patients with dantrolene medication, and the clinical response of dantrolene was similar as in hiPSC-CMs from the same patients; dantrolene

**67**

lular K+

encoding K+

**2.3 LQT type 2 (LQT2)**

*Modelling of Genetic Cardiac Diseases*

**2.2 LQT type 1 (LQT1)**

ing α subunit of potassium (K+

*DOI: http://dx.doi.org/10.5772/intechopen.84965*

abolished or markedly reduced arrhythmias in patients and their hiPSC-CMs with certain mutation in *RYR2*, while it did not have any clinical effect with hiPSC-CMs or with other *RYR2* mutations [31]. Furthermore, an antiarrhythmic drug, flecainide, used to treat CPVT1 patients [34] was able to reduce the Ca2+ irregularities under adrenergic stimulation in CPVT1 hiPSC-CMs [30, 35]. CPVT2 patients harboring homozygous CASQ2-G112 + 5X mutation in *CASQ2* gene showed the rapid polymorphic VT under exercise stress test [36]. Adult rat ventricular myocytes were studied to understand the effect of *CASQ2* mutation in ECC, demonstrating that mutated CMs exhibited spontaneous extrasystolic Ca2+ elevations and delayed afterdepolarization (DADs) upon adrenergic stimulation [36]. Later, hiPSC-CM model harboring CASQ2-G112 + 5X mutation emulated these phenotypic features of disease, and AAV9-based gene delivery effectively prevents the development of adrenergic-induced DADs and triggered arrhythmias in CPVT2 hiPSC-CMs [37].

LQT type 1 (LQT1) is caused by loss-of-function mutation in *KCNQ1* gene encod-

(IKs). LQT1 is responsible for 30–35% of all LQTS cases [38]. LQT1 is characterized by prolongation of QT interval in ECG, which could lead to SCD due to VT, typically torsades de pointes [39]. hiPSC-CMs derived from LQT1 patients faithfully recapitulated the clinical hallmark by showing prolonged action potential duration (APD) which is analogous to QT duration in ECG, and reduced IKs current densities are held responsible for abnormal repolarization [40–42]. ML277, an IKs activator, increased the IKs amplitude by enhancing the activation of IKs, thus resulting in shortening of APD in LQT1 hiPSC-CMs [40]. In addition, adrenergic stimulation in LQT1 hiPSC-CMs induced the early afterdepolarization (EAD) [42], which is similar to arrhythmias triggered in LQT1 patients by exercise or emotional stress [39]. Clinically, β-blockers were effective in minimizing the risk of cardiac events in LQT1 patients [43]. Similar antiarrhythmic effect of β-blockers has been observed in LQT1 hiPSC-CMs [42]. Furthermore, hypokalemia is the electrolyte disturbance caused by lower K+

blood serum, which aggravates the QT prolongation and facilitates the development of hypokalemia-induced torsades de pointes in LQT1 patients [39, 44]. We successfully developed and mimicked these disease phenotypes in LQT1 hiPSC-CMs carrying G589D or IVS7-2A > G mutation in *KCNQ1* gene. Additionally, lowering the extracel-

relaxation [46] observed in our video image-based software analysis [47].

LQT type 2 (LQT2) is an LQTS subtype, which is caused by loss-of-function mutations in *KCNH2* gene also known as human ether-a-go-go-related gene (*hERG*)

 channel mediating rapid delayed rectifier K current (IKr). LQT2 is responsible for approximately 25–30% of all LQTS cases [38]. Similar to LQT1, LQT2 patients also exhibit the prolongation of QT interval and torsades de pointes. As in LQT1 hiPSC-CM model, LQT2 hiPSC-CMs also recapitulated clinical phenotypes by displaying longer APD resulted from reduced IKr current densities and enhanced EAD following the adrenergic stimulation [48–50]. Our early study of LQT2 hiPSC-CMs carrying R176W mutation in *KCNH2* gene demonstrated the reduced IKr current densities, prolonged repolarization, and increased arrhythmogenicity although the donor is an

 concentration prolonged APDs and induced the formation of EADs in LQT1 hiPSC-CMs [45]. Both G589D- and IVS7-2A > G-specific LQT1 hiPSC-CMs displayed longer APD and higher Ca2+ abnormalities in baseline; G589D hiPSC-CMs demonstrated prolonged contraction, while IVS7-2A > G hiPSC-CMs showed impaired

) channel mediating slow delayed rectifier K+

current

level in

#### *Modelling of Genetic Cardiac Diseases DOI: http://dx.doi.org/10.5772/intechopen.84965*

*Visions of Cardiomyocyte - Fundamental Concepts of Heart Life and Disease*

**2. Channelopathy phenotypes in hiPSC-CMs**

testing. However, the human sample exhibits some of the major challenges: there is limited supply of human cardiac biopsies, and it involves complex procedures and ethical issues. In addition, these cardiac biopsies are typically obtained from the end stage of cardiac diseases; hence it is not possible to understand the mechanism of cardiac diseases [20, 21]. These obstacles are mostly overcome by the groundbreaking discovery of reprogramming adult somatic cells into induced pluripotent stem cells (iPSCs) [22, 23] which can be differentiated into cardiomyocytes (CMs) (hiPSC-CMs) [24–26]. The main advantages of hiPSC-CMs are iPSCs can be generated at any period of a patient's life, they have unlimited supply, and these retain the same genetic information as the donor, i.e., hiPSC-CMs are patient specific (**Figure 2**). These are superior features of hiPSC-CMs to the conventional in vitro modeling of cardiac diseases. In addition, hiPSC-CMs can be cultured for several months, which enable us to study acute and chronic effect of mutation and drugs on CMs. Thus, hiPSC-CMs not only provide the platform to investigate the mutation-specific mechanism but also assist to anticipate drug response on an individual basis and guide us to personalized

Channelopathy cardiac diseases are caused by mutations in cardiac ion channels located in the cellular membrane or organelles. Mutations in ion channels result in misbalance of fine-tuning ion exchange during excitation-contraction coupling (ECC), which could lead to cardiac arrhythmias and SCD in the worst case. The main cardiac channelopathies are CPVT, LQTS, BrS, and short QT syndromes (SQTS) [27]. These cardiac channelopathies have been extensively studied using

**2.1 Catecholaminergic polymorphic ventricular tachycardia (CPVT)**

CPVT is an inherited cardiac disease with the prevalence of about

1:5000/10,000. This disease is characterized by premature ventricular contraction and/or polymorphic ventricular tachycardia (VT) induced by adrenergic stimulation in response to emotional stress or physical exercise in structurally normal heart. Over 150 mutations in ryanodine receptor type 2 (*RYR2* gene) are responsible for ~ 55% of CPVT type 1 cases (CPVT1), and mutation in calsequestrin 2 (*CASQ2* gene) CPVT accounts for 3–5% CPVT type 2 (CPVT2) cases [28, 29]. In addition, mutations in calmodulin (CALM1) genes and in triadin (TRDN) have been reported causing CPVT. *RYR2*, *CASQ2*, CALM1, and TRDN are involved in ECC, and mutation in any of these genes results in elevated intracellular Ca2+, which leads to abnormal Ca2+ handling and arrhythmias [28, 29]. In consistency with clinical phenotype, many hiPSC-CM model had demonstrated the exacerbation of electrophysiological and Ca2+ handling abnormalities upon adrenergic stimulation [26, 30–32]. Furthermore, Zhang and colleagues had modeled hiPSC-CMs harboring CPVT1-associated F2483I mutation in *RYR2* gene and demonstrated that CPVT1 hiPSC-CMs had longer and wandering Ca2+ sparks and smaller sarcoplasmic reticulum Ca2+ content [32]. Later on, the same group corrected this mutation using clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9) gene editing technique and showed that this mutation is causative rather than associative to the disease [33]. hiPSC-CM model for CPVT has also been used in studying the efficacy of various drugs. Previously we had directly compared the clinical results from CPVT1 patients with dantrolene medication, and the clinical response of dantrolene was similar as in hiPSC-CMs from the same patients; dantrolene

**66**

medicine in future.

hiPSC-CMs and described below.

abolished or markedly reduced arrhythmias in patients and their hiPSC-CMs with certain mutation in *RYR2*, while it did not have any clinical effect with hiPSC-CMs or with other *RYR2* mutations [31]. Furthermore, an antiarrhythmic drug, flecainide, used to treat CPVT1 patients [34] was able to reduce the Ca2+ irregularities under adrenergic stimulation in CPVT1 hiPSC-CMs [30, 35]. CPVT2 patients harboring homozygous CASQ2-G112 + 5X mutation in *CASQ2* gene showed the rapid polymorphic VT under exercise stress test [36]. Adult rat ventricular myocytes were studied to understand the effect of *CASQ2* mutation in ECC, demonstrating that mutated CMs exhibited spontaneous extrasystolic Ca2+ elevations and delayed afterdepolarization (DADs) upon adrenergic stimulation [36]. Later, hiPSC-CM model harboring CASQ2-G112 + 5X mutation emulated these phenotypic features of disease, and AAV9-based gene delivery effectively prevents the development of adrenergic-induced DADs and triggered arrhythmias in CPVT2 hiPSC-CMs [37].