*2.1. Model*

I adopted Alaoui and Penta's EDR model for theoretical predictions [13]. In this model, players follow an endogenous reasoning process that determines the strategic bound in a particular context. With added structure on beliefs, the model is able to predict a player's actual level of play in any game that could use a *k*-level iterative best response reasoning process. The main benefit of using this model is that the structure of the model allowed me to conduct a comparative statics exercise on a player's reasoning process. One of the main goals of this study was to conduct a comparative static exercise on the cost side. Below, I provide more detailed descriptions of some key features of this model. These features are relevant to the experimental design and predictions for this paper.

A player's cognitive bound is a mapping from the incremental cost of reasoning (*c*(*k*)) and the incremental value of reasoning (*v*(*k*)) at each level to the intersection of the two terms.

$$\kappa(\upsilon, \mathfrak{c}) = \min \{ k \in \mathbb{N} \mid \upsilon(k) \ge \mathfrak{c}(k) \text{ and } \upsilon(k+1) < \mathfrak{c}(k+1) \} \tag{1}$$

A player reaches their cognitive bound at the *k*th level by having a value of reasoning for that level exceeds cost of reasoning, but their cost–benefit analysis no longer supports the one-higher level (i.e., *k* + 1) of reasoning. Further denote the cognitive bound of player *i* as ¯ *ki*, where:

$$
\vec{k\_i} = \kappa (v\_{i\prime} c\_i). \tag{2}
$$

According to Alaoui and Penta, the value of reasoning is affected by the payoff of the game [13]. The cost of reasoning is an endogenous characteristic of an individual, which is largely related to their cognitive or reasoning ability. In this paper, I take their assumption on the value of reasoning and continue to assume that the payoff is the only incentive for players to apply logical reasoning in the games. I provide a further discussion on the cost of reasoning. Beyond an individual's endogenous ability, the strategic environment (such as cognitive load) provides many challenges for a person in applying strategic reasoning, which alters the cost of reasoning.

A player's belief is represented as a tuple. Since the game in my design is symmetric in payoffs, a player's belief can be restricted to the beliefs about the cost of reasoning. Therefore, the first element of the tuple, *ci*, represents player *i*'s own cost of reasoning. The second element is player *i*'s beliefs of his opponent's (player *j*) cost of reasoning, denoted as *ci j* . The last element *c ij i* is player *i*'s second-order belief, which is their belief about player *j*'s belief of themselves. Any higher-order beliefs could be nested to the first- and second-order beliefs; therefore, a player's belief is represented as:

$$t\_i = (c\_{i\prime}c\_{j\prime}^i c\_i^{lj}).\tag{3}$$


**8. Conclusions and Future Work**

Two promising fundamental functions have been proposed for working with generalizations of Segerdahl's process: (a) the scale derivative **w** Czarna et al. (2017) and (b) the integrating factor *I* Avram and Usabel (2008), and they are shown to be related via Thm. 1.

Segerdahl's process per se is worthy of further investigation. A priori, many risk problems (with absorbtion/reflection at a barrier *b* or with double reflection, etc.) might be solved by combinations of the hypergeometric functions *U* and *M*.

However, this approach leads to an impasse for more complicated jump structures, which will lead to more complicated hypergeometric functions. In that case, we would prefer answers expressed in terms of the fundamental functions **w** or *I*.

We conclude by mentioning two promising numeric approaches, not discussed here. One due to Jacobsen and Jensen (2007) bypasses the need to deal with high-order hypergeometric solutions by employing complex contour integral representations. The second one uses Laguerre-Erlang expansions—see Abate, Choudhury and Whitt (1996); Avram et al. (2009); Zhang and Cui (2019). Further effort of comparing their results with those of the methods discussed above seems worthwhile.

**Author Contributions:** The authors had equal contributions for conceptualization, methodology, validation, formal analysis, investigation, writing—original draft preparation, review and editing, and project administration.

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

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


*3.2. FTIR Spectroscopy and Solubility Measurements*

Figure 3 shows the FTIR spectra for all six new phases obtained in this investigation. FTIR provides a fingerprint sign of each compound and wealthy information about the noncovalent interactions between acceptor and donor groups. Peak shifts can be found in the bands of the functional groups involved in the hydrogen bonds, namely, the carbonyl –C=O functional group of the amine group of the lidocaine and lidocaine hydrochloride molecule and –OH groups in the polyphenol molecules. The shifts in lidocaine's –C=O

group stretching vibration occurred from 1661 cm−1 to 1623–1620 cm−1 for the new crystalline forms obtained, and from 1680 cm−1 to 1677–1670 cm−1 for lidocaine hydrochloride. These shifts demonstrate the hydrogen bonding between the –C=O group of lidocaine molecules and the –OH groups of the polyphenols [7], which as further confirmed by the structure solution.

**Figure 2.** Stability in ageing conditions (40 ◦C and 75% relative humidity) for the three **(lidhcl)** new forms.

**Figure 3.** FTIR spectra of (**a**) (**lid**) and (**b**) (**lidhcl**) multicomponent forms.

In order to measure the solubility of the new (lidhcl) forms, FTIR measurements were performed to avoid the overlapping observed in the UV spectra, due to the ability of FTIR to show us isolated peaks from each component, allowing us to construct a calibration curve with the intensity of an unambiguous peak of (lidhcl).

A calibration curve was built with different (lidhcl) concentrations [18,19] obtaining linear models with *R*2 values greater than 0.99 (Figure 4), which were used to calculate the apparent solubility. The peak used for the calibration curves was the area between 1712 cm−1 and 1612 cm−1 corresponding to (lidhcl), where there was no interference of any of the used coformers.

**Figure 4.** Calibration curve of (**lidhcl**) concentrations ranging from 20 to 400 mg/mL.

The constructed calibration curve is shown in Figure 4, where the model's excellent agreement with the experimental concentrations can be appreciated. Each concentration was prepared and measured in duplicate to assure reproducibility, and the mean value is represented.

Table 2 presents the apparent solubilities calculated from FTIR data for each new (lidhcl) phase.

**Table 2.** . Solubilities in (mg/mL) of **(lidhcl)** multicomponent forms obtained from FTIR data.

The solubility decreased in the new phases with respect to reported values for **(lidhcl)** [9], generating **(lidhcl)** forms with an interesting path to modulate solubility.


**6. Tips of direct surgery for recurrent aneurysms after endovascular coil embolization**

When choosing open clipping of a recurrent cerebral aneurysm after initial coil embolization, it is critical to confirm by preoperative cerebral angiography that there is sufficient space between the neck and the coil mass to allow for clip application. (**Figure 3a** and **b**) Toyota et al. reported that a remnant neck height of the aneurysm more significant than 2 mm is a prerequisite for surgery if the coil mass within the aneurysm is not removed [37]. Also, Waldron et al. reported that open cerebral aneurysm clipping without removing the coil mass in the aneurysm is unsuitable when the coil width and compaction height ratio exceeds 2.5 and a wedge angle greater than 90 degrees [38]. Confirming these findings by preoperative cerebral angiography is essential.

Perioperative stroke complications have been reported to be higher in patients undergoing intraoperative coil mass extraction [23]. Coil extraction was performed in 3/111 patients (2.7%), all of whom suffered a postoperative stroke [39]. The effects of coil embolization of cerebral aneurysms on the vital structure of the aneurysm, mother vessel, branches, and cranial nerves have been attributed to inflammatory and degenerative change mechanisms. A coil mass within the aneurysm causes inflammation and degeneration, resulting in extrusion of the coil mass outside the aneurysm, invasion of the surrounding vital structure, inflammatory adhesions, protrusion of the coil loop into the mother vessel, and scarring of the vessel wall. Surgical coil mass extraction in this condition is likely to cause steno-occlusion of the parent artery and damage to the vital structure. These can lead to postoperative stroke and neurological

#### **Figure 3.**

*(a) and (b): Schema of the morphological parameters of recurrent aneurysms after endovascular coil embolization. (a): The gray two-direction arrow indicates a coil width (C), and the black two-direction arrow indicates either a compaction height (H) or a remnant neck width (RNH). (b): The angle shown in the figure indicates a wedge angle. Aneurysms with RNH greater than 2 mm or a C/H ratio greater than 2.5 with a wedge angle greater than 90 degrees are unsuitable for direct clipping surgery without coil removal.*

*Perspective Chapter: Role of Direct Surgery for Recurrent Aneurysms after Endovascular Treatment DOI: http://dx.doi.org/10.5772/intechopen.112076*

#### **Figure 4.**

*a (upper) and b (lower): Schema of the neck clipping of the recurrence. (a): When a single aneurysm clip is applied to the aneurysm neck, atherosclerotic or inflammatory wall thickening (small black arrow) and coil loop inside (small white arrows) often prevent complete closure of the neck (thick white arrow). (b): The tandem clipping method, in which a fenestrated aneurysm clip is applied to the far side aneurysm neck, skipping the near side aneurysm neck, and an additional clip is used to close the near side aneurysm neck, is extremely useful in the above situation, as it provides complete closure of the neck.*

sequelae. Therefore, in the case of open clipping of recurrent cerebral aneurysms after initial coil embolization, coil mass extraction should be avoided as much as possible to ensure a safe procedure with a low rate of surgical complications.

Even in cases where preoperative imaging has shown sufficient space for clip application in the recurrent cerebral aneurysm, intraoperative findings may indicate that the room is smaller than expected during surgery. This phenomenon may be due to thrombus formation caused by coil mass in the aneurysm or thickening of the aneurysm wall due to inflammatory changes. In addition, a coil loop in the neck of the aneurysm may prevent complete occlusion of the neck when a single clip is applied (**Figure 4a**). The tandem clipping method has been reported to help deal with incomplete occlusion of cerebral clips due to stiff neck aneurysm walls and coil loops (**Figure 4b**) [40]. In this technique, a fenestrated aneurysm clip is applied to the far side aneurysm neck, skipping the near side aneurysm neck, and an additional clip is used to close the near side aneurysm neck. This technique is beneficial in narrow aneurysm neck spaces, stiff aneurysm neck walls, and the presence of coil loops in the neck, as complete occlusion of the aneurysm neck can be achieved without coil mass extraction. On the other hand, for large or giant cerebral aneurysms that recur after initial coil embolization and are at high risk for open aneurysm clipping, mother vessel occlusion with intracranial or extracranial bypass may be helpful [38, 40]. By this surgical strategy, direct manipulation of the aneurysm is no longer necessary, and it can be a safe treatment for recurrent cerebral aneurysms after coil embolization of deep locations like the upper basilar artery system or when there is already a high mass effect on the vital structures.

### **7. Illustrative case presentations**

**Case 1:** A 65-year-old woman presented with Hunt and Kosnik grade 1, Fisher group 3 subarachnoid hemorrhage. Her digital subtraction angiography (DSA) revealed a right internal carotid artery-posterior communicating (IC-Pcom) artery aneurysm. She underwent coil embolization. The aneurysm was completely obliterated. Her postoperative course was uneventful, and she was discharged home without a neurological deficit. Six months after initial treatment, follow-up DSA showed the remarkable recurrence of the aneurysm with coil compaction (**Figure 5a**). Thus, she underwent additional clipping surgery for the recurrent IC-Pcom aneurysm.

**Intraoperative findings** (Video 1 https://1drv.ms/f/s!AnNEb0cDdygmSdOw3kIA3vy\_9kf?e=pgxew1): Coil mass inside the aneurysm dome were seen through not only the wall of the dome but also through the wall of the neck. First, we applied a straight-type titanium clip, but the tip slipped toward the Pcom, so we removed this clip. Then, we applied a fenestrated Elgiloy clip in partial occlusion of the far side aneurysm neck. Indocyanine-green video angiography (ICG-VA) showed patency of ICA, Pcom, and anterior choroidal artery and nonfilling of the aneurysm. We added a straight Elgiloy clip in parallel fashion to secure the complete obliteration of the aneurysm.

Postoperative CT showed no complication and complete obliteration of the aneurysm (**Figure 5b** and **c**). Her postoperative course was uneventful, and she was discharged home without a neurological deficit.

**Case 2:** A 63-year-old woman presented with Hunt and Kosnik grade 2, Fisher group 3 subarachnoid hemorrhage. Her DSA revealed a right IC-Pcom artery aneurysm. She underwent coil embolization. The aneurysm was completely obliterated. Her postoperative course was uneventful, and she was discharged home without a neurological deficit. Forty-one months after initial treatment, follow-up DSA showed the remarkable recurrence of the aneurysm with fundal migration (**Figure 6a**). Thus, she underwent additional clipping surgery for the recurrent IC-Pcom aneurysm.

Intraoperative findings (**Video 2** https://1drv.ms/f/s!AnNEb0cDdygmSdOw3kIA3vy\_9kf?e=FQyE2p): Coil mass inside the aneurysm dome were seen through the dome's wall. And fundal migrated coil loops compressed the right oculomotor nerve. We applied a straight-type titanium clip to the neck of the aneurysm and supposed this to be complete obliteration. We incised the dome with microscissors to release the oculomotor nerve from compression by the coil loops. Major bleeding was seen from the incised aneurysm dome. To stop bleeding, we applied a temporary clip to the proximal ICA. The bleeding was controlled completely. Then we applied an additional fenestrated clip and removed the temporary clip.

Postoperative CT showed a thin epidural hematoma and complete aneurysm obliteration (**Figure 6b** and **c**). Her postoperative course was uneventful, and she was discharged home without a neurological deficit.

#### **Figure 5.**

*a-c: The imaging studies of illustrative case 1. (a): Follow-up DSA (lateral view of right ICGA) 6 months after initial coil embolization showed remarkable recurrence of the treated IC-Pcom aneurysm (white arrow). (b) and (c): Postoperative plain CT and CT angiography showed no complication and complete obliteration of the aneurysm.*

*Perspective Chapter: Role of Direct Surgery for Recurrent Aneurysms after Endovascular Treatment DOI: http://dx.doi.org/10.5772/intechopen.112076*

**Figure 6.**

*a-c the imaging studies of illustrative case 2. (a) Follow-up DSA (lateral view of right ICGA) 41 months after initial coil embolization showed remarkable recurrence of the treated IC-Pcom aneurysm (white arrow). (b) and (c) postoperative plain CT and CT angiography showed thin epidural hematoma and complete aneurysm obliteration.*

**Case 3:** A 43-year-old man presented with Hunt and Kosnik grade 3, Fisher group 3 subarachnoid hemorrhage. His DSA revealed a left IC-Pcom artery aneurysm. He underwent coil embolization. The aneurysm was completely obliterated. His postoperative course was uneventful, and he was discharged home without a neurological deficit. Eighteen months after initial treatment, follow-up DSA showed the remarkable recurrence of the aneurysm with regrowth, and he underwent additional stentassisted coil embolization for the aneurysm. However, 30 months after the second treatment, follow-up MRI and DSA revealed the recurrence as a partially thrombosed

#### **Figure 7.**

*a-f: The preoperative (a-c) and postoperative (d-f) imaging studies of illustrative case 3. (a) Silent MRA 30 months after the second treatment. (b) T1-weighted MRI. (c) 3D rotational angiography. The imaging studies showed recurrent partially thrombosed giant left IC-Pcom aneurysm (white arrow). (d) Postoperative diffusionweighted MRI. (e) and (f): Postoperative DSA anterior-posterior and lateral view. The imaging studies showed no ischemic complication and complete aneurysm obliteration with preservation of normal arteries.*

giant aneurysm (**Figure 7a**–**c**). Thus, he underwent additional clipping surgery for the recurrent IC-Pcom aneurysm.

Intraoperative findings (Video 3 https://1drv.ms/v/s!AnNEb0cDdygmSsspZ6yOvo4Mle1?e=hlee0e): The stent was inserted in the left proximal intracranial ICA, but proximal control at this site was confirmed feasible. Left Pcom origin was observed at the proximal neck of the aneurysm. Coil mass inside the aneurysm dome and stent strut were seen through the wall of the dome and ICA. The left anterior choroidal artery and perforators from Pcom firmly adhered to the dome of the aneurysm. The adhesion was detached with sharp dissection. While performing temporary occlusion in the proximal intracranial ICA, first, we applied a fenestrated titanium clip in partial occlusion of the far side aneurysm neck. ICG-VA revealed the residual flow into the aneurysm through the near side of the neck of the aneurysm. We added a straight titanium clip in a parallel fashion to secure the complete obliteration of the aneurysm. ICG-VA showed complete obliteration of the aneurysm and preservation of the parent artery and branches.

Postoperative imaging studies showed complete aneurysm obliteration without complication (**Figure 7d**–**f**). His postoperative course was uneventful, and he was discharged home without a neurological deficit.


**A. Appendix**

*The Influences of Hearing and Vision on Egocentric Distance and Room Size Perception… DOI: http://dx.doi.org/10.5772/intechopen.102810*

#### **Table 11.**

**Ethics statement**

**Acknowledgements**

improvements.

**A. Appendix**

**120**

**Conflict of interest**

According to the funding institution (Deutsche Forschungsgemeinschaft) an ethical approval is not required, since the respective indications do not apply [125]. The study was conducted under the ethical principles of the appropriate national

This work was carried out as a part of the project "Audio-visual perception of acoustical environments", funded by the Deutsche Forschungsgemeinschaft (DFG MA 4343/1-1) within the framework of the research unit SEACEN, coordinated by Technische Universität Berlin and Rheinisch-Westfälische Technische Hochschule Aachen, Germany. We thank the staff of the performance rooms for their friendly cooperation, the Berlin Budapest Quartet (Dea Szücs, Éva Csermák, Itamar Ringel, Ditta Rohmann) and actress Ilka Teichmüller for their performances, Alexander Lindau, Fabian Brinkmann, and Vera Erbes for the in-situ acquisition of the rooms' acoustic and visual properties, Mina Fallahi for the geometric picture editing, Annika Natus, Alexander Haßkerl, and Shamir Ali-Khan for the 3D video shooting and post-production, and all test participants. Finally, we thank the two anonymous

reviewers for critically reading the manuscript and suggesting substantial

**Measure** *Content A room* **off GH JC KH KO RT KE** Mean Music (n = 50) off — 10.87 6.85 10.25 10.52 11.80 15.47

Speech (n = 38) off — 9.96 5.91 9.56 10.33 10.19 12.45

*V room*

GH 7.46 10.79 7.19 10.23 10.13 10.41 14.41 JC 7.18 10.51 7.32 10.28 11.07 10.60 13.85 KH 8.07 9.92 7.25 9.89 10.72 10.63 13.76 KO 6.74 10.63 7.44 9.84 10.17 10.01 13.12 RT 5.71 9.19 7.26 9.60 9.92 9.77 13.09 KE 12.91 11.16 8.43 11.24 11.22 11.61 15.23

GH 7.10 8.88 6.11 8.82 8.87 9.37 11.52 JC 8.07 8.53 6.46 8.98 9.04 9.09 11.39 KH 6.67 8.62 6.00 8.69 8.72 8.67 11.26 KO 5.90 8.28 5.67 8.31 8.38 8.70 10.68 RT 5.30 7.67 6.03 8.12 8.30 8.03 10.62 KE 11.07 9.72 7.07 9.72 9.92 9.94 12.23

The authors have no conflict of interest to declare.

professional society (Deutsche Gesellschaft für Psychologie) [126].

*Advances in Fundamental and Applied Research on Spatial Audio*

*Descriptive statistics of* perceived source distance *(D).* ^

**References**

[1] Cabrera D, Nguyen A, Choi YJ. Auditory versus visual spatial

[2] Kuusinen A, Lokki T. Auditory distance perception in concert halls and

the origins of acoustic intimacy. Proceedings of the Institute of Acoustics. 2015;**37**(3):151-158

final thoughts on the subject, documented. Acoustics. 2019;**1**(3): 561-569. DOI: 10.3390/acoustics1030032

[4] Stevens JC, Marks LE. Crossmodality matching of brightness and loudness. Proceedings of the National Academy of Sciences of the USA. 1965;**2**:

[5] Thomas GJ. Experimental study of the influence of vision on sound localization. Journal of Experimental

[6] Howard IP, Templeton WB. Human Spatial Orientation. London: Wiley;

[7] Gardner MB. Proximity image effect in sound localization. The Journal of the Acoustical Society of America. 1968; **43**(1):163. DOI: 10.1121/1.1910747

[8] Mateeff S, Hohnsbein J, Noack T. Dynamic visual capture: Apparent auditory motion induced by a moving visual target. Perception. 1985;**14**: 721-727. DOI: 10.1068/p140721

[9] Kitajima N, Yamashita Y. Dynamic

capture of sound motion in

Psychology. 1941;**28**:163-177. DOI: 10.1037/h0055183

407-411

1966

**123**

[3] Hyde JA. Discussion of the relation between initial time delay gap (ITDG) and acoustical intimacy: Leo Beranek's

impression: A study of two auditoria. In: Barrass S, Vickers P, editors. Proc. of ICAD 04-Tenth Meeting of the Int. Conf. on Auditory Display. Sydney, Australia: International Community for Auditory Display (ICAD); 2004

*DOI: http://dx.doi.org/10.5772/intechopen.102810*

three-dimensional space. Perceptual and Motor Skills. 1999;**89**(3):1139-1158. DOI: 10.2466/pms.1999.89.3f.1139

[10] Kohlrausch A, van de Par S. Audiovisual interaction in the context of multimedia applications. In: Blauert J, editor. Communication Acoustics (Chapter 5). Berlin: Springer; 2005. pp. 109-138. DOI: 10.1007/3-540-27437-5\_5

[11] Shams L, Kamitani Y, Shimojo S. Visual illusion induced by sound. Cognitive Brain Research. 2002;**14**: 147-152. DOI: 10.1016/S0926-6410(02)

[12] Andersen TS, Tippana K, Sams M. Factors influencing audiovisual fission and fusion illusions. Cognitive Brain Research. 2004;**21**:301-308. DOI: 10.1016/j.cogbrainres.2004.

[13] Vatakis A, Spence C. Audiovisual synchrony perception for music, speech, and object actions. Brain Research. 2006;**1111**(1):134-142. DOI: 10.1016/j.

[14] MacDonald J, McGurk H. Visual influences on speech perception process. Perception & Psychophysics. 1978;**24**:253-257. DOI: 10.3758/

[15] Beerends JG, de Caluwe FE. The influence of video quality on perceived audio quality and vice versa. Journal of the Audio Engineering Society. 1999;**47**:

[16] Larsson P, Västfjäll D, Kleiner M. Auditory-visual interaction in real and virtual rooms. In: 3rd Convention of the

[17] Larsson P, Väljamäe A. Auditoryvisual perception of room size in virtual environments. In: Proc. of the 19th Int.

EAA. Spain: Sevilla; 2002

00069-1

*The Influences of Hearing and Vision on Egocentric Distance and Room Size Perception…*

06.004

brainres.2006.05.078

BF03206096

355-362

**Table 12.**

*Descriptive statistics of* perceived room size *(*^ *S).*


**2. Post-COVID-19 conditions**

Although respiratory symptoms make up the majority of COVID-19's clinical presentation due to the SARS-CoV-2 infection, other conditions like pulmonary (such as pneumonia or dyspnea with exertion), gastrointestinal (such as anorexia or diarrhea), neurological (such as headache, anosmia, or dizziness), or even cardiac ones (such as ischemia, arrhythmias, or myocarditis) may also manifest. It is now well established that nearly a third of patients worldwide will have persistent symptoms for weeks and months, sometimes up to a year after being diagnosed with COVID-19 [7]. These results, particularly for patients who had severe symptoms and spent many weeks in intensive care, are comparable to the recovery from SARS in 2003. But COVID-19 is different because a large portion of those who do not need to be hospitalized seem to relapse, persist, or manifest new symptoms months after the initial infection [8].

When SARS-CoV and MERS-CoV outbreaks occurred in the past, people who recovered from these viral illnesses were left with lingering symptoms like extreme fatigue, a lower quality of life, protracted shortness of breath, and behavioral health issues. These symptoms have put a significant burden on the local health-care systems where the outbreak occurred. Similarly, despite scientific evidence that SARS-CoV-2 replication stops four weeks after the commencement of infection (on the basis of sampling viral isolates from the respiratory tract, not the nasopharyngeal/oropharyngeal sample), a collection of distinct clinical symptoms known as post-COVID-19 syndrome has been reported in a minority of patients who have recovered from COVID-19 caused by SARS-CoV-2 [9]. A meta-analysis including 4828 post-COVID-19 patients showed that symptoms and post-acute sequelae of SARS-CoV-2 can persist for weeks to months after the infection [10].

Persistent symptoms after the COVID-19 illness are called long-term COVID or post-acute sequelae of COVID-19. Some individuals who have contracted the virus that causes COVID-19 may go on to develop post-COVID conditions (PCC) or long COVID as a result of their infection. Long-haul COVID, post-acute COVID-19,

post-acute sequelae of SARS CoV-2 infection (PASC), long-term effects of COVID, and chronic COVID are only a few of the terms used to describe post-COVID problems [11].

The hallmark of post-acute COVID-19 syndrome is the continuation of clinical symptoms for longer than four weeks following the beginning of acute symptoms [9]. The term "post-COVID conditions" was created by the Center for Disease Control (CDC) to characterize health issues that last longer than four weeks after getting COVID-19. The following conditions fall under this category: multi-organ COVID-19 effects, impacts of COVID-19 treatment/hospitalization, and extended COVID or persistent post-COVID syndrome (PPCS) [9].

The multi-organ effects of COVID-19 include clinical manifestations involving the cardiovascular, pulmonary, renal, and neuropsychiatric organ systems, although the duration of these multi-organ effects is unclear [9, 13].

The long-term "effects of treatment or hospitalization for COVID-19" are similar to other severe infections. These include post-intensive care syndrome (PICS), which consists of impairment of cognition, psychological health, and physical function in an intensive care unit survivor. It can also include muscle weakness, problems with thinking and judgment, and symptoms of PTSD. Many patients with these complications caused by COVID-19 recover over time [9, 14].

In addition to morbidity and mortality in the acute phase, post-acute health problems and consequences have also been recorded in persons who have survived COVID-19. According to the review, up to 80% of patients with COVID-19 continue to complain of health problems after the acute infection and more than 50 side effects have been reported [15]. Post-acute COVID-19 was categorized as follows by Nalbandian et al. based on the severity of symptoms following COVID-19 infection: After the initial acute episode, subacute or persistent COVID-19 symptoms can last up to 12 weeks, and chronic or post-COVID syndrome symptoms start to show up after that. This should not, however, be attributed to a different diagnosis [16].

A large observational cohort study from 38 US hospitals assessed the outcomes of 1250 COVID-19 survivors 60 days after hospital discharge using medical record abstraction and telephone surveys. During the study period, 6.7% of patients died, while 15.1% of patients required readmission. Out of a total of 488 patients who completed the telephone survey, 32.6% of patients reported persistent symptoms including 18.9% with new or worsening symptoms. In this study, 159 individuals reported experiencing cardiopulmonary symptoms (e.g. cough or dyspnea), including 92 who experienced new or worsening symptoms and 65 who experienced irreversible loss of taste or smell. Fifty-eight patients reported new or deteriorating difficulties carrying out their everyday tasks. Among the 195 patients who had jobs prior to being admitted to the hospital, 117 were able to go back to work, while 78 were unable to do so due to continued health issues or job loss. Almost half of all patients (238 of 488) reported being emotionally affected by their health, and 28 sought mental health care after discharge [17].

Similar findings were obtained in studies conducted in Europe. In a populationbased prospective cohort study conducted in Spain to determine post-COVID-19 complications and risk factors among patients six months after SARS-CoV-2 infection, in a sample of 484 patients, it was shown that 160 patients (33.1%) experienced at least one post-COVID-19 problem after six months, and 47 of them (29.5%) sought medical help. Hair loss, exhaustion, loss of taste or smell, and headache were the most prevalent long-lasting effects. The complication's risk factors included female gender, age over 35 years, current smoker, and exposure to COVID-19. A third of patients had persistent symptoms compatible with long-term COVID-19 syndrome [18]. In 143 patients who were discharged from the hospital after recovering from acute COVID-19 at a median time of up to 60 days following the onset of symptoms, a post-acute outpatient clinic established in Italy revealed that 87.4% of patients had persistent symptoms. The most frequently reported symptoms were fatigue (53.1%), dyspnea (43.4%), joint pain (27.3%), and chest pain (21.7%), and 55% of patients continued to experience three or more symptoms. In this study, a decline in quality of life was noted in 44.1% of patients [19]. Also, a study conducted in Brazil confirmed the impact of COVID-19 on worsening health-related quality of life and mental health in COVID-19 patients three months after hospital discharge, compared to that before the onset of COVID-19 symptoms [20].

Other studies have reported similar findings. Results of a prospective follow-up study of 110 survivors in the United Kingdom, 8–12 weeks after hospital admission [21], and 277 survivors in Spain, 10–14 weeks after the disease onset [22], as well as survey studies of 100 UK COVID-19 survivors, 4–8 weeks after discharge [23], and 120 patients discharged from a hospital in France, 100 days after admission [24], showed that fatigue, dyspnea, and psychological distress, such as PTSD, anxiety, depression, and loss of concentration and sleep disorders, observed in approximately 30% or more of study participants at follow-up.


**2. γ-Secretase controls Notch signaling**

Notch is a family of evolutionarily conserved type 1 membrane proteins with a mass of about 300 kDa [31] that mediates fates of numerous cells in both invertebrates and verte‐ brates [16, 17]. For example, cells expressing the major ligand Delta inhibit the neural differ‐ entiation of neighboring Notch-expressing cells during neurogenesis. Disruption of or disorder in Notch signaling leads to developmental defects or cancer in mammals [18].

While *Drosophila* has only one Notch gene, four Notch isoforms (Notch1 to 4) have been identified in mammals. The typical Notch protein contains 36 tandem epidermal growth fac‐ tor (EGF)-like repeats in its extracellular domain, and six tandem ankyrin-like (CDC10) re‐ peats, a nuclear localization signal, and a PEST sequence in its intracellular domain [31]. The 11th and 12th EGF-like repeats are essential for binding to its ligands [32]. Notch is cleaved in the trans-Golgi network, apparently by furin-like covertase, and is expressed on the cell surface as a heterodimer [33].

**Figure 1.** Notch signaling pathway. Notch proteins are expressed on the cell surface as heterodimers after cleavage at the S1 site by furin. The binding of Notch to the ligand triggers sequential proteolytic cleavage of the regulated intra‐ membrane proteolysis (RIP). When Notch binds to the ligand, Notch is cleaved at the S2 site in the juxtamembrane region by TACE or ADAM protease. Next, the remaining protein stub is further cleaved by γ-secretase at the S3 and S4 sites within the transmembrane domain and NICD is released from the membrane. Then, NICD translocates into the nucleus and binds to the CSL together with MAML. The resultant CSL–NICD–MAML complex removes co-repressors (Co-R) from CSL transcription factor and recruits a co-activator (Co-A), resulting in conversion from repressor to activa‐

γ-Secretase–Regulated Signaling and Alzheimer's Disease

http://dx.doi.org/10.5772/54230

63

tor. Finally, the complexes of CSL-NICD-MAML-Co-activators promote transcription of the target genes.

on neighboring cells and trigger sequential proteolytic cleavage. Finally, the intracellular do‐ main (ICD) of Notch (NICD) is released from the cell membrane by γ-secretase; NICD then translocates into the nucleus where it modulates gene expression through binding to transcrip‐

Recently, more than five dozen type 1 transmembrane proteins, including Notch and APP, have been reported as substrates for γ-secretase [21]. The ICDs of these proteins are also re‐ leased from the cell membrane [13-15, 22]. Furthermore, it has been shown that some of these ICDs exist in the nucleus. These processes are very similar to those involved in Notch signaling. Thus, the common enzyme γ-secretase modulates the proteolysis and turnover of putative signaling molecules; this suggests that mechanisms similar to the Notch signaling pathway may widely contribute to γ-secretase–regulated signaling [13-15, 23]. Indeed, it has been shown that the ICD of APP (AICD), which is released from the cell membrane by γsecretase, also translocates to the nucleus [24-26] and may function as a transcriptional regu‐

To test the hypothesis that APP has a signaling mechanism similar to that of Notch, we es‐ tablished embryonic carcinoma P19 cell lines that overexpressed AICD [29], which may mimic signaling mechanisms. Although neurons differentiated from these cell lines, AICD expression induced dynamic changes in gene expression profile and neuron-specific apopto‐ sis [30]. These results suggest that APP also has a signaling mechanism, which may be close‐

In this chapter, we first summarize current research progress regarding Notch, APP, and γsecretase. We also focus on the signaling hypothesis; γ-secretase–regulated mechanisms similar to Notch signaling may widely play roles in signaling events involving type 1 trans‐ membrane proteins, including APP. Next, we review recent evidence supporting the exis‐ tence of APP signaling. Furthermore, we discuss the possibility that APP signaling is

Notch is a family of evolutionarily conserved type 1 membrane proteins with a mass of about 300 kDa [31] that mediates fates of numerous cells in both invertebrates and verte‐ brates [16, 17]. For example, cells expressing the major ligand Delta inhibit the neural differ‐ entiation of neighboring Notch-expressing cells during neurogenesis. Disruption of or disorder in Notch signaling leads to developmental defects or cancer in mammals [18].

While *Drosophila* has only one Notch gene, four Notch isoforms (Notch1 to 4) have been identified in mammals. The typical Notch protein contains 36 tandem epidermal growth fac‐ tor (EGF)-like repeats in its extracellular domain, and six tandem ankyrin-like (CDC10) re‐ peats, a nuclear localization signal, and a PEST sequence in its intracellular domain [31]. The 11th and 12th EGF-like repeats are essential for binding to its ligands [32]. Notch is cleaved in the trans-Golgi network, apparently by furin-like covertase, and is expressed on the cell

tion factors. Therefore, γ-secretase plays a central regulatory role in Notch signaling.

lator [27, 28]. These observations suggest the existence of APP signaling.

ly related to AD.

62 Understanding Alzheimer's Disease

involved in the onset and progression of AD.

surface as a heterodimer [33].

**2. γ-Secretase controls Notch signaling**

**Figure 1.** Notch signaling pathway. Notch proteins are expressed on the cell surface as heterodimers after cleavage at the S1 site by furin. The binding of Notch to the ligand triggers sequential proteolytic cleavage of the regulated intra‐ membrane proteolysis (RIP). When Notch binds to the ligand, Notch is cleaved at the S2 site in the juxtamembrane region by TACE or ADAM protease. Next, the remaining protein stub is further cleaved by γ-secretase at the S3 and S4 sites within the transmembrane domain and NICD is released from the membrane. Then, NICD translocates into the nucleus and binds to the CSL together with MAML. The resultant CSL–NICD–MAML complex removes co-repressors (Co-R) from CSL transcription factor and recruits a co-activator (Co-A), resulting in conversion from repressor to activa‐ tor. Finally, the complexes of CSL-NICD-MAML-Co-activators promote transcription of the target genes.

*Drosophila* has two different Notch ligands, Delta [34] and Serrate [35]. In mammals, two families of Notch ligands, Delta-like protein family (Dll1, 3, and 4) [36-38] and Jagged family (Jagged 1 and 2) [39, 40], have been identified. The extracellular domains of all these ligands also contain variable number of EGF-like repeats, e.g., *Drosophila* Delta has nine, while most vertebrate Deltas have eight, and *Caenorhabditis elegans* Lag-2 has two repeats. All these li‐ gands share a single copy of a 2nd cysteine-rich conservative motif called the DSL (**D**elta: **S**errate: **L**ag-2) domain [41], which is essential for binding to Notch [42].

expressed in neurons) or presence (APP-751 and APP-770) of the Kunitz protease inhibitor

γ-Secretase–Regulated Signaling and Alzheimer's Disease

http://dx.doi.org/10.5772/54230

65

As described below, APP undergoes sequential proteolytic cleavage reactions to generate the extracellular fragment, intracellular fragment (AICD), and Aβ fragment that is located in the membrane-spanning region. Note that both the extracellular fragment and AICD are generated at the same time as Aβ. Extensive post-translational modifications of APP, such as

Mammals have two other members of APP family called APP-like protein 1 (APLP1) and 2 (APLP2) [56]. APLP1 expression is restricted to neurons. On the other hand, expression of APLP2 is detected in many tissues, although it is highly enriched in the brain. These APP family proteins share conserved domains, such as the E1 and E2, in the extracellular region. The E1 domain contains several subdomains, such as a growth factor-like domain and a metal-binding motif [57]. The E2 domain has a coiled coil dimerization motif and may bind

Interestingly, the amino acid sequence of the Aβ fragment is not highly conserved and is unique to APP; on the other hand, the highest degree of sequence conservation is found in the ICD not only within the APP homologues [29] but also within the APP family [9]. This strong sequence conservation most likely reflects functional importance of the ICDs in the

**Figure 2.** Schematic domain structure of APP. APP protein family shares the conserved E1 and E2 domains in their extracellular region. The E1 domain contains N-terminal growth factor-like domain (GFLD) and copper-binding do‐ main (CuBD). The E1 domain is linked via acidic domain to the carbohydrate domain including E2 domain, which con‐ sists of RERMS sequence and central APP domain (CAPPD). E2 domain is followed by the Aβ region, and the intracellular domain (AICD) which is the most conserved region. Although the Kunitz protease inhibitor (KPI) domain

is present at the indicated site in APP-751 and APP-770, APP-695 lacks this domain.

glycosylation, phosphorylation, and tyrosine sulfation, have been observed.

(KPI) domain toward the N-terminus of the protein [55].

proteoglycans in the extracellular matrix [58].

APP family proteins.

As shown in Fig.1, in the canonical Notch signaling pathway, ligands bind to the extracellu‐ lar domain of Notch proteins on neighboring cells and trigger sequential proteolytic cleav‐ age reactions; this is called the regulated intramembrane proteolysis (RIP) mechanism [43]. Precise steps of Notch processing are mentioned in section 4.2 of this chapter. In brief, first, Notch is cleaved within the juxtamembrane (JM) domain by metalloproteases to remove most of the extracellular region [44, 45]. Next, the remaining protein stub is further cleaved by γ-secretase within the transmembrane (TM) domain and NICD is released from the membrane [46-48]. The released NICD translocates to the nucleus and controls the expres‐ sion of certain genes through binding to transcription factors. Thus, γ-secretase plays a cen‐ tral regulatory role in Notch signaling.

Members of the CSL transcription factor family (CBF1/RBP-jκ in mammals, Su(H) in *Droso‐ phila*, and Lag-1 in *C. elegans*) are major downstream targets of Notch signaling [19]. NICD binds to CSL transcription factors, and six tandem ankyrin-like repeats in NICD are essen‐ tial for binding to CSL transcription factors [49]. NICD also binds to Mastermind-like pro‐ teins (MAML family in mammals) [50], thus forming the CSL-NICD-MAML complex. The formation of these complexes results in removal of co-repressors from CSL and recruitment of co-activators, such as P/CAF and P300 [50, 51]. Through this process, the CSL complex is converted from a transcriptional repressor to an activator. Finally, these complexes bind to the *cis*-acting DNA sequences of target genes, such as Hes (Hairy/Enhancer of split in *Droso‐ phila*), which encode the basic helix-loop-helix (bHLH) transcription factors, and promote their transcription [52].


**3.2 General important points should always be noticed**

**133**

**Figure 5.**

*Airway Management in Head and Neck Pathology DOI: http://dx.doi.org/10.5772/intechopen.94498*

with some special FiO2 settings).

ETT intubation in those cases.

B. **Jet Venturi ventilation:**

1 cm down to the level of the stenosis.

laryngoscope or attached to laryngoscope.

laryngoscope) or infra glottic jet ventilation.

*Tritube: (Tritube, Ventinova medical, Eindhoven, the Netherlands).*

3.**In case of using surgical LASER:** a LASER-protected tube must be used. In case we use jet ventilation or apneic technique (both are safe for the LASER

4.**In case of the patient has a tracheostomy tube:** 5.5–6.0 mm Endo Tracheal Tube (ETT) inserted at the tracheal stoma into the trachea, **LASER** protected ETT should be used in case we use LASER. Using (apneic technique) if airway surgery is performed distal to the tracheal stoma site, applying frequent (reinsertion technique) of stomal ETT to give Oxygen in between the treatment.

5.Mask induction with anesthetic agents is the suitable method of airway management for endoscopic treatment of **subglottic/tracheal stenosis**, or using total intravenous anesthesia (TIVA) then using jet ventilation. Avoid using

6.In case of scar formation because of **tracheostomy** was done in a patient with subglottic/tracheal stenosis, then the airway interning should be done at least

In general, lesions present at the anterior 2/3 (membranous vocal folds) of the larynx can be easily exposed and treated with size 5.5 mm or smaller micro laryngeal ETT. Lesions at the posterior 1/3 (vocal processes and posterior commissure/ arytenoids region) of the larynx we can use one of those techniques (**Figure 5**):

A. **Oral intubation** using a small long ETT size: 5.0 mm or 5.5 mm microlaryngoscopy tube (MLT) with a length (30 cm length) with adult tube's balloon that makes good airway sealing. Or using Tritube (**Figure 3**), an ultrathin endotracheal tube (outer diameter 4.4 mm/inner diameter 2.4 mm).

1.Supra-glottic jet Venturi needle (**Figure 6**) Applied through port within

2.Tracheal Jet ventilation (where the tip of the jet is near the tip of the

**3.3 Anesthesia technique used in case of micro-laryngoscopy (MLS)**

*Special Considerations in Human Airway Management*

**2.5 Airway endoscopy and trans-nasal tracheoscopy (TNT)**

**3. Airway management for benign laryngeal surgery**

**3.2 General important points should always be noticed**

another plans become necessary.

It is a form of indirectly visualizing the airway and larynx in which the clinician does not directly view the lower part of the airway or larynx [4]. Instead, it is

*Example of fiber optic tracheoscopy on a tracheal stenosis patient under local anesthesia showing the carina*

Working with our anesthesia colleagues at one place which is the upper airway passages is one of the most important (and often neglected) aspects of successful laryngeal operation. Lack of cooperation and pre-surgical planning with the anesthesiology team can make simple micro-laryngoscopy case into an airway crisis [1]. Benign laryngeal tumors are more common than the malignant one. Phono-surgery is used to excise most of those tumors whether is a vocal cord cyst, polyp, nodule, etc.

1.**Different plans + proper equipment's**: A good management plan for taking care of the patient's airway should be negotiated with the anesthetist before going to the surgery. An excellent plan (plan A), also another strategy (plans B and C) must be ready so that the airway management is algorithmic and automatic, as opposite to unplanned reaction. Before taking the patient into the operating theater, both the operating surgeon and the anesthesia team must have the right equipment in the room, opened, and "ready to use" if

2.**Positioning**: for optimal laryngoscopy exposure the patient put in the "sniffing positioning," head extended on the neck, and the neck flexed on the chest [6].

visualized with fiber optic or digital laryngoscopes inserted trans-nasally. The images from the scope can be displayed on a monitor for the clinician, patient and others to view at the time of the procedure; it can also be recorded. Experienced clinicians can also perform it on a wake tracheoscopy using local anesthesia on cooperative patients. They are an essential tool for identifying and planning strategies for management of difficult airway cases [5] (**Figure 4**).

**132**

**3.1 Introduction**

**Figure 4.**

*(a) and stenosed part of the trachea (b).*


*5.1. Pluripotent Stem Cells in Cardiovascular Disease Modeling*

The use of PSCs to model cardiac disease in vitro has become highly attractive, especially after the introduction of iPSCs [107]. This is mainly because of inadequacies of other models in terms of sampling, propagation, and maintenance, as for human primary cardiomyocytes, or their ability to fully recapitulate physiological properties of human CMs, as in rodent models. Considering the relative difficulty in cloning and genetically modifying human ESCs, most established models of CVDs are iPSC-based [107]. The feasibility in sampling and propagation of iPSCs, as well as advances in reprogramming protocols, which later adopted nonintegrating genomic approaches to deliver the reprogramming factors, has greatly increased their popularity [159]. Patient-specific iPSC-CMs have enabled the study of genetic variants underlying several CVDs and establish a phenotype–genotype understanding of not only monogenic, but rather complex and difficult-to-model genetic variants (e.g., chromosomal deletions or translocations), and, most important, model congenital

heart disease (CHDs) in newborns [160,161]. As a result, several patient-derived iPSCs lines have been developed to model CVDs. The first of such was reported by Carvajal-Vergara et al. in 2010 for LEOPARD syndrome, an autosomal dominant developmental disorder characterized by hypertrophic cardiomyopathy [162]. Ever since, several other cell lines have been reported, mainly modeling cardiac channelopathies, (e.g., long QT syndromes), cardiomyopathies of wide etiology spectrum (e.g., dilated, hypertrophic, arrhythmogenic, Barth syndrome, and Pompe-disease-associated), and infectious myocarditis [163–177].

Despite the previously discussed disparities in structural and electrophysiological characters of iPSC-derived and adult CMs, these studies have shown that patient-specific iPSC-CMs recapitulate their corresponding disease phenotypes. For example, whole-cell patch-clamp analyses of different long QT syndrome (LQTS) patient-derived iPSC-CMs showed typically prolonged APs, decreased rectifier potassium currents IK, increased late sodium currents INaL, and impaired voltage-dependent inactivation of the L-type channels (LTC), due to malfunctions in corresponding proteins of potassium (KCNQ1, KCNH2 in LQTS1 and 2), sodium (SCN5A in LQTS3), and calcium (CaV1.2 in LQTS8 or Timothy syndrome) channels, respectively. Moreover, these patient-specific models demonstrated great utility for pharmacological screening of several drugs with disease-modifying abilities, leading to both novel and/or personalized therapeutic strategies (reviewed in [160]).

Finally, patient-specific iPSC-derived non-CMs were also generated, for example, of SMCs or endothelial cells. A more recent example is an elegant publication by Gu et al., utilizing iPSC-derived endothelial cells from patients with autosomal-dominant mutations in *BMPR2* associated with familial pulmonary arterial hypertension (FPAH) [178]. In their study, comparing symptomatic patients with unaffected carriers highlighted important modifiers of the BMP-receptor pathway, as well as differentially expressed genes, which imparted protection against FPAH. Their findings were of great importance as to the identification of multiple genetic factors affecting disease penetrance, which could be therapeutically targeted to modify disease progression and severity.

Importantly, the previous example behooves an important consideration when conducting studies on patient-specific iPSCs for CVD modeling, which pertains to the identification and/or the availability of proper control lines. This is because, even among patient-matched donor cohorts, genetic variability can still confound the analysis of the disease phenotype, especially in the presence of disease modifiers, or when the genotype–phenotype is less conspicuous [169,179]. In such cases, it is possible to rely on more than one control cell line—albeit a laborious approach. Alternatively, the patient's iPSC-CMs can be compared to those from a healthy sibling, thus limiting genetic variability [171]. However, recently developed computational in silico models of iPSC-CMs and their optimization by Paci and colleagues have provided an unprecedented approach to this issue, enabling simulation and calibration of over a thousand diseased or control iPSC-CM models [180–182]. Finally, in case of monogenetic diseases, an isogenic cell line created by correction of the disease-causing mutation in the patient iPSCs by means of gene-editing approaches can serve as the best control cell line (discussed below). An elegant example was reported in a study by Bellin and colleagues, where they used iPSC-CMs from LQTS2 patients with a distinct mutation in potassium channel KCNH2, and compared it to an isogenic control upon correction of the genetic mutation [183]. Furthermore, they reproduced the study model in human ESC-CMs, where they introduced the same mutation, and recapitulated the disease phenotype, thus generating two genetically distinct isogenic pairs of LQTS2 and control lines.

#### *5.2. Pluripotent Stem Cells in Pharmaceutical Screenings*

Since their first introduction, iPSC-CMs have become attractive for drug testing, antiquating the hERG test, which utilizes cell lines that stably express the human ether-a-go-go-related gene (hERG) *KCNH2* encoding the IKr channel involved in cardiac repolarization. Whole-cell patch-clamp screening for compounds that block the IKr current serves as a good marker of cardiotoxicity, as such blockade leads to the prolongation of the QT interval, i.e., ventricular repolarization, resulting in potentially fatal ventricular tachycardia called Torsade de Pointes [184]. Since the actual risk for cardiac toxicity is not confined to a certain channel and/or mechanism, iPSC-CMs are hence more representative in typifying cardiac toxicity to drugs. Furthermore, recent introduction of automated patch-clamp (APC) devices, all-optical cardiac electrophysiology with novel optogenetic actuation, and video microscopy have all revolutionized drug screening in iPSC-CMs and tissue constructs, enabling high-throughput testing platforms for hundreds of samples and/or drugs, thus creating a wealth of information in short time [185–188]. Furthermore, comprehensive in vitro proarrhythmic Assay (CIPA) has recently emerged as a powerful model to predict cardiac toxicity by integrating the knowledge from both in vitro and recently developed in silico computational models (http://cipaproject.org/about-cipa/) [189]. However, as discussing this is beyond the scope of this review, we refer the reader to the cited work by Paci et al.

#### *5.3. Genetic Modification of Pluripotent Stem Cells*

The advent of genome-editing methods has incited great progress in PSC research. Exploiting the cell's inherent DNA-repair mechanisms, such as nonhomologous end-joining (NHEG) or homologous recombination (HR), has long been used to introduce small but disruptive mutations to target genes, either by insertion or deletions of base pairs, also known as "Indels". The discovery and later advances of nucleases that can more specifically target desired sequences, such as zinc-finger nucleases (ZFNs) or transcription activator-like effector nucleases (TALENs), have enabled the study of several disease causing mutations [190–192]. Many PSC-lines have been generated by using this technology for both disease modeling and even clinical applications [193–196]. Vector-mediated delivery of sequence-specific nucleases along with a homologous DNA template to patient-derived iPSCs leads to the excision of targeted locus and, by virtue of cellular homology directed repair (HDR) system, can be corrected by the homologous template with the desired genetic modification. A prominent example is the combination of ZFNs and piggyBac technology which could achieve a biallelic correction of a disease-causing mutation in human iPSCs [197]. In a recent study by Karakikis et al., they reported the use of TALENs to correct gene mutations in patients with hereditary heart failure [198]. These patients harbor an amino acid deletion mutation (R14del) in the coding region of the phospholamban (PLN) gene, which is an important regulator of cardiac calcium cycling in the sarcoplasmic reticulum (SR). They display a phenotype of dilated cardiomyopathy, hypertrophy, episodic ventricular arrhythmia, and overt HF by middle age [199,200]. Skin-derived iPSCs from these patients were isolated, edited, and CM-differentiated, where further analyses showed reversal of the disease's phenotype. Nevertheless, engineering of sequence-specific ZFNs or TALENs, as well as achieving their robust delivery for this purpose, can be laborious and technically challenging, let alone high in cost [192,194,201].

In recent years, CRISPR/Cas9 has emerged as the new horsepower of genome-editing technology, overshadowing ZFNs and TALENs [202]. The system, first described in prokaryotes as part of their adaptive immune system, relies on an RNA-guided endonuclease (Cas9) that localizes to complementary DNA sequences, where it creates double-strand break amenable for correction by the cell's endogenous HR. Provided that a homologous sequence is available, desired gene modifications can be introduced [202]. Indeed, CRISPR/Cas9 has been zealously received by cell biologists as an attractive tool for SC research [203]. In cardiovascular biology, CRISPR/Cas9 was successfully applied to patient-derived iPSC to target disease-causing mutations of CVDs [204–206]. A recent study demonstrated the utility of CRISPR/Cas9 in phenotypic characterization of iPSC-CMs from patients with arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) [207]. In this patient cohort, mutations in the *SCN5A* encoding the Nav1.5 sodium channel protein led to the phenotype, which could be reversed in this study upon editing with CRISPR/Cas9.

A study published earlier this year by Seeger and colleagues made use of genome-editing techniques to create isogenic iPSC lines from patients with heterozygous mutations in the myosin-binding protein C3 (*MYBPC3*), which is deemed as the underlying cause of hypertrophic cardiomyopathy (HCM) [208]. Their results refuted previous hypotheses of either MZBPC3 haploinsufficiency or truncated poison peptide as the underlying cause of HCM. However, they were able to provide evidence for chronic activation of the nonsense-mediated decay (NMD) as the initial pathogenic trigger that leads to dysregulated gene expression and aberrant calcium signaling upon MYBPC3 mutations.

The aforementioned examples give a great promise to SC therapy of CVDs. One might also envisage the possibility of autologous cell transplantation of iPSC-derived CMs with rectified mutations to ameliorate or even cure disease conditions. However, great challenges remain as to the validation of these technologies, let alone deciding on a safe and effective clinical setting for PSCs delivery to treat CVDs. The next chapter outlines recent advances in preclinical research on SC-based therapy for CVDs.

#### **6. Translational Potential of PSCs in Cardiovascular Regenerative Therapy**

Harnessing the multifaceted potential of SCs for effective therapeutic purposes to treat CVDs is the ultimate goal of the above-introduced laborious efforts of scientists over the past decades. Provided that SC-derived CMs are sufficiently propagated, differentiated, and maturated, their application to the diseased myocardium spans a wide spectrum of delivery methods, from intravenous administration to direct myocardial injection. Nevertheless, several factors are to be considered with regard to engraftment of transplanted cells and integration, as well as functional contribution to host myocardium, electromechanical coupling between graft and host CMs, and long-term survival. The aforementioned limitations have long been challenges to preclinical and translational applications of SC therapy in general, and in cardiac regenerative therapy in particular. The following summarizes advances made in the realm of preclinical and translational research with PSCs over the past decades, in light of examples from small and large animal models and up until the first clinical initiatives.

#### *6.1. Pluripotent Stem Cells in Rodent Models*

Earlier studies attempted to engraft human ESC-derived cardiomyocytes in rodent models and reported transient functional improvement in cardiac parameters [209,210]. However, poor engraftment and survival of transplanted cells has been a challenge in these settings. Laflamme et al. utilized pro-survival factor cocktail to limit CM death upon engraftment in infarcted rat heart and reported positive outcomes [108]. To overcome poor engraftment and survival issues, Masumoto and colleagues developed a layered-sheet assembly of three cardiovascular cell populations, namely CMs, endothelial cells, and vascular mural cells, differentiated from mouse ESCs and transplanted into nude-rat model of MI. The transplanted sheets were reported to ameliorate infarct size and improve cardiac function; however, such benefits were shown to be attributed to paracrine-mediated neovascularization and not to actual contribution of transplanted cells [211]. Despite these results, the same group of scientists from Kyoto continued to optimize the stacked-sheet approach, and they recently reported successful long-term survival of engrafted cells through insertion of gelatin hydrogel microspheres between each cardiovascular cell sheet [212]. Analogously, human iPSC-CMs have recently demonstrated favorable therapeutic outcomes when injected in infarcted myocardia of mice. Interestingly, however, the engraftability and survival of those cells depended heavily on the maturation stage [142].

Importantly, electromechanical coupling between the graft and host myocytes is a rather crucial consideration to avoid ventricular arrhythmia. In a Guinea pig model, Shiba et al. reported successful engraftment of human ESC-derived CMs with a 1:1 electrophysiological coupling and improved mechanical function of injured hearts [213].

#### *6.2. Pluripotent Stem Cells in Large-Animal Models*

#### 6.2.1. Porcine Models

Swine models were featured in the earliest attempts of cell therapy for heart disease. A number of features make the pig an attractive translational model. These include a heart weight-to-body ratio that is equal to a human's and a similar sinus rate (~90 bpm) [214]. ESC-derived cardiomyocytes have been functionally tested in a swine model of complete atrioventricular block as biologic pacemaker for the treatment of bradycardia [215]. Hereof, Kehat et al. reported survival and functional integration of the transplanted cells, which were able to pace the porcine ventricle with complete heart block [215]. In another porcine model of acute MI, Ye and colleagues used a mixture of cardiovascular cell populations—this time from human iPSCs origin—loaded on a three-dimensional fibrin patch containing IGF-1 (insulin-like growth factor 1) and reported functional integration and significant improvements of several cardiovascular parameters [216]. More recently, Kawamura et al. took a rather unprecedented approach to enhance survival and engraftment of transplanted human iPSC-CMs by combining cell-sheets with pedicle omentum flap as a source of angiogenic factors and reported enhanced engraftment, survival, and therapeutic outcome in a porcine model of ischemic cardiomyopathy [217,218]. The aforementioned tissue sheet technology from Kyoto was also recently applied to a porcine model of MI, where a heterogeneous mixture of cardiovascular cell populations differentiated from human iPSCs and reported functional restoration of the infarcted hearts and attenuated remodeling [219]. Another study from 2018 by Gao and colleagues reported the application of human iPSC-derived fabricated cardiac muscle patches (hCMPs) composed of CMs, smooth muscle and endothelial cells, reprogramed from cardiac fibroblasts and maturated in dynamic culture conditions [220]. They transplanted these patches in infarcted pig hearts and demonstrated significant improvements upon histological and functional analyses. Altogether, these results highlight the importance of co-administration of nonmyocyte cardiac cells, which provide paracrine and angiogenic support equally important to both host and graft tissue. Furthermore, they highlight the superiority of modern tissue engineered scaffolds over direct application of cells. Finally, these studies corroborate the utility of swine models for translational cardiovascular research.

#### 6.2.2. Non-Human Primate Models

The utility of non-human primates (NHPs) in regenerative medicine has long been appreciated, especially in transplantation medicine [221]. In this regard, certain macaque species (e.g., *Macaca fascicularis* or Mauritian Cynomolgus macaque) are a valuable preclinical model to study allogenic transplantation of iPSCs [222]. This is because they exhibit limited diversity in their major histocompatibility complex (MHC) genes, which are distributed only among seven haplotypes and are structurally identical to those in humans [223,224]. Indeed, allogenic transplantation of iPSC-CM among MHC-matched Cynomolgus monkeys was shown in a study by Shiba et al. to be immune-tolerable, and improved cardiac contractile function upon MI [224]. Matching MHC antigens between donors and recipients was shown by others to reduce immunogenicity upon allogenic transplantation of iPSC-CM in the Cynomolgus macaque [225].

NHPs continue to provide unmatched insights to PSC-therapy of CVDs in late-translational studies. In a recently published elegant work by Chong et al., human ESC-CMs were utilized in the pigtail macaque (*Macaca nemestrina*) as a model of ischemia-reperfusion injury [226]. They reported significant re-muscularization of the infarcted areas, structural and functional integration of the grafted cells via establishment of adherent junctions, and electromechanical coupling, typified by synchronous calcium transients. Such promising results were slightly relegated by the presence of arrhythmia in the grafted animals—albeit nonfatal.

#### **7. Pluripotent Stem Cells in First Human Trials**

The aforementioned successes in late-translational studies with large animal models, as well as the advances made in tissue engineering and grafting techniques, paved the way to the first clinical application of PSC therapy in cardiac settings, which was recently reported in a case study by Menasche et al., using ESC-derived cardiac progenitors [227]. They used the ESC I6 line, which was enriched in vitro by culturing on clinical-grade irradiated human foreskin fibroblasts as feeder cells. Cardiac commitment was then achieved by bone morphogenic protein-2 (BMP-2) and a specific tyrosine kinase inhibitor of the fibroblast growth factor receptor (FGFR), and then confirmed by the

expression of the cardiac specific transcription factor Isl-1, as well as the stage-specific embryonic antigen-1 (SSEA-1), which was used for cell purification by immunomagnetic sorting. The cells were embedded in a fibrin scaffold patch and surgically implanted in the infarcted area of a 68-year-old woman patient with severe heart failure. The three-month follow-up showed functional integration of the patch, evident by electrocardiography, and overall symptomatic improvement marked by enhanced left-ventricular ejection fraction (LVEF), with no complications of arrhythmia, tumors, or immunosuppression-related adverse events. The results were encouraging, and the Parisian group conducted a larger-scale study, wherein six patients received cellularized patches of ESC-derived committed cardiac progenitors [228]. Their one-year follow-up demonstrated safety and tolerability of the grafted cells, with no detected tumors. Moreover, they reported modest symptomatic improvements, as well as in different cardiac parameters.

As for iPSCs, a group of scientists from Osaka have reported their granted permission to pursue with their clinical application last year and the results are yet to be reported [229].

#### **8. Conclusions and Remarks**

Stem cells are a novel source of cells which might be used as a screening tool for pharmaceutical developments. Here, single cells on iPSC status, as well as differentiated cardiomyocyte progenitors, might be used. In addition, engineered heart tissue displays a second model situation for screening of novel therapeutic options, before applying in animal experiments or clinical trials. Since these systems are based on human sources of cells, testing in these model situations might enhance safety and side effect prediction in novel approaches of cardiovascular therapies. Utilization of stem cells in patients suffering from cardiovascular disease is a second interesting field with great potential. The allogenic transplantation of stem cells requires only a modest immunosuppression and might improve cardiac function and thereby survival, as well as quality of life, in patients suffering from cardiac conditions, e.g., heart failure. However, the clinical potentials, as well as the potential side effects, need to be investigated in clinical trials before establishing stem-cell-based therapy as a standard of care in cardiovascular patients. In summary, stem cells, especially induced pluripotent stem cells, have wide therapeutic potential, but need to be characterized and investigated in more detail in preclinical, as well as clinical, trials to understand in more detail their potentials and risks.

**Author Contributions:** M.S., writing—original draft and visualization; R.H., writing—review and editing.

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

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

### **Abbreviations**

#### **References**

1. WHO. Cardiovascular Diseases (CVDs). Available online: http://www.who.int/en/news-room/fact-sheets/ detail/cardiovascular-diseases- (accessed on 1 June 2019).

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

*Review*


**Simona Roxana Pătărlăgeanu 1, Costel Negrei 1, Mihai Dinu 1,\* and Roxana Chiocaru 2**

Received: 7 March 2020; Accepted: 29 April 2020; Published: 6 May 2020

**Abstract:** This paper focuses on the current environmental issues, more specifically the amount of greenhouse gases humanity is being confronted with at the moment. The research was carried out on a niche of the topic, namely on the carbon footprint of public buildings. The concept of a sustainable university is new and insufficiently explored, and as part of the environmental metabolism, it influences anthropic sustainability in a directly proportional manner.  The ecological effects may be mitigated by the academic community through green urban design. The ecological performance can be expressed in an economically efficient manner, which can, at the same time, create a precious channel of communication within the entire academic community though volunteering for sustainability. Moreover, this research has identified several solutions for optimizing the carbon footprint, which do not hinder the necessary economic development. Within the current context, when most economic activities are leading to ecological collapse, sustainability should be reprioritized with the help of the academic society, through the examples offered by applied research. The premises of this research were represented by bibliometric analyses and the results obtained have proven its importance, as well as the importance of certain scenarios involving solutions for improving the metabolism of nature.

**Keywords:** sustainability; sustainable university; ecological collapse; climate change; green vertical facades