*2.2. Naproxen Extraction*

Commercialized Proxen 500 mg tablets were ground into a fine powder using an electric grinder. The Proxen powder obtained is added to a 3M HCl solution and stirred for 24 h then left to stand for 1 h. Naproxen (NPX) or (S)-2-(6-methoxynaphthalen-2-yl) propionic acid, which is very poorly soluble in water precipitates, the additives dissolve, and the precipitant is then recovered by filtration. The extracted NPX powder is washed several times with water and then dried under a vacuum at 25 ◦C to constant weight. In order to remove the residual matter from the organic phase, the dry precipitate obtained is

dissolved in chloroform and then transferred to a separating funnel containing an equivalent amount of distilled water. The whole is then stirred until the complete dissolution of Naproxen. The two phases are finally separated by settling. This process was repeated three times to ensure the purification of the product. Pure Naproxen is then extracted from the isolated organic phase by evaporating chloroform at room temperature (25 ◦C) using a rotary. The melting point of the pure NPX white crystals obtained, measured by DSC analysis, indicates 166 ◦C, which agree with the literature [34].

### *2.3. Preparation of NPX/pHEMA and NPX/pHPMA*

NPX/pHEMA and NPX/PHPMA composites containing 2, 5, 7, and 10 wt% of NPX were prepared in situ by free radical polymerization at 25 ◦C in the presence of NPX using camphorquinone as a photoinitiator. Using known amounts distillated under reduced pressure of HEMA and HPMA monomers, camphorquinone and NPX were weighed with precision and placed in a Teflon pan traversed with a stream of nitrogen U. These mixtures are irradiated throughout the reaction time by means of UV light coming from a UV lamp with a wavelength of 380 nm and a power of 13.3 MW. A solid film deposed in the Teflon pan is obtained indicating the completion of the polymerization reaction. To remove all traces of residual monomer encrusted in the film obtained, the Teflon pan and polymer film set are placed in a vacuum oven maintained at 40 ◦C until constant mass. The aggregated NPX particles deposited or glued to the film surface are removed by washing three times with distillated water. Two series of NPX/pHEMA and NPX/pHPMA mixtures containing 2, 5, 7, and 10 wt% of NPX content are prepared by this same method, and the preparation conditions are summarized in Table 1.

**Table 1.** Preparation conditions of NPX/pHEMA and NPX/pHPMA composites.

#### *2.4. Characterization*

The FTIR spectra of NPX powder, PHEMA homopolymer, NPX/PHEMA, and NPX/ PHPMA composites films were performed in the wavenumber range 400–4000 cm−1 on a Nicolet 6700 FT-IR from the company Thermo Scientific. A 30,000–200 cm−1 diameter diamond-like Smart orbit crystal reflector, supplied by the same company, was used to accomplish this task. The DSC thermograms of drug, polymer, and their mixtures were performed on a Shimadzu DSC-60 (Japan) previously calibrated with indium. An amount of 8–10 mg of NPX powder or film samples were deposited in an aluminum pan and then closed, being placed in the DSC analysis cell. All samples were scanned from −40 to +240 ◦C under nitrogen gas atmosphere at a heating rate of 20 ◦C·min−1. All the thermograms taken from the second scan run revealed no traces of polymers or drug degradation. The *Tg* value of pure constituents or their mixture was taken precisely as the median point on the thermogram indicating the variation in the heat capacity versus the temperature. The *Tm* value was taken exactly at the top of the endothermic peak. The surface morphology of NPX particles, polymers, and their composites were examined by scanning electronic microscope using a JEOL JSM-6360LV SEM (Tokyo, Japan) at an accelerating voltage of 10 kV. The surface and cross-sections of samples were sputter-coated with a thin layer of gold prior and was imaged at a magnification range of 300–3000 nm. The crystalline

structures of NPX powder, polymers, and mixtures were examined by XRD analysis on an X-ray diffractometer (Rigaku D/max 2000) equipped with a Cu anode tube. The applied voltage was 40 kV and a generator current of 100 mA. All samples were examined at 2*θ* = 5◦–80◦ at a scanning rate of 1.0◦·min−1.

A U-2910 spectrophotometer manufactured by Hitachi Company was used to measure ultraviolet and visible light absorbance of NPX released. Absorbance was measured using quartz cuvettes with a side length of 1 cm. The wavelength corresponding to the maximum absorbance of NPX was 230 nm. The released NPX concentration was deduced from a linear calibration curve indicating the change in absorbance versus concentration.

#### *2.5. Cell Culture and Proliferation Assessment*

The human oral cancer cell line Ca9–22 cells were cultured at 37 ◦C and 5% CO2 in RPMI-1640 medium (Thermo Fisher Scientific, Burlington, ON, Canada), supplemented with L-glutamine, 5% fetal bovine serum (FBS, Gibco) provided by the same company, and 1% penicillin/streptomycin solution (Sigma-Aldrich, Oakville, ON, Canada). Cell proliferation was bi-evaluated using two MTT and LDH tests as described by Semlali et al. 2021 [35,36] and Contant et al. 2021 [37]. For the MTT assay, 105 Ca9-22 cells per well containing the sample were seeded in 24-well plates for 24 h. After adhesion and growth of the cells, the culture medium is replaced by a new one containing a solution of MTT at 5 mg·ml−1 in PBS and left for 3 h at 37 ◦C in the dark. Then, the cells were lysed with HCl, 0.05 N, in 1 mL of isopropanol. Addition of 100 μL of analysis buffer to test wells from 96-well microplates was required to measure absorbance at 550 nm by an iMark reader (Bio-Rad). The percentage of viable proliferating cells was determined using Equation (1)

$$\text{Cell}.viability(\%) = \frac{OD\_T - OD\_B}{OD\_C - OD\_B} \times 100,\tag{1}$$

where *ODT*, *ODB,* and *ODC* are the optic densities of the treated cell, blank, and control, cell respectively.

Adhesion positive control was the plate for the treated cultured tissues. The negative control for cell adhesion was the plate for untreated cultured tissue. LDH assay was realized by the LDH Cytotoxicity Detection Kit from Roche, which allows to directly quantify the cell death in culture based on the measurement of lactate dehydrogenase released into growth media. As described in our previous work [35,36], 105 cells per well were seeded in 24-well plates containing NPX/pHEMA and NPX/pHPMA with different compositions. After adhesion for 24 h, 50 μL of each supernatant was transferred in triplicate into a 96-well plate and supplemented with 50 μL reconstituted substrate mixture. Then, the plates were incubated for 30 min at room temperature in the dark until the yellow color developed, before reading at 490 nm with an xMark microplate absorbance spectrophotometer (Bio-Rad, Mississauga, ON, Canada). Triton X-100 (1%) was used as a positive control for LDH, and the negative one was obtained with untreated cells. LDH release activity was calculated using Equation (2)

$$\text{LDH.activity}(\%) = \frac{ABS\_p - ABS\_{\text{nc}}}{ABS\_{pc} - ABS\_{\text{nc}}} \times 100,\tag{2}$$

where *ABSp*, *ABSnc*, and *ABSpc* are the absorbance of drug-carrier system, positive control, and negative control, respectively.

#### *2.6. Swelling Properties*

The swelling behavior of pHEMA and pHPMA hydrogels was studied on samples of thin films of dimensions 3 cm × 3 cm and thickness varying between 2.20 and 2.56 mm. Each film sample of determined mass (*mo*) was placed in 50 mL of an aqueous solution at known pH (1 or 7) and maintained at 37 ◦C, then stirred (260 rpm) until the swelling equilibrium was reached. The mass of the medium absorbed at each time interval (*mt*) is

obtained by weighing the film after delicately wiping the droplets deposited on the two surfaces using tissue paper. The swelling degree of pHEMA and pHPMA film samples was determined from Equation (3).

$$S(wt\% ) = \frac{m\_l - m\_o}{m\_o} \times 100 \,\text{\AA} \tag{3}$$

#### *2.7. Density Measurements*

Polymer density values were determined at 25 ◦C using a pycnometer, in which cyclohexane was used as a non-solvent and Equation (4) [38]:

$$\rho = \frac{m\_p \times \rho\_{\rm chx}}{m\_p + m\_{pc} + m\_T} \,\prime \tag{4}$$

where *mp* is the mass of the polymer, *mpc* is the mass of the pycnometer with cyclohexane, and *mT* is the mass of the pycnometer with cyclohexane and polymer. *ρchx* is the density of cyclohexane (0.78 g·cm−3). Each experiment was triplicated, and the density was taken from the average arithmetic values obtained.

#### *2.8. In Vitro Release Dynamic of NPX*

The "in vitro" release dynamic of NPX from the NPX/pHEMA and NPX/pHPMA drug carrier systems was investigated at body temperature (37 ◦C) in aqueous media of pH1, 3, 5, and 7. NPX released was monitored for 72 h, in which 0.5 mL of the solution was withdrawn after each time interval, then dosed by UV analysis. The accumulative drug release percent ADR (wt %) at time *t* was calculated at chosen time intervals using the following equation:

*ADR*(*wt*%) = *mt* × 100 *mo* , (5)

where *mt* and *mo* are the total mass of NPX released at a certain time *t* and the initial mass of drug loaded in the polymer.

#### **3. Results and Discussions**

#### *3.1. Characterization*

#### 3.1.1. FTIR Analysis

A comparison between the FTIR spectra of the NPX/pHEMA composites with those of their components shown in Figure 1 reveals, for the composite absorption bands, that they are practically similar to those of pure pHEMA. The NPX/pHEMA spectra exhibits a shift in the broad absorption band of the hydroxyl group vibrations from 3472 to 3463 cm−1 and a shift of the sharp absorption band attributed to the carbonyl group (C=O) vibration of pHEMA from 1727.62 cm−1 to 1724.32 cm−1. It is well known that the position of the vibration peak of the carbonyl group suggests that most of the carboxylic acid groups are associated with the intermolecular hydrogen bonds formed between the HEMA derived moieties and the acid groups [39–41]. In addition, the wide absorption band in the spectral region 3200–3600 cm−1 corresponding to the vibrations of the OH group also confirms that hydrogen bonds form in the structure of poly(2-hydroxyethyl methacrylateco-acrylic acid). The deconvolution in Lorentzian peaks of the hydroxyl absorption band between 2600 cm−1 and 4000 cm−1 (Figure 2) also reveals the appearance of a new band at 3274.54 cm−1, attributed to the vibration of the hydrogen bond between hydroxyl group of pHEMA and carbonyl group of NPX. On the carbonyls side, the deconvolution of the absorption band between 1600 cm−1 and 2000 cm−1 (Figure 3) reveals another new absorption band at 1723.5 cm−1, thus confirming this finding. Similar results are also observed for NPX/pHPMA composites in Figure 4. Indeed, the comparison between the FTIR spectra of the NPX/pHPMA composites with that of its pure polymer reveals a small shift in the absorption band of the carbonyl group of pHPMA toward the lower wave number (from 1724.12 cm−1to 1727.34 cm−1) and in the hydroxyl group (from 3400.07 cm−1

to 3395.36 cm−1). These facts are without doubt attributed to a dynamic caused by the hydrogen bond interactions leading to a miscibility of these two components.

**Figure 1.** Comparative FTIR spectra of: (**A**) pure NPX; (**B**) NPX/pHEMA2; (**C**) NPX/pHEMA5; (**D**) NPX/pHEMA7; (**E**) NPX/pHEMA10; (**F**) virgin pHEMA.

**Figure 2.** Deconvolution in Lorentzian of the FTIR spectra of pure pHEMA and NPX/pHEMA7 composite spectra between 4000 cm−1 and 2600 cm−1.

**Figure 3.** Deconvolution in Lorentzian curves of the FTIR spectra of pure pHEMA and NPX/pHEMA7 composite spectra between 2000 cm−1 and 1600 cm−1.

**Figure 4.** Comparative FTIR spectra of: (**A**) pure NPX; (**B**) NPX/pHPMA2; (**C**) NPX/pHPMA5; (**D**) NPX/pHPMA7; (**E**) NPX/pHPMA10; (**F**) virgin pHPMA.

#### 3.1.2. XRD Analysis

The crystalline structure of NPX in the pHEMA and pHPMA polymer matrices was investigated by X-ray diffraction, and the results obtained are gathered with their pure components in Figures 5 and 6, respectively. As shown in Figure 5, the XRD pattern of pure NPX aggregated powder shows its highly crystalline structure and nature through the distinct peaks at 6.5◦, 12.4◦, 16.6◦, 19◦, 20◦, 22.5◦, 24◦, and 28.6◦ 2*θ*, which are in good agreement with the literature [42,43]. The XRD spectra of pHEMA and pHPMA reveal an amorphous structure. The XRD motif of the NPX/pHEMA7 composite shows no new crystallinity signals or those characterizing the crystallinity of pure NPX; as for the pure polymer, this material exhibits a completely amorphous structure. Similar results are also obtained for the NPX/pHPMA systems as shown in Figure 6. This indicates that the NPX drug is uniformly distributed in its molecular level inside the polymer matrix.

**Figure 5.** X-ray diffraction spectra of NPX, pHEMA, and NPX/pHEMA systems with different NPX contents.

#### 3.1.3. DSC Analysis

The DSC thermograms of pure NPX, pHEMA, and NPX/pHEMA systems with different NPX contents are shown in Figure 7. The thermal curve of pure pHEMA shows a glass transition temperature (*Tg*) at 86 ◦C, which agrees with that of the literature [44], while the pure Naproxen shows, through its thermal plot, a sharp endothermic peak at 166 ◦C characterizing its melting temperature [34]. The NPX/pHEMA thermograms reveal a small shift in the *Tg* of pHEMA toward the low temperatures and a complete disappearance of the NPX melting peak. This reveals a uniform distribution of the NPX filler in the polymer matrix in its molecular level, in which Naproxen loses its crystallinity, thus confirming the results obtained by FTIR and XRD analysis. The decrease in the *Tg* value of the polymer in the mixture is probably due to the increase of the free volume between the polymer chains caused by the insertion of MPX molecules between them, thus promoting the chain sliding.

**Figure 6.** X-ray diffraction spectra of NPX, pHPMA, and NPX/pHPMA systems with different NPX contents.

**Figure 7.** DSC thermograms of pure NPX, pHEMA, and NPX/pHEMA7 systems.

The thermal analysis of NPX/pHPMA systems by DSC technique led to the results of Figure 8. As it can be observed on the thermogram of pure pHPMA, a transition appears at 83 ◦C characterizing the glass transition of this polymer [45]. Concerning the NPX/pHEMA system, as for the system containing PHEMA carrier, a shift was shown in the Tg of the pHPMA from 82 ◦C to 54 ◦C as the NPX content in the mixture increased. A complete disappearance of the transition characterizing the fusion of the NPX is also observed on the thermograms of NPX/pHPMA mixtures, except that containing 10 wt% of NPX content, in which a weak transition at 131 ◦C attributed to the melting point of excess of NPX aggregates.

**Figure 8.** DSC thermograms of NPX, pHPMA, and NPX/pHPMA systems with different NPX contents.

#### *3.2. Cells Adhesion and Toxicity*

As shown in Figure 9 (in blue), the NPX/pHEMA drug-carrier system with different NPX contents presents, in general, a good adhesion compared to the negative (untreated tissue culture plate) and positive (tissue culture plate treated for cell adhesion) control used in this study. However, the NPX amount incorporated in the pHEMA in drug carrier system seemed to not significantly affect the cell adhesion when the Ca9-22 cells were treated with naproxen. These results were also confirmed by the LDH assay (Figure 10 in green). In addition, NPX/pHEMA drug carrier systems, as well as the pure pHEMA, induce low cytotoxicity compared to the negative and positive controls (2% Triton). Comparable results were also observed when the pHEMA was replaced by pHPMA in the drug carrier system, regardless of the range of the composition investigated (Figure 9 in blue and Figure 10 in green).

**Figure 9.** Effect of NPX content in NPX/pHEMA and NPX/pHPMA drug carrier systems on Ca9-22 cells adhesion

**Figure 10.** Effect of NPX content in NPX/pHEMA and NPX/pHPMA drug carrier systems on Ca9-22 cells cytotoxicity.


**3. Results**

#### *3.1. Study Population*

A total of 121 patients were included in this study: 61 in the intervention group and 60 in the control group. An overview of patient characteristics, underlying diagnoses for LTx, comorbidities and immunosuppression is shown in Tables 1 and 2. There were only small di fferences in the demographic data between the study groups. The control group had a more extended warm ischemic time (*p* = 0.005). As coimmunosuppression, patients received mycophenolate mofetil (MMF) at a daily dose of 1000 (500–2000) mg (LCPT) and 1500 (500–2000) mg (control), everolimus at 2.0 (0.5–5.0) mg (LCPT) and 2.0 (2.0–4.0) mg (control), prednisolone at 5.0 (5.0–7.5) mg (LCPT and control) and sirolimus at 1.0 mg (control). In the intervention group, 45 patients su ffered from chronic kidney disease (CKD, categories 2–4). The control group showed a similar distribution in 39 LT recipients. No patients were in CKD category 5 or on dialysis. In the absence of kidney biopsies, the underlying renal disease remained unclear. The median interval between transplantation and study onset was 2.8 (0.1–20.8) years in the intervention group and 6.6 (0.2–16.5) years in the control group (*p* < 0.001). The reasons for a conversion from standard-release Tac to LCPT were CNIT (n = 7), neurotoxicity (n = 5) and prevention of side e ffects via better bioavailability of LCPT (n = 49).

**Table 1.** Patient characteristics.

Statistics: Values shown as mean ± standard deviation or number (percentage). a *t*-test, b Fisher's exact test. LCPT, once-daily MeltDose® tacrolimus; Tac, tacrolimus; LTx, liver transplantation; BMI, body mass index; CIT, cold ischemic time; WIT, warm ischemic time; CMV, cytomegalovirus.

**Table 2.** Underlying diagnoses for LTx, comorbidities and immunosuppression at study start.

Statistics: Values shown as number (percentage). All *p*-values from Fisher's exact tests. LCPT, once-daily MeltDose® tacrolimus; Tac, tacrolimus; LTx, liver transplantation; NASH, nonalcoholic steatohepatitis; CKD, chronic kidney disease (categories set with reference to [23]). MMF, mycophenolate mofetil; CNIT, calcineurin inhibitor nephrotoxicity.

#### *3.2. C*/*D Ratio*

At study start (baseline), the C/D ratio in the intervention group was comparable to that in the control group (1.68 (0.30–13.45) vs. 1.76 (0.38–7.40) ng/mL×1/mg, respectively; *p* = 0.362, Table 3). During the 12-month evaluation period, no significant changes in the C/D ratio were observed in the control group. After 12 months, the median C/D ratio was approximately at the baseline level (1.75 (0.49–6.40) ng/mL × 1/mg; *p* = 0.847). In the control group, there was a slight decrease in both the daily Tac dose at study end compared with that at baseline (2.5 (0.5–10.0) vs. 2.8 (0.5–10.0) mg, respectively; *p* = 0.084), as well as in the median Tac trough level (4.7 (1.5–14.3) ng/mL at study onset to 4.1 (1.6–15.6) ng/mL after 12 months; *p* = 0.082). However, the di fferences in both cases were not significant.

In contrast, the C/D ratio in patients switched to LCPT was 50% higher 12 months after conversion than that at baseline (2.52 (0.58–6.40) vs. 1.68 (0.30–13.45) ng/mL × 1/mg, respectively; *p* < 0.001). A significant increase in the C/D ratio was already observed in this group 3 months after study onset (2.03 (0.33–13.60) ng/mL × 1/mg; *p* = 0.008). Regarding the daily Tac dose, a significant reduction of 33.3% was observed after 12 months compared with that at baseline (2.0 (0.4–7.8) vs. 3.0 (1.0–22.0) mg, respectively; *p* < 0.001)). Moreover, the Tac trough level was significantly reduced at study end (4.4 (2.2–11.8) vs. 6.0 (1.5–26.9) ng/mL at study onset; *p* < 0.001).

To confirm that conditions were stable before study onset, C/D ratios, Tac doses and trough level 3 months before enrolment were also obtained. There were no significant di fferences between the groups at t−3 (Table 3) nor between study start and 3 months earlier within a group. Patients in the intervention group showed similar median C/D ratio compared with that at baseline (1.44 (0.24–6.20) vs. 1.68 (0.30–13.45) ng/mL × 1/mg, respectively; *p* = 0.204). Daily Tac dose differed significantly due to single outlier values shortly after transplant (3.0 (0.5–12.0) (t−3) vs. 3.0 (1.0–22.0) (t0) mg; *p* = 0.049), while Tac trough level showed no considerable differences (5.0 (2.4–15.3) (t−3) vs. 6.0 (1.5–26.9) (t0) ng/mL; *p* = 0.722).

No significant differences were detectable in the control group between baseline and 3 months before: C/D ratio (1.69 (0.40–9.20) vs. 1.76 (0.38–7.40) ng/mL × 1/mg, respectively; *p* = 0.626), Tac daily dose (2.5 (0.5–9.0) vs. 2.8 (0.5–10.0) mg, respectively; *p* = 0.362) and Tac trough level (4.4 (1.5–14.7) vs. 4.7 (1.5–14.3) ng/mL, respectively; *p* = 0.742).

**Table 3.** Tacrolimus concentration/dose (C/D) ratio, daily dose and blood trough concentration.

To confirm that conditions were stable before enrolment, values 3 months prior to study onset are given for all patients who had already undergone liver transplantation (n = 54 vs. 58). In the intervention group (LCPT), values 3 months before and the day before the first LCPT intake (study onset) were determined when s-r-Tac was administered. LCPT, once-daily MeltDose® tacrolimus; Tac, tacrolimus; s-r-Tac, standard-release tacrolimus. *p*-values from Mann–Whitney U-test.

As shown in Figure 2, the C/D ratio at study end was significantly higher in patients on LCPT than in the control group (2.52 (0.58–6.40) vs. 1.75 (0.49–6.40) ng/mL × 1/mg, respectively; *p* = 0.009). The median Tac trough level and the daily dose were significantly higher in the intervention group at study onset (Table 3). After 12-month follow-up, the Tac dose in the LCPT group was significantly reduced compared with that in the control group (2.0 (0.4–7.8) vs. 2.5 (0.5–10.0) mg, respectively; *p* = 0.047). However, the Tac trough level was comparable in the two groups at study end (4.4 (2.2–11.8) vs. 4.1 (1.6–15.6) ng/mL, respectively; *p* = 0.283).

**Figure 2.** Boxplots of C/D ratio among patients receiving LCPT (dark grey) or standard-release Tac (light brown) at baseline and 3, 6, 9 and 12 months later. There were significant differences between the two study groups at 6 and 12 months after conversion. *p*-values reflect differences between the groups at each time point.

#### *3.3. Renal Function*

At baseline (study onset, t0), patients in the control group had a higher mean eGFR than patients switched to LCPT (Figure 3), although the difference (ΔeGFR) was not significant (*p* = 0.157). However, mean ΔeGFR in patients on LCPT had significantly improved at 6 months after conversion (*p* = 0.029). In contrast, patients on standard-release Tac showed a significant decline of mean ΔeGFR 9 months after study initiation (*p* = 0.006). Over the 12-month evaluation period, mean ΔeGFR continued to improve significantly in patients receiving LCPT (*p* = 0.001), whereas mean ΔeGFR continued to deteriorate in the control group (*p* < 0.001). In a pairwise comparison between the groups, eGFR values did not differ significantly (Supplementary Table S1).

**Figure 3.** Glomerular filtration rate (eGFR; mL/min/1.73 m2) over time and the difference from baseline at each time point (ΔeGFR ± SEM) in each study group. Improved renal function with a significantly increased mean ΔeGFR was already observed 3 months after conversion to LCPT (dark grey). *p*-values reflect comparison of ΔeGFR between the study groups.

While absolute eGFR values are meaningful to only a limited extent, eGFR slope (ΔeGFR) relative to the baseline can be used as additional empirical support (Table 4). Three months before study onset, there were no significant differences within the study groups relative to baseline. In the intervention group, renal function increased 6 months after conversion (*p* = 0.029). In contrast, LT recipients in the control group showed a significant decline of eGFR 9 months after study initiation (*p* = 0.006). Over the 12-month evaluation period, renal function continued to significantly improve in patients receiving LCPT (*p* = 0.001), whereas eGFR continued to deteriorate in the control group (*p* < 0.001).

**Table 4.** Slope analysis (ΔeGFR) of glomerular filtration rate (eGFR; mL/min/1.73 m2).

The "estimate" value describes the difference between the respective time point and the baseline (ΔeGFR). A negative value shows a decline and a positive value an improvement of eGFR. LCPT, once-daily MeltDose® tacrolimus; Tac, tacrolimus; *p*-values within a group are relative to the baseline.

In further analysis, the eGFR values of the patients suffering from diabetes mellitus and arterial hypertension were compared between the groups.

At every time point, patients with diabetes mellitus had significantly lower eGFR than patients without it, regardless of the study group (Table 5). However, eGFR among diabetic patients recovered in a manner similar to that of nondiabetics upon switching to LCPT. In contrast, renal function deteriorated in patients maintained on standard-release Tac in a similar fashion, regardless of diabetes.

**Table 5.** Glomerular filtration rate (eGFR; mL/min/1.73 m2) in diabetic and nondiabetic patients.

LCPT, once-daily MeltDose® tacrolimus; Tac, tacrolimus; t0 to t12, time points (months). eGFR values shown as mean ± standard deviation. *p*-values from *t*-test.

Patients with arterial hypertension in both study groups had a lower mean eGFR than patients with normal blood pressure at each time point (Table 6). However, renal function recovered in patients treated with LCPT and deteriorated in those maintained on standard-release Tac over the course of the study, regardless of the presence of arterial hypertension.

**Table 6.** Glomerular filtration rate (eGFR; mL/min/1.73 m2) in patients with and without arterial hypertension.

LCPT, once-daily MeltDose® tacrolimus; Tac, tacrolimus; t0 to t12, time points (months). eGFR values shown as mean ± standard deviation. *p*-values from *t*-test.

Multivariable analysis was performed to identify independent predictors of alterations in renal function expressed as ΔeGFR (Supplementary Table S2). Conversion to LCPT was the only identified independent predictor of significant changes in eGFR.

#### *3.4. Liver Function*

During the entire follow-up, we monitored the graft function (Table 7). LT recipients in the LCPT group showed significantly lower serum bilirubin concentrations than the control group at all time points. However, the median values in both study groups remained within the lower part of the normal range throughout the course of the study. Regarding the parameters ALT and INR, no differences were observed between the groups.

**Table 7.** Assessment of liver function over time in each study group.

LCPT, once-daily MeltDose® tacrolimus; Tac, tacrolimus; ALT, alanine transaminase; INR, international normalized ratio; *p*-values from Mann–Whitney U-test.

## **4. Discussion**

The present study shows that the conversion of LT recipients from standard-release Tac to LCPT was beneficial in regard to renal function. This may be due to the improved bioavailability of LCPT which led to a significant increase in C/D ratio.

Notably, the median daily Tac dose declined by 33.3% among LT recipients after conversion. A dose reduction of approximately 30% with a comparable area under the curve (AUC) was reported in recent studies of KT and LT recipients [20,24,25]. In those studies, this finding was also attributed to the greater bioavailability of LCPT.

In our cohort, the median C/D ratio among LT recipients who switched to LCPT had increased by 50% at 12 months after conversion. The C/D ratio among patients maintained on standard-release Tac remained unchanged over the 12-month period. In accordance with these data, Franco et al. described a 35% increase in the C/D ratio among KT recipients after conversion from IR-Tac and a 83.3% increase among those who were switched from ER-Tac to LCPT [26]. In the study by Rostaing et al., KT recipients had a 20% higher C/D ratio 12 months after conversion to LCPT and a 24.4% higher C/D ratio 24 months after conversion [27]. In contrast, Kami ´nska et al. showed that the C/D ratio of KT recipients converted from IR-Tac to ER-Tac did not change significantly [28]. To our knowledge, the present study is the first to describe a significant increase in the C/D ratio after a switch to LCPT among LT recipients.

In a previous study, we explored the impact of the C/D ratio on renal function after kidney transplantation (KTx) [14]. Fast metabolizers, defined as patients with a C/D ratio < 1.05 ng/mL × 1/mg, showed a strong association with decreased renal function compared with slow metabolizers in a 24-month follow-up. Similar results were confirmed among LT recipients in a 36-month follow-up study [13]. In that cohort, the cut-o ff value for fast metabolizers was defined as a C/D ratio < 1.09 ng/mL × 1/mg. In a 5-year follow-up, KT recipients with a lower Tac C/D ratio showed a higher risk of renal impairment as well as higher mortality rates [17]. Recently, several studies confirmed these findings [19,29,30] and a further negative impact of fast Tac metabolism on increased kidney allograft rejection rates and BK virus infections was demonstrated [17,18,31].

Given these results, we postulated that a higher C/D ratio after conversion to LCPT is associated with nephroprotection. Surprisingly, we already observed significant improvement of renal function 6 months after conversion. Twelve months after conversion, the mean ΔeGFR was 4.7 mL/min/1.73 m2 higher than at baseline. In contrast, eGFR had deteriorated significantly in patients maintained on standard-release Tac 9 months after study onset and ΔeGFR had decreased by 4.3 mL/min/1.73 m2 at 12 months.

After conversion to LCPT, the median trough level declined from 6.0 ng/mL at study onset to 4.6 ng/mL (month 3) without a subsequent decrease until month 12. A lower Tac trough level in the LCPT group has already been reported in a prospective study, although the same target trough level was given [27]. Alongside better bioavailability of LCPT, trough level reduction might be another reason for the increase in renal function. However, median trough levels did not vary considerably between subsequent time points (t3–t12) while renal function showed further recovery. Notably, median Tac trough levels were also slightly reduced in the control group (t0–t12), although eGFR showed further decline over the 12-month follow-up. Therefore, we postulate that improvement of bioavailability and a reduced peak Tac level after conversion to LCPT are factors more relevant to the increase in eGFR than the reduction in Tac trough levels alone.

As an explanation for the nephroprotective potential of LCPT, Schütte-Nütgen et al. hypothesized that a lower daily Tac dose results in a lower peak serum concentration (Cmax), which in turn reduces the side e ffects of Tac overdosing within the first hours after drug intake [17]. In a review article on LT recipients, Baraldo reported that LCPT had a similar AUC after 24 h and a similar minimal blood concentration (Cmin), but had a significantly lower Cmax and a smaller Cmax/Cmin fluctuation ratio when compared with IR-Tac [32]. In addition, Bunnadaprist et al. postulated that there is a reduced cumulative Tac dose in KT recipients receiving LCPT [33]. In a recent study, we also showed that fast metabolizers with a C/D ratio < 1.05 ng/mL × 1/mg had significantly higher Tac blood concentrations than slow metabolizers 2 h after Tac intake [16]. In the same study, we showed that a low C/D ratio was significantly associated with acute CNIT. Although renal biopsy is not routinely performed in LT recipients, we can assume that patients converted to LCPT su ffered less frequently from CNIT. In contrast, Kamar et al. reported similar renal function in de novo KTx recipients who were randomized to LCPT or ER-Tac in a 4-week follow-up [34]. Notably, Cmin and AUC0–24 were slightly higher in the LCPT group (at days 3, 7 and 14), a fact that might have influenced the results.

In the current study, the control group had an increased warm ischemic time (WIT) compared with the intervention group (~5 min). Prolonged cold and warm ischemic times can be associated with long-term allograft dysfunction [32]. Nevertheless, at the beginning of our study, the liver function parameters ALT and INR did not di ffer between the groups and median bilirubin was within the normal range. In a study by Laskey et al., increasing WIT during LTx was associated with a lack of renal recovery in the presence of pretransplant subacute kidney injury [35]. It was concluded that minimization of WIT could potentially avoid renal replacement therapy or the need for subsequent kidney transplantation. At the study start in our cohort, the control group showed even higher eGFR values despite increased WIT compared with the intervention group. Notably, the control group had a more extended interval between LTx and study onset than patients switched to LCPT (6.6 (0.2–16.5) vs.2.8 (0.1–20.8) years, respectively).

In regard to the Tac formulations used before study onset, IR-Tac was administered more frequently than ER-Tac in the intervention group and vice versa in the control group. A recent study on pharmacokinetics in a large transplant cohort showed similar Tac trough levels and bioavailability between these two formulations [36]. Notably, C/D ratio as well as C/D intrapatient variability was reported not to change considerably during conversion from IR-Tac to ER-Tac in KT recipients [28]. These findings justify our and others' approach of including patients taking either one of these formulations [19,29].

In the current study, patients su ffering from diabetes mellitus or arterial hypertension had reduced renal function. Interestingly, patients who were switched to LCPT (median C/D ratio increased from 1.68 to 2.52 ng/mL × 1/mg) showed considerable recovery of eGFR independent of the presence of both conditions. In accordance with these findings, Bardou et al. showed that slow Tac metabolizers (C/D ratio > 1.8 ng/mL × 1/mg) were less likely to su ffer from diabetes and hypertension after LTx [37].

Finally, we recognize that our study has limitations due to its retrospective design and the limited sample size from a single-centre. In addition, in this study, we cannot provide Tac Cmax, C2 (2 h after Tac intake) nor AUC, although higher Cmax or C2 could potentially induce higher CNIT. Therefore, we can only hypothesize that, after conversion to LCPT, lower C2 was a more relevant factor to the improvement of renal function than trough level reduction. Further investigations should also include data on the concentrations of di fferent Tac metabolites, which could be responsible for adverse e ffects, such as CNIT, infections and myelotoxicity [38,39]. Furthermore, given the retrospective design of this study, the study beginning in the control group had a wide range from March 2017 until August 2018 and the time period from LTx to the beginning of the study was significantly increased compared with that in the intervention group. The longer Tac exposure in the control group might have had a negative influence on renal function in this cohort. However, at t0, the control group showed even higher eGFR values than patients converted to LCPT (70.6 ± 19.3 vs. 65.3 ± 21.1, respectively).

Another limitation of the study is that the reasons for conversion to LCPT in our study were taken only from the clinical reports from our Outpatient Transplant Clinic. In addition, in contrast to the case for KTx recipients, renal biopsy is not routinely performed in LT recipients which limits our ability to analyse CNIT before study onset.


**2. Materials and methods**

any change in the state of climate that is reflected in shifts in mean climatic variables over extended periods, typically decades or longer [3]. Climate change may result from natural internal processes within the climate system [4] or variations in natural or persistent anthropogenic external variability [5]. Climate results from fluctuations in the mean state or other climate statistics on temporal scales beyond those of individual weather events [6]. Climate change impacts, such as increased water shortages due to persistent droughts, present a threat to wildlife resources and consequently wildlife-dependent livelihoods in Africa. Thus, managing wildlife resources populations requires an understanding of the nature, magnitude and distribution of current and future climate impacts [7]. The term wildlife collectively refers to all forms of undomesticated flora and fauna found in terrestrial or aquatic environments [8]. In the present study, wildlife resources refer to faunal or floral species with potential benefit to human kind. Climate change has greater impacts on livelihoods of people in developing countries due to their low adaptive capacity [9]. However, this chapter focuses on terrestrial and semi-aquatic vertebrates, specifically mammals and their habitats. In Southern Africa, wildlife species promote the lives and livelihoods of local communities, particularly those living adjacent to protected areas [10]. Such communities generate revenue from activities such as eco-tourism, safari and consumptive hunting and bush meat trading. Therefore, there are huge economic losses associated with floods, droughts and wildfire. Against this backdrop, different mechanisms have been put in place to encourage climate change adapta-

2 Selected Studies in Biodiversity

tion and mitigation in the biodiversity sector at global, regional and national levels [11].

Africa, Southern Europe, Middle East, Southeast Asia and Australia [16].

The Intergovernmental Panel on Climate Change [12] projects a rise in surface temperature over the twenty-first century under all assessed emission scenarios. Global averaged temperatures are projected to increase by between 0.15 and 0.3°C per decade [13]. The frequency and intensity of heat waves are also likely to be more frequent and prolonged [14]. Many regions are likely to experience more frequent and extreme precipitation events. According to the Intergovernmental Panel on Climate Change (IPCC) [15], there is high likelihood that climate-related extremes, such as heat waves, droughts, floods, cyclones and wildfires, will expose some ecosystems, rendering them vulnerable to climate variability. Climate models project increased aridity and persistent droughts in the twenty-first century for most of

According to the Intergovernmental Panel on Climate Change [17], climatic changes are occurring at a faster rate than expected, particularly in Southern Africa. The IPCC predicts a 10 to 24% in mammalian species in sub-Saharan Africa national parks [13]. Climate change due to natural variability is therefore affecting terrestrial biological systems. Diffenbaugh and Field noted that there has been significant rise in temperatures of terrestrial ecosystems due to global warming [18]. Climate change directly affects ecosystems through seasonal changes in rainfall and temperature and indirectly through other disturbances such as fire and drought [11]. The IPCC [12] also notes that generally there is high confidence that several terrestrial, freshwater and marine species have shifted their geographic ranges, seasonal activities, migration patterns, abundances and species interactions in response to climate change. Based on available scientific literature, the IPCC Fourth Assessment Report (AR4) also reports that there is medium confidence that terrestrial ecosystems could have faced some impacts in Africa in recent decades due to climate change [12]. Additional threats from extensive land use and degradation, changes in frequency and severity of extreme events and interactions with other stresses [18] further threaten the resilience of terrestrial ecosystems. However, the This study adopted both quantitative and qualitative methods to obtain data on the effects of climate change and variability on mammalian species on selected national parks in Zimbabwe. We conducted a review existing literature (1980–2015) from peer-reviewed journal articles, books, edited book chapters, electronic academic theses, technical reports from Google Scholar, Scopus and Web of Science covering issues on climate change, climate variability and wildlife resources in Southern Africa. Data were also obtained from unpublished internal scientific reports and management plans from the Zimbabwe Parks and Wildlife Management Authority (ZPWMA) and other and external reports from policy documents related to biodiversity and climate change in Southern Africa. Technical reports and public publications from key stakeholders, such as the International Union for Conservation of Nature (IUCN), World Wide Fund for Nature (WWF) and African Wildlife Foundation (AWF), were also reviewed. The following keywords or phrases were used: "climate change," "climate variability," "impacts/effects," "wildlife," "mammals," "biodiversity," "ecosystems," "drought," "rainfall," "temperature," "herbivores," "global environmental change," "plants," "flora" and "fauna" with a combination of "AND" between the keywords to retrieve the relevant literature.

Secondary data on rainfall, temperature and large mammal surveys for the period 1960–2015 were also collected from published and unpublished reports from the Zimbabwe Parks and Wildlife Management Authority. Secondary data were either adopted or used to plot graphs showing large mammal trends in relation to rainfall and temperature. Data were collected between August 2016 and February 2017. Literature obtained from all the documents was categorised under the following themes: (1) historical trends in selected mammals in relation to climate variables (1980–2015), (2) predicted and observed impacts of climate change and variability on wildlife resources in selected protected areas in Southern Africa and (3) implications for conservation. For further analysis, case studies from Zimbabwe (**Figure 1**) were used to highlight impacts of climate change on wildlife resources.

**3. Results and discussion**

game hunting activities.

This section highlights predicted and observed trends in rainfall and temperatures across Southern Africa and their implications on wildlife resources. Impacts of climate change on selected wildlife species and some of the predicted and observed impacts of climate change and variability are subsequently highlighted for selected protected areas. Implications in

Impacts of Climate Change and Climate Variability on Wildlife Resources in Southern Africa...

http://dx.doi.org/10.5772/intechopen.70470

5

Global warming has caused a gradual reduction in annual rainfall across Africa over the past 50 years [24]. Projections of precipitation and runoff in Africa suggest a decrease of up to 10% in precipitation in most of Southern Africa (including Zimbabwe) by 2050 [28]. This reduction could reduce the distribution and availability of both food and surface water for animal species [24]. Rainfall is the main climatic factor governing herbivore population dynamics across Africa [29, 30]. African wildlife resources are therefore likely to be subjected to climate warming due to rising temperatures and extreme events, such as droughts and floods [27]. Thus, climate-induced extreme events threaten some of the large protected areas that have

The IPCC [3] predicts that Africa is projected to have 'above-average' climate change in the twenty-first century. Thus, global warming will have the greatest effects on biodiversity in the continent [32]. Chamaille-Jammes et al. [33] noted that climate change is predicted to affect both the mean annual rainfall and its seasonal distribution over the African continent. Thus, climate warming has a potential to directly affect wildlife resources through shifts in onset and duration of rainy seasons and drought on wildlife species, reduction in species distribution ranges, alteration in abundance and diversity of mammals, changes in calving and population growth rates, changes in juvenile survival of most ungulates and changes in species richness of birds and mammals [27]. Consequently, these changes in wildlife species abundance and distribution will have direct serious negative impacts on ecotourism and

Southern Africa is characterised by highly variable climatic conditions associated with fluctuating temperature and rainfall. The region is prone to frequent and intense El Nino-Southern Oscillation (ENSO) events, leading to widespread drought in some areas and widespread flooding in others [34]. According to the IPCC [12], mean seasonal temperatures are predicted to increase, and El Nino-Southern Oscillation effects, fires and severe weather anomalies are more likely to be more common in Southern Africa. Projections of precipitation and runoff in Africa suggest a decrease of up to 10% in precipitation in most of Southern Africa by 2050 [28]. Most of Southern Africa is prone to extreme events such as droughts and floods [35] and climate warming, which are the major climate change factors that are likely to affect wildlife resources in Southern Africa [27]. Climate can affect mammalian populations indirectly by excessive temperatures or rainfall, through bottom-up effects on food plant productivity or top-down effects on predator efficiency [36]. Bottom-up control mechanisms are based on the

view that herbivore populations are limited by forage quality and quantity [37].

terms of adaptation and mitigation for conservation are also discussed.

**3.1. Rainfall and temperature trends: implications for wildlife resources**

been designated to conserve much of Africa's magnificent biodiversity [31].

**Figure 1.** Location of the largest protected areas (used as case studies) in Zimbabwe.


**Conflict of interest**

No conflict of interest is present.

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**1. Introduction**

From the first generation (1G) that were introduced in 1979 by Nippon Telegraph and Telephone (NTT) to today's fifth generation (5G), mobile communication networks are constantly improving the speed and efficiency of bandwidth usage to support various applications with diverse requirements such as latency, high data rates and real-time support for random traffic demands [1].

The increasing number of not only smart phones, tablets and laptops but also the huge number of other devices such as IoT (Internet of Things) nodes, wearable devices for healthcare will demand significant challenges in 5G systems to manage a huge amount of devices and connections [2]. Besides, the exponential growth of mobile video services (e.g., live video streaming, online video gaming, mobile TV) requires wider bandwidth and higher spectral efficiency than that of 4G systems [3].

Such a huge volume of data traffic and connections will lead to 5G systems to use new and higher frequency bands [4]. Some other factors such as ultra-low latency (less than one millisecond), fast-tracking will also be considered in the design of 5G system architecture. 5G systems support radio connections and end-to-end network connectivity at ultra-high speed, lower latency, higher reliability and massive connectivity [5].


3.5.1. Effects of Unit Microstructure

Fatigue cracks generally occur at heterogeneous nucleation sites, such as inclusions, pores, or soft spots in microstructures [28]. It can be seen from Figure 15 above that there was more spheroidal graphite in the matrix of nodular cast iron. Similar to a hole, the graphite among the metallic matrix has no intensity. During thermal cycling, cracks are always initiated at the graphite phase, and the main crack often propagates along the graphite and matrix between the nearest graphite. Therefore, the presence of graphite reduces the thermal fatigue resistance of the material matrix to a certain extent. However, no graphite phase was found in the units, hence no bridge connection was produced between the main crack and the microcracks, whose growth depends on graphite, i.e., the crack propagation route was cut off by the unit. Therefore, the units had better thermal fatigue resistance and prevented cracks from propagating. In addition, laser remelting can refine the grains of the structure and improve the unit strength, which prevents crack propagation.

### 3.5.2. E ffects of the E ffective Size and Distance of Units

Schematic diagrams of di fferent-sized units which prevented crack propagation are shown in Figure 17 (Figure 17 A1–D1 is the sample without thermal cycling, and Figure 17 A2–D2 is the sample after 2000 thermal cycles). Since the units had similar surface areas, the depth determined the size of its cross-sectional area. A greater depth of a unit indicates a greater bridging degree of a crack and also a larger cross-sectional area of the strengthened unit; however, the existence of microcracks or holes in the unit reduces the e ffective depth and cross-sectional area. In addition, the existence of such defects greatly reduces the strength of the unit. Under the action of thermal stress, thermal fatigue cracks will continue to expand into the unit, preventing the unit from e ffectively locking cracks. Therefore, when the laser energy density was increased, the unit with a larger e ffective size completely bridged the cracks, and since there were no cracks or holes in the interior, it had a larger tensile strength and could withstand larger thermal tension caused by thermal cycling. This allowed it to greatly reduce the thermal tension on the cracks, thus e ffectively preventing cracks from propagating.

**Figure 17.** Schematic description for cracks blocked by units with di fferent sizes. ( **A**1–**D**1) before thermal cycles; ( **A**2–**D**2) after thermal cycles.

Since thermal stress was uniformly distributed on the crack, the total force of the thermal stress on the crack depends mainly on the crack length. The smaller the distance between adjacent units, the shorter the length of the crack that is blocked by the unit, and therefore the smaller the resultant thermal stress on the crack. In addition, a smaller unit spacing increases the number of units that can share thermal stresses. When subjected to the same magnitude of thermal stress, the crack in specimens with a smaller unit spacing was subject to less thermal stress, and the cracking speed was slower. In summary, a smaller unit spacing resulted in a better crack blocking e ffect.


**1. Introduction**

A phenomenon called serration—stress fluctuations in a saw-tooth shape—occurs when a uniaxial test (e.g., tensile test) is performed on aluminum alloys containing solute atoms in the solid solution [1–4]. Dynamic strain aging gives rise to the Portevin–Le Chatelier effect [3,5,6]. One of the manifestations of this effect is the serration that occurs when dislocations are pinned or released from the atmosphere of a solute atom. For example, in Al–Mg alloys (5000 series aluminum alloys), significant serrations are generated, as well as a strain pattern similar to a Luder's band on the surface of the alloy, thereby impairing its appearance. Many studies on the serration phenomena have been conducted, primarily involving the 5000 series aluminum alloys. Existing studies identified serration behavior as an interaction between dislocations and the solute Mg [3,5,6]. This phenomenon was reported to be affected by strain rate and temperature because the velocity of dislocation motion and the diffusion rate of Mg are affected by the strain rate and the temperature, respectively [3,5].

Previous studies have demonstrated that the serration behavior varies with change in the strain rate. The classifications of serrations are briefly summarized below [7]. Figure 1 shows an example of variations in the serration behavior with a change in strain rate [8]. It shows an A-type stress fluctuation that repeatedly rises and falls (the wavy stress fluctuation at a relatively high strain rate, . εA) and B-type saw-tooth-like stress fluctuations generated at intermediate strain rate (. εB, . εA+B). Serrations sometimes occur as a result of a combination of behaviors. For example, an A + B-type—a combination of A and B-type stress—has been reported. The serrations observed at low strain rates ( . εC) are classified as C-type stress (the CA and CB types depending on the frequency of fluctuation), in which the fluctuations are irregular. At a strain rate above . εA or below . εC, serrations do not occur, rather, a smooth stress–strain relationship is obtained.

**Figure 1.** Examples of different serration behaviors for different strain rate regimes [8].

To date, serrations have been evaluated primarily by uniaxial assessments such as compression and tensile tests. The strain rate in the uniaxial test is defined by the following equation:

$$
\dot{\kappa}\_u = \frac{d\_{\ell u}}{d\_t},
\tag{1}
$$

where . ε*u* is the strain rate and ε*u* is the strain during the uniaxial test. However, present authors [8] have, through experiment, proved that serration phenomena can be evaluated through indentation tests. Through continuous measurement of the load and the corresponding displacement, while loading and unloading in an indentation test, some mechanical properties that cannot be obtained through hardness tests could possibly be evaluated [9]. Therefore, the indentation test is also a vital non-destructive test for metals. The mechanical properties obtained through indentation test are evaluated using the loading curvature *C*, as shown below:

$$P = \text{Ch}^2,\tag{2}$$

where *P* and *h* are the load and displacement during indentation, respectively. The loading curvature is given by the following relational expression:

$$\mathbf{C} = f\begin{pmatrix} \mathbf{E}\_r \ \mathbf{Y}\_r \ n\_r \ \alpha\_r \ T\_r \ \dot{\varepsilon}\_i \end{pmatrix} \tag{3}$$

where *E* is Young's modulus, *Y* the yield stress, *n* the work hardening index, α the indenter angle, *T* the temperature, and . ε*i* the strain rate of the indentation. Doerner and Nix [10] proposed the following empirical equation for determining the value of . ε*i* using a triangular pyramid indenter with the same area to depth ratio as the Vickers pyramid:

$$
\dot{x}\_i = k \left( \frac{\dot{h}\_l}{h} \right) \tag{4}
$$

where *k* is the material constant and . *h* is the displacement velocity. This equation does not include the effect of indenter angle. It should be noted here that the concepts of the representative stress (σ*r*) and strain (ε*R*) for indentation analysis in order to normalize the load–displacement curves [11–13]. In the indentation test, σ*r* is the flow stress of the uniaxial test at a particular strain ε*r*. The representative strain is given the formula [13]:

ε*R* = 0.0638 cot α, (5)

where α is a half-apex angle. For example, ε*R* is approximated as 0.023 when using a conical indenter with a half-apex angle of 70.3◦ (described later). Therefore, Equation (4) expresses the strain rate at this representative strain value.

The . ε*i* has the same dimension as . ε*u*, however, the definition of strain rate for indentation tests differs from that of uniaxial tests. The strain rate of indentation is distributed inside the test materials in a complex manner [14]. Thus, existing studies have proposed the concept of effective strain rate, . ε*e*, to consider the effect of the distribution of the strain rate on the indentation [15–17]. The effective strain rate is given by the formula: .

$$
\dot{\varepsilon}\_c = \beta \left( \frac{\dot{h}}{h} \right) \tag{6}
$$

where β is a material constant. Equations (4) and (6) have the same form. However, it has been shown that the value of β correlates the strain rate in indentation with that in uniaxial tests [15–17].

Previous studies [8,18] indicated that the serration phenomenon in indentation could possibly be evaluated using the concept of effective strain rate. Indentation is performed using a sharp indenter, hence, a complicated deformation field is generated in the test material, whose deformation mechanism has only recently been clarified [19]. Through the indentation tests, it was also discovered that there is a test evaluation limit called critical strain [19] and that serrations could be used as an index to evaluate this effect [18]. However, these previous studies were conducted only in an ambient temperature environment, hence, the effect of temperature on the serration behavior during indentation was not investigated. There are many unknown factors that could affect the behavior of the serrations obtained from indentation.

Until now, the strengths of most metals are evaluated through uniaxial tensile tests. However, next-generation metals are expected to have micro- and nano-scale properties. Therefore, there is a need to adopt such tests as an indentation test that can non-destructively evaluate the strength of a small area with accuracy comparable to that of uniaxial tests. In this study, we extend our previous work [8,18] to clarify the effects of testing temperature on the serration behavior during indentation tests. The microstructural changes in the Al–Zn–Mg–Cu alloy (7075 aluminum alloy) due to natural and artificial aging were employed [20]. In addition, indentation was established as a new method of evaluating material properties through the evaluation of the serration behavior related to the microstructure.

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

#### *2.1. Specimen*

A 7075 aluminum alloy (hereafter referred to as the 7075 alloy) specimen was used in this study. Table 1 lists the chemical composition of the 7075 alloy. The dimensions of the cylindrical specimen were 40 mm (diameter) and 40 mm (height), and the end face was finished by lathing. The solution

treatment was conducted at 753 K for 3600 s, followed by water-cooling. Indentation tests were performed on this solution-treated specimen.

**Table 1.** Chemical composition of the investigated 7075 alloy (wt%).

#### *2.2. Indentation*

#### 2.2.1. Testing Conditions

A universal testing machine (Instron, series 5982, Norwood, MA, USA) attached with a jig was used for the indentation. Approximately 1 mm was loaded into the lathe-machined surface of the specimen as milli-indentation. A conical indenter (Figure 2a) made of a WC–Co superalloy was used. The radius of the tip (referred to as the roundness) of the indenter was 8.63 μm. The indenter angle was measured to be 141.02◦ using a laser microscope. This angle is almost equal to that of a conical indenter (140.6◦) that has the same indentation projection area at the same indentation depth as those of the Berkovich indenter (a triangular pyramid with a ridge angle of 115◦; see Figure 2b). In the existing studies, the effects of the roundness of indenters up to the initial stage of indentation (approximately 1 μm) were reported, where the roundness of the indenter was approximately 10 μm. However, the effect was obtained to be negligible when the indentation was higher than 1 μm [21]. In this study, we assumed an indentation of 1 mm. Therefore, the effect of the roundness of the indenter was minimal.

**Figure 2.** Schematic diagram of a cone-type indenter: (**a**) conical type and (**b**) Berkovich (triangular pyramid) type. Here α is the indenter angle and the shaded section is the projection area.

In this study, the temperature at which the Guinier–Preston (GP) zones and the η phase precipitate in 7075 alloys was adopted as the testing temperature (described later). First, a temperature of 343 K at which GP zones have been confirmed to precipitate (or nucleate) [20,22] was chosen. The η phase has often precipitated under the condition of aging at 393 K for 24 h (T6 temper). In this study, however, a temperature of 443 K at which the η phase has been reported to precipitate within a short time (600 to 3600 s) [20] was adopted. Figure 3 shows a schematic diagram of the indentation test at high temperatures. A home-built electric furnace was used. A thermocouple was attached to the specimen surface and was controlled to a predetermined temperature using a temperature controller (CHINO, SY2111, Tokyo, Japan). The arrival times at 343 and 443 K were approximately 1800 and 3600 s, respectively. The specimen was held for 900 s at each of the temperatures, after which indentation was performed. Furthermore, the specimen was held at 77 K in a liquid nitrogen where the low-temperature indentation was performed. At this temperature, aging takes place at a very low rate. Unlike the high-temperature test, the low-temperature indentation test was performed in a container using waterproof paper. The specimen was also indented at room temperature (293 K), thus, the test was performed in four different temperature environments. For each of the temperature conditions,

the test preparation was initiated within 300 s of the solution treatment. Therefore, the effect of natural aging was small, except at room temperature.

**Figure 3.** A schematic diagram of milli-indentation at high temperatures (343 and 443 K).

#### 2.2.2. Indenter Control

The control methods for indenters can be classified into two: loading rate and displacement rate controls. In this study, a commercial universal testing machine was used, hence, displacement rate control was employed. During indentation, the strain rate was varied by varying the displacement rate (see Figure 4). Previous studies have shown that serrations are being affected by the diffusion of Mg in the solid solution [5]. The time required for Mg atoms in the solid solution to sufficiently pin stagnant dislocations is given by the following equation [23,24]:

$$t\_a = \left(\frac{C\_1}{3C\_0}\right)^{\frac{3}{2}} \frac{kTb^2}{3DLI\_m},\tag{7}$$

where*C*1 is the concentration of solid solution atoms required for serrations to occur, *C*0 the concentration of solid solution atoms in the material, *k* the Boltzmann's constant, *T* temperature, *D* the diffusion rate, and *Um* the binding energy between the solid solution atoms and dislocations. When clusters and GP zones are formed by aging, the amount of Mg in the solid solution decreases, and accordingly, *C*0 decreases. *D* also increases as temperature increases [25,26]. It is difficult to measure the diffusion rate of solute atoms at low temperatures. Therefore, we predicted using the following equation:

$$D = D\_0 \exp\left(-\frac{Q}{RT}\right),\tag{8}$$

where *D*0 is the frequency factor, *Q* is the activation energy and *R* is the gas constant. Table 2 shows the prediction of the diffusion rate of solute atoms in aluminum at the testing temperature [27–29]. Hence, to promote the diffusion of solute atoms, a constant displacement is maintained. As shown in Figure 4, the displacement was held constant at two different values, for 20 s each, during the indentation test.

**Table 2.** Prediction of the diffusion rate of solute atoms in aluminum at the testing temperature using Equation (8).

**Figure 4.** A schematic diagram of the time history of displacement.

#### **3. Results**

Figure 5 shows the load–displacement relationships for all the tests at different temperatures. For the tests at temperatures above room temperature, the load increased with an increase in temperature regardless of the change in the displacement rate. This increase in the load is attributed to the precipitates formed by artificial aging. At 77 K, a decrease in the displacement rate lowered the increase rate of the load as the displacement increased as compared to the other temperatures.

The effective strain rates for the indentations were calculated using Equation (6). Herein, β = 0.1 was used, based on previous studies [8,18]; the effective strain rate–displacement relationship is shown in Figure 6. The effective strain rate under displacement rate control, given by Equation (6), decreased as the displacement increased. The indentations were performed at three different rates by varying the indenter speed (see Figure 4). A wide range of effective strain rates (from 10−4 to 100 s−1) was obtained during the indentations. There was no significant difference in the effective strain rate, even when the testing temperature changed. Aluminum alloys are known to have high strength–strain rate sensitivity at lower temperatures [30,31]. This implies that at extremely low temperatures, the strength of aluminum alloys decreases as the strain rate decreases. Therefore, during the cryogenic indentation, the decrease in the increase rate of the load was as a result of the decrease in the effective strain rate with increasing displacement.

**Figure 5.** Load–displacement curves at 77, 293, 343, and 443 K. The change in displacement rate during indentation is shown in Figure 4.

**Figure 6.** Effective strain rate–displacement relationship at each testing temperature.

We calculated the loading curvature-displacement relationship from the load–displacement relationship using Equation (2) (see Figure 7). The effect of temperature and strain rate on the indentation was confirmed by the change in the loading curvature. When testing temperatures greater than the room temperature, the loading curvature was observed to increase as the temperature increased regardless of the change in the displacement rate. At 293 and 343 K, an increase in the loading curvature was observed after holding as compared with that before holding. By contrast, there was a decrease in the loading curvature after holding for the test conducted at 77 and 443 K. At 77 K, not only the time in which the displacement rate increases but also the loading curvature decreases with increasing displacement.

**Figure 7.** (**a**) Loading curvature-displacement curves at 77, 293, 343, and 443 K; (**b**) enlarged view. The change in displacement rate during indentation is as shown in Figure 4.

#### **4. Discussion**

#### *4.1. E*ff*ect of Aging on the Material Strength*

The precipitation process of 7075 alloys established in previous research is as follows: [32–34].

Supersaturated solid solution (ssss) → vacancy-rich clusters (VRC) → GP zone → η → η. (9)

A cluster or GP zone is an aggregate of atoms with a diameter of the order of nanometres. Herein, η denotes the metastable phase, whereas η denotes the stable phase. Clusters and GP zones are formed during the natural and artificial aging, indicating an increase in material strength. After additional aging, η precipitates and the strength of the material reaches its climax (peak aged). However, as η continues to grow and η starts to precipitate, there is a decrease in the strength of the sample (over-aged). Therefore, increasing the testing temperature increases the strength as a result of the formation of precipitates until the peak-age condition is reached. The amount of solute atoms is also decreased. The succeeding sections discuss each testing temperature based on the above findings.

#### 4.1.1. Temperature of 77 K

In [15], when the load was held during indentation at room temperature, the loading curvature after holding was obtained to be higher than that before the holding. This is attributed to the fact that an amount of solute atoms in solid solution diffuses into the dislocations during the holding period, thereby, causing the pinning of dislocations. At 77 K, which is a very low-temperature environment, the above-mentioned precipitation process was not observed and the sample probably remained in a solid solution. Therefore, solute atoms were in the solid solution during the indentation. However, there was no increase in the loading curvature after holding. This indicates that solute atoms have not segregated (or diffused) to the dislocations and pinned them in this low-temperature environment because solute atom diffusion is very slow as shown in Table 2, i.e., no time for diffusion.

#### 4.1.2. Temperature of 293 and 343 K

In agreement with the results obtained in a previous study [18], an increase in the value of the loading curvature after holding was observed at 293 and 343 K as a result of dislocation pinning caused by the diffusion of solute atoms into the dislocations. At 343 K, the GP zone formed inside the material as a result of aging. However, a sufficient number of solute atoms to cause dislocation pinning was expected to be retained in the solution.

#### 4.1.3. Temperature of 443 K

In contrast to the results obtained in the tests conducted at 293 and 343 K, at 443 K, the value of the loading curvature after holding was smaller than that before holding. This could be attributed to the fact that the amount of solute atoms must have been significantly reduced by aging, hence, the dislocation pinning effect was less likely to occur.

#### *4.2. E*ff*ect of Testing Temperature on the Serration Behavior*

Figure 8 shows an enlarged view of the region of the curve that is indicated by the arrow in Figure 7b as a means to investigate the details of the loading curvature-displacement relationship. Serrations were observed at 293 and 343 K but not at 77 and 443 K. The effective strain rate was approximately 7 × 10−4 s−1 at all testing temperatures (see Figure 6). It has been stated that the serrations observed in uniaxial tests were affected by the strain rate and the testing temperature. To discuss the effect of testing temperature on the serration behavior observed in this study, the testing temperatures related to indentation were investigated.

**Figure 8.** Enlarged view of Figure 7b: (**a**) 77 K, (**b**) 239 K, (**c**) 343 K, and (**d**) 443 K.

#### 4.2.1. Temperature of 77 K

At 77 K, no fluctuation was observed in the loading curvature, as shown in Figure 8a, hence, serrations did not occur at this temperature. Because serrations occur at room temperature, it is assumed that the effect was not due to strain rate. This indicates that aging does not occur at very low temperatures. It also shows that there was barely any interaction between the dislocations and solute atoms.

#### 4.2.2. Temperature of 293 K

As shown in Figure 8b, there was a significant fluctuation in the loading curvature. The occurrence of B-type serrations was confirmed (see Figure 1). There was a large number of solid solution atoms in the sample as 293 K corresponds to the early stage of aging. Consequently, there was a tendency for interaction between dislocations and solute atoms in solid solution to occur. Thus, the occurrence of a significant number of serrations was confirmed.

#### 4.2.3. Temperature of 343 K

At 343 K, as shown by the arrow in Figure 8c, the interval at which the loading curvature dropped is greater than that at 293 K (see Figure 8b). Therefore, A- and B-type serrations (see Figure 1) were confirmed to have occurred. As the strain rate was constant, other causes of change in the serration phenomenon were observed. These are inferred to be the formation of GP zones, thus, a decrease in the number of solute atoms, and the increase in testing temperature. When the amount of solute atoms decreases, the chance of interaction with dislocations decreases, hence, serrations barely occur.

#### 4.2.4. Temperature of 443 K

No serration was observed at 443 K because of the formation of η and the increase in the strength of the material. The amount of solute atoms was, therefore, greatly reduced compared with other testing temperatures. Thus, the interaction between dislocations and solid solution atoms was less likely to occur. In addition, the diffusion rate of solute atoms at 443 K is higher than that at 343 K, hence, it is inferred that the remaining solid solution solute atoms were pinned to dislocations and deviates from the conditions at which serrations could occur. Therefore, it is believed that the interaction between dislocations and solid solution atoms does not appear in the loading curvature.

#### **5. Conclusions**

In this study, to clarify the effect of temperature change on the resulting serrations during indentation tests, we performed milli-indentations on an aluminum alloy (7075 alloy) at various temperatures. The serration phenomenon during indentation was varied by controlling the number of precipitated phases based on the effect of natural and artificial aging. This variation was as a result of the interaction between dislocations and the solid solution atoms observed under the different testing temperatures and strain rate on indentation, similar to that observed in previously reported uniaxial test results. Therefore, the serration phenomenon can be investigated via sharp indentation tests, which is considered valuable as a non-destructive testing technique for evaluating the dynamic strain aging of next-generation metals.

This study focused on the interaction between dislocations and the number of solute atoms based on the varying temperature and strain rate in indentation tests. Thus, the effect of the number of solute atoms and the testing temperature cannot be separated. Therefore, qualitative evaluations (e.g., microstructure evaluation by transmission electron microscopy or X-ray diffraction) to study the conditions that may separate these effects is recommended for future studies.

**Author Contributions:** H.Y. and T.K. conceived, designed, and performed the experiments; N.O. considered from research results; H.Y. and T.K. drafted this paper. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by The Light Metal Educational Foundation, Inc., Japan.

**Acknowledgments:** The authors would like to thank Hikaru Ootani for assisting with the conduction of the indentation test.

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

#### **References**

© 2020 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/).


**2.2. pH of the bath**

The effect of pH on particle incorporation is dependent on the nature of the particles. For instance, when MoS2 particles are used as the incorporate particles, the effect of pH value on the particle content in deposit was insignificant. Nevertheless it can be concluded that increasing pH decreased the amount of particles present in the coating [6]. However, incor‐ porated particles are significantly decreased when pH is below 2 in Al2O3-Ni coating pair [11]. In addition, decreasing pH is preferred to manage the internal stress. Low pH values, less than 5, are selected to obtain acceptable stress levels [10]. Increase in pH of the solution may lead to discharge of hydroxyl ions instead of nickel dissolution and oxygen evolution [10] resulted in high internal stresses. It is also concluded that the internal stress was increased when the pH was increased from 2 to 4 in MoS2 – Ni system [12].


*Trends in the Applications of Spongins*

The history of studies on the chemistry, molecular biology, biochemistry, and bioinspired materials science of spongins remains relevant today, partly due to the poorly understood basis of ecological disaster in the case of sponge diseases, but mostly due to recent progress in the direct applications of sponge skeletons as 3D spongin scaffolds in tissue engineering and biomimetics. Additionally, the marine ranching of bath sponges worldwide is a crucial factor in the adoption of spongins as renewable naturally prestructured proteinaceous scaffolds.

The spongin-based skeletons of bath sponges appear to possess a number of unique and useful properties, which had been exploited long before such scientific fields as tissue engineering and bioengineering were proposed. As reviewed by Szatkowski et al. [86], from the 18th century commercial bath sponges were valued in medicine due to their softness, high compressive strength, ability to retain shape, and high sorption rates. For these reasons, they were used as compression bandages for pressing open sinuses, in overcoming strictures of body passages (including the rectum), for dilation of the cervix uteri [86–90], and in the form of sponge tents applied in the uterus to expand the cavity and enable examination. More intriguingly, fragments of sponge skeleton were used as small prostheses in early "plastic surgery" [91]. Revolutionary results were obtained by Hamilton in 1881. In a paper entitled "*On sponge-grafting*" [92], he reported the following case. A woman underwent surgery for removal of a mammary tumor, during which a large area of skin was removed. The skin was replaced with a thin slice of an aseptic sponge skeleton, which ten days after the surgery was observed to be vascular, and three months later was covered with epithelial tissue (Figure 5).

**Figure 5.** Sketch of a fragment of spongin framework (**b**) surrounded by a great number of living cells (**a**,**c**) in a sponge-grafting application (adapted from [92]).

Today, spongin-based scaffolds are actively used in diverse applications related to tissue engineering. Positive results have been reported with human osteoprogenitor cells on the skeleton of *S. officinalis* [46], with osteoblast-like MG-63 cells growing on spongin from *Hymeniacidon sinapium* [93] and with mouse primarily osteoblasts on spongin from *Callyspongiidae* marine demosponges [49]. Recently, Nandi et al. [51] have proposed that the skeleton of the marine sponge *Biemna* sp.—alone and in combination with growth factors—is a promising biomaterial for bone repair and bone augmentation.

Besides applications in the biomedical field, spongin-based scaffolds have been successfully used as adsorbents of diverse dyes [94,95] and as supports for enzyme immobilization [96]. It was recently shown that spongins are thermostable up to 260 ◦C [86,97,98]. This property opens the door for applications of spongin-based scaffolds with 3D architecture in such novel scientific disciplines as Extreme Biomimetics [99], with the aim of developing novel advanced composite materials.

#### **3. Collagen IV and Related Proteins in Sponges**

It is now well established that collagens are key to the structural integrity and biomechanical properties of various tissues of Metazoans.  Collagen IV networks have a polygonal architecture that endows basement membranes (BMs) with a tensile strength sufficient to protect tissues from mechanical stress, in addition to serving as important regulators of the dynamic events associated with cell adhesion, signaling, and survival [100]. According to the modern view [101], only the presence of the collagen IV gene was precisely correlated with the emergence of BMs in animals. Thus, the triple helical collagen IV was required for the development of BMs.

BMs underlie the epithelia in Metazoa from sponges to humans [102]. Interestingly, until 1996, basement membrane structures and type IV collagen were known to be present in all multicellular animal species except sponges. In Porifera, BMs are associated with the basal surfaces of polarized epithelial cells [103]. After the first report on the identification of type IV collagenous sequences in the homoscleromorph sponge *Pseudocorticium jarrei* by cDNA and genomic DNA [103], this collagen has been found in diverse poriferans. For example, in corresponding transcriptome data from a calcareous sponge (*Sycon coactum*) and another homoscleromorph sponge (*Corticium candelabrum*), two new type IV collagen genes were found in each [104]. Homologs of important components

of basement membrane genes, including type IV collagen, have been found in the Demospongiae *Spongilla lacustris*, *Ircinia fasciculata*, and *Chondrilla nucula* [105]. The discovery of type IV collagen in Calcarea and Demospongiae is very important, because nowhere in this group has a BM-like structure been noted. The presence of type IV collagen in glass sponges (Hexactinellida) remains to be detected. Polyclonal antibodies have detected type I (but not type IV) collagen in the anchoring spicules of the *Hyalonema sieboldii* glass sponge [3].

The relationship between type IV collagen and the so-called spongin short chain collagen (SSCC) [106] is still under investigation [101]. SSCC has been considered as ancestral to type IV collagen [107]. Like type IV collagen, SSCC also has NC1 domains which produce the globular heads particular to type IV collagen and which are required for assembly of the unique scaffold of the BM (see for review [104]). It is suggested that collagen IV and its spongin variant are primordial components of the extracellular microenvironment, where collagen IV especially was a key player in the evolution of epithelial tissues in Metazoa, including sponges, due to the transition to multicellularity [101].

Interestingly, collagen IV from the demosponge *Chondrosia reniformis* has recently been patented as a source of special membranes for biomedical applications [108]. The collagen was isolated with an extraction solution of 100 mM Tris-HCl, 10mM EDTA, 8 M urea, and 100 mM 2-mercaptoethanol, rendering the protein in the form of a precipitate. This was used for the development of stable and non-cytotoxic type IV collagen membranes, which can be applied in tissue engineering and regenerative medicine approaches for epithelial repair, regeneration, or replacement. The technology includes the re-epithelialization of any single and stratified epithelium, with emphasis on the skin.

#### **4. Fibrillar Collagens in the Mesohyl of Demosponges**

The mesohyl includes a noncellular colloidal mesoglea with embedded collagen fibers, spicules, and various cells, being as such a type of mesenchyme. It is currently debated whether the mesohyl and pinacoderm layers in sponges are true tissues [109]. Collagens serve several functions in sponges [27,106,110]. The formation of mesohyl certainly involves the activity of fine fibrils made of fibrillar collagen. The collagen fibrils both mediate cell–matrix interactions via membrane receptors and provide the structure of the extracellular matrix (ECM), a situation observed in vertebrates. The increase in the structural diversity of fibrillar collagen chains, their different forms of maturation, and interactions with other ECM components appeared during the process of evolution [111]. The diversity of sponges which contain high amounts of fibrillar collagen within their mesohyl has been described previously (see for review [110–112]).

Fibrillar bundles, formed by the association of several hundred collagen fibrils, have been observed in diverse species of *Tethya*, *Chondrosia*, *Chondrilla*, *Jaspis*, and *Suberites* (see for details [112,113]). The densely packed bundles of collagen fibrils are secreted exclusively by the highly polarized lophocyte cells [43,111]. These are actively moving cells, pulling behind them a bundle of regularly arranged collagen fibrils.

Another kind of collagen-producing cell has been discovered in the mesohyl of the demosponge *Suberites domuncula* [114,115], in which the expression of collagen genes is controlled by silicate and myotrophin [116]. SEM observations have revealed the complex collagen network surrounding the spicules within the mesohyl of adult specimens (Figure 6).

Collagen fibers have also been identified in the mesohyl of the demosponge *Haliclona rosea* [116]. Collagen has also been reported in the mesohyl of such Calcarea sponges as *Leucosolenia* sp. and *Leucandra* sp. [117].

Collagen fibril content is also high in the external asexual buds that occur in *Tethya lyncurium* [118]. Similarly, the buds of *T. sychellensis* contain a dense collagen matrix [119]. Buds consist of cellular masses that sprout out from the surface of adults and are able to develop into new functional individuals [119].

Recently, special attention has been focused on fibrillar collagens in the mesohyl of *C. reniformis*. This species is the only sponge which has been experimentally proven to contain a dynamic collagenous

mesohyl capable of stiffening upon being manipulated [120]. It was shown that the different physiological states recorded in laboratory experiments are expressions of the mechanical adaptability of the collagenous mesohyl of *C. reniformis*, and suggest that stiffness variability in this sponge is under cellular control [121].

**Figure 6.** SEM view through the collagenous mesohyl of the demosponge *S. domuncula*. Layers of collagen fibrils (**A,B**) are a result of the activity of the unique collagen-producing cells which are seen to line up along the surface of the spicules (**C**–**E**). The line of cells (**A**) can move from left to right along the spicule, depositing a rough, nanofibrillar collagenous layer in their wake (**C**) (see also [114]).


*<IP_ADDRESS>. Direct cortical stimulation (DCS)*

For this technique, electrodes are applied directly to the cortical/subcortical surface. Direct cortical stimulation (DCS) to identify the primary motor cortex (PMC) is accomplished using paired electrodes. Stimulation is performed using 4–6 pulse trains at 500 Hz (the reason we denote this paradigm high frequency; this technique is also known as multipulse, which is misleading), with biphasic pulses of 150–200 μs in the duration/phase. Motor evoked poten‐ tials are assessed using pairs of subdermal needles spaced approximately 2 cm apart that are inserted into the contralateral muscles, but surface electrodes attached to the skin can also be used. Depending on the location of the tumour, it is customary to use the following muscles: the orbicularis oculi, orbicularis oris, deltoid, brachial biceps, extensor digitorum carpal flexor, abductor pollicis brevis, abductor digiti minimi, quadriceps, tibialis anterior and abductor hallucis.

Stimulation is initiated at 4 mA and increased continuously in increments of 1–2 mA until a stable compound muscle action potential (CMAP) is recorded, at a minimum amplitude of 30 μV or until an upper limit of 30 mA is achieved without eliciting a CMAP [5,6].

An alternative strategy entails the use of Ojemann's stimulation or low-frequency stimula‐ tion, which consists of a 50–60 Hz train that is 3–5 seconds in length and has a pulse width as high as 0.5 ms [37,38].

Although a systematic comparison between both strategies remains to be performed, it is important to be aware that neither electrical thresholds nor muscle response or electrical safety are equivalent.

In this sense, it is important at this point to consider some electrophysiological variables concerning patient safety. The effect of any type of electrical stimulation over the neural tissue is mediated by the total amount of charge applied to the system and the duration of applica‐ tion [39]. The electric current (*i*, in mA) is defined as follows:

$$i\left(t\right) = \frac{dq}{dt}\tag{8}$$

where *q* is the charge (in μC) and *t* is time (in ms). Thus, the total charge applied during time

*tpw* (time of pulse width) can be calculated from Eq. (8) as *q*(*t*)= *∫* 0 *t pw i*(*t*)*dt*. For square pulses (which are the most common), the integral equals the amplitude × duration, e.g.:

$$q\left(t\right) = \mathbf{i} \times \mathbf{t}\_{\text{pw}}\tag{9}$$

However, this expression only provides information about the charge/pulse. Therefore, to elucidate the total charge administered to the tissue (*q*total), we must multiply by the number of pulses (*N*) as follows:

$$q\_{\text{total}} = \mathbf{i} \times \mathbf{t}\_{pw} \times \mathbf{N} \tag{10}$$

Another relevant feature concerning safety is the maximum charge density (*ρ*max), which is defined as *q*max/*A* (μC/cm2 /phase), where *A* is the area (usually in cm2 ). This parameter directly depends on the size and shape of the stimulation electrode.

A comparison of these magnitudes is provided in **Table 1** for three different stimulation paradigms.

HF, high frequency.

In some tumours located in the brainstem, cervical or upper thoracic cord, it can be useful to use a D wave recording rather than MEP. This wave reflects the travel wave of CNAP from the lateral cortico-spinal tract and must be recorded through electrodes placed in the imme‐ diacy of the spinal cord [32]. This technique is very useful because it can be performed under total neuromuscular relaxation and elicited by only one pulse. However, it cannot be used for vascular pathology of the spinal cord and must be cautiously considered when tumours are

There is a false dispute, in our opinion, regarding the superiority of constant-current over constant-voltage stimulation. Of course, there are benefits and flaws of both techniques, but safe and reliable monitoring can be performed using voltage stimulation. Moreover, using a current of 61 mA with a pulse width of 500 μs [35], the total charge applied is 30.5 μC. However, a current of 340 mA over 50 μs [36] supplies a charge of 17 μC, which is approximately half of

For this technique, electrodes are applied directly to the cortical/subcortical surface. Direct cortical stimulation (DCS) to identify the primary motor cortex (PMC) is accomplished using paired electrodes. Stimulation is performed using 4–6 pulse trains at 500 Hz (the reason we denote this paradigm high frequency; this technique is also known as multipulse, which is misleading), with biphasic pulses of 150–200 μs in the duration/phase. Motor evoked poten‐ tials are assessed using pairs of subdermal needles spaced approximately 2 cm apart that are inserted into the contralateral muscles, but surface electrodes attached to the skin can also be used. Depending on the location of the tumour, it is customary to use the following muscles: the orbicularis oculi, orbicularis oris, deltoid, brachial biceps, extensor digitorum carpal flexor, abductor pollicis brevis, abductor digiti minimi, quadriceps, tibialis anterior and abductor

Stimulation is initiated at 4 mA and increased continuously in increments of 1–2 mA until a stable compound muscle action potential (CMAP) is recorded, at a minimum amplitude of 30

An alternative strategy entails the use of Ojemann's stimulation or low-frequency stimula‐ tion, which consists of a 50–60 Hz train that is 3–5 seconds in length and has a pulse width as

Although a systematic comparison between both strategies remains to be performed, it is important to be aware that neither electrical thresholds nor muscle response or electrical safety

In this sense, it is important at this point to consider some electrophysiological variables concerning patient safety. The effect of any type of electrical stimulation over the neural tissue is mediated by the total amount of charge applied to the system and the duration of applica‐

μV or until an upper limit of 30 mA is achieved without eliciting a CMAP [5,6].

tion [39]. The electric current (*i*, in mA) is defined as follows:

located near the anterior horns.

218 Neurooncology - Newer Developments

the total charge supplied with longer pulses.

*<IP_ADDRESS>. Direct cortical stimulation (DCS)*

hallucis.

high as 0.5 ms [37,38].

are equivalent.

\*The surface is calculated from a 1.3 mm diameter disk electrode.

\*\*The surface is calculated from a 1 mm diameter spherical electrode, assuming that only 1/4th of the surface is in contact with the cortex.

**Table 1.** Comparison of magnitudes.

Although there are no well-defined limits forthe above-mentioned magnitudes, from the table we can observe that Ojemann's technique is the paradigm with the highest *q*total/train, and the stimulation for awake craniotomy has the highest *ρ*max.


Acknowledgements

This project was undertaken with the financial support of the Government of Canada, Federal Department of Environment and Climate Change, the Research and Development Corporation (RDC) of Government of Newfoundland and Labrador (now InnovateNL), and Natural Sciences and Engineering Research Council of Canada (NSERC) under Grant to B. Salehi (NSERC RGPIN-2015-05027). The SAR imagery was provided by the Canada Center for Mapping and Earth Observation and Environment and Climate Change Canada. Field data were collected by various organizations, including Ducks Unlimited Canada, Government of Newfoundland and Labrador Department of Environment and Conservation, and Nature Conservancy Canada. The authors thank these organizations for the generous financial supports and providing such valuable datasets.


*Review* **Dual Antiplatelet Therapy with Parenteral P2Y12 Inhibitors: Rationale, Evidence, and Future Directions**

**Giulia Alagna 1, Paolo Mazzone 2, Marco Contarini 2 and Giuseppe Andò 1,\***

**Abstract:** Dual antiplatelet therapy (DAPT), consisting of the combination of aspirin and an inhibitor of the platelet P2Y12 receptor for ADP, remains among the most investigated treatments in cardiovascular medicine. While a substantial amount of research initially stemmed from the observations of late and very late stent thrombosis events in the first-generation drug-eluting stent (DES) era, DAPT has been recently transitioning from a purely stent-related to a more systemic secondary prevention strategy. Oral and parenteral platelet P2Y12 inhibitors are currently available for clinical use. The latter have been shown to be extremely suitable in drug-naïve patients with acute coronary syndrome (ACS), mainly because oral P2Y12 inhibitors are associated with delayed efficacy in patients with STEMI and because pre-treatment with P2Y12 inhibitors is discouraged in NSTE-ACS, and in patients with recent DES implantation and in need of urgent cardiac and non-cardiac surgery. More definitive evidence is needed, however, about optimal switching strategies between parenteral and oral P2Y12 inhibitors and about newer potent subcutaneous agents that are being developed for the pre-hospital setting.

**Keywords:** dual antiplatelet therapy; P2Y12 inhibitors; acute coronary syndrome; clopidogrel; prasugrel; ticagrelor; cangrelor; selatogrel; zalunfiban