**3. Routine use of VS in elective and emergency surgeries**

VL and DL have been used for decades in both elective and emergency intubations. The introduction of VL has shifted the paradigm of airway management [50]. VS was first introduced by Shikani in patients (both adults and children) undergoing routine otolaryngologic procedures [6] and has since been applied in various clinical scenarios, including difficult and emergency airway [12, 51]. The VS technique has been studied in adults in supine position [52–56], in lateral decubitus position [57], and in pediatric patients with difficult airway [30, 58]. It has also been used during awake intubation [53, 59] and for diagnostic bronchoscopy [60]. VS has also been used by emergency room physicians [61] and emergency medical technicians in the prehospital airway management [62].

#### **Figure 11.**

*Presence of mucus and saliva interfering with laryngeal views obtained by VS technique during tracheal intubation. A morbidly obese 42-year-old woman with BMI 46.6 kg/m2 (165 cm, 127 kg) underwent fasciotomy and debridement. Past history included sleep apnea and snoring. LQS score grade 1. The intubation time (for demonstration) is 22 s. (Also see the Supplementary Materials Video S9).*

In 2010, VS was introduced into our department. At that time, most of our staff still used DL and some tried VL (e.g., GlideScope®) [63, 64]. We established a formal airway training course in DL, VL, and VS use for the staff and novice operators using cadaver and mannequins. Currently, we have more than 22 sets of VS from four different brands in our 21 operating rooms. Although it is not mandated to use VS, VS prevails among all the available intubating tools we have. We regularly survey the novice users and trainees to evaluate their performance and obtain feedback. Notably, VS is used in more than 90% of tracheal intubations (**Table 2**).

**Figure 12** demonstrates the routine use of VS for tracheal intubation in an elective surgery. We have previously reported on the application of VS in patients undergoing an emergency surgery during the COVID-19 pandemic [65–70] (see **Figures 13** and **14**). The coverall personal protective equipment (PPE) and plastic sheet barrier did not interfere with smooth tracheal intubation using VS in COVID-19 patients. In both clinical scenarios (**Figures 12**–**14**), VS technique provides a swift, and safe tracheal intubation, while protecting the intubating provider from secretions as he/she does not have to put his/her face close to the patient's mouth.

**Table 3** summarizes the strengths and weakness of VL and VS. The clinical performance of VS is usually evaluated in many aspects, including (1) insertion in the oropharynx; (2) visualization of the epiglottis; (3) advancement in the glottic aperture; (4) maneuverability of the stylet; and (5) adverse events such as dental trauma, soft tissues damages, autonomic overstimulation, aspiration, hypoxia, etc. In both normal airway and difficult airway scenarios, VS shows its advantages with shorter intubating time [13, 71–74], less autonomic stimulation [55], and shorter learning curve [75].

#### **Figure 12.**

*A typical case of routinely applying VS technique for tracheal intubation in elective surgery. An 89-year-old woman with BMI 25.3 kg/m2 (150 cm, 57 kg). The intubation time (from lip to trachea) is 7 s. (Also see the Supplementary Materials Video S10).*

#### **Figure 13.**

*A typical case of routinely applying VS for tracheal intubation during emergency surgery. A 26-year-old woman with BMI 21.3 kg/m2 (150 cm, 48 kg) underwent an emergent orthopedic surgery due to multiple trauma during COVID-19 pandemic in 2020. Combined use of VS and a piece of plastic sheet as a protective barrier was noted. The two anesthesiologists wore PPE during tracheal intubation procedure. The anesthesia assistant was performing jaw-thrust maneuver. (Also see the Supplementary Materials Video S11).*

#### **Figure 14.**

*Same patient as in Figure 13. Close-up views from the VS video LCD screen. (A and B) Views of oropharynx and larynx. (C) Epiglottis. (D and E) Views of glottis and vocal cords. (F) Entry into trachea. The intubation time was 12 s. (Also see the Supplementary Materials Video S12).*

*the degree of relative comparison between use of VL and VS.*

#### **Table 3.**

*Comparison between videolaryngoscopes (VL) and video-assisted intubating stylet (VS).*


**4. Conclusions**

In conclusion, this article provided a novel concept of two-step control of the release rate of pH-sensitive drugs. Additionally, the results of the drug in vitro release profiles proved that both the dissolution-rate controlling step created by the sub-layer containing CA and the diffusion-rate

controlling step developed by the ADEC coating showed significant effects on the release rate of LXP. In addition, the amount of the acid modifier in the optimal formulation, which accounted for only approximately 3% of the total preparation weight, was dramatically decreased compared with other formulations containing acidic modifiers. The in vivo studies revealed that this novel two-step control system could achieve a more stable and sustained release plasma concentration of LXP compared with the immediate release tablet.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1999-4923/11/6/260/s1, Figure S1: pH solubility profiles of loxoprofen at 25 ◦C. Figure S2: Contour plots showing the effects of (**A**) *X*1 and *X*2, (**B**) *X*2 and *X*3, and (**C**) *X*1 and *X*3 on the response *Y1*. Figure S3: Contour plots showing the effects of (**A**) *X*1 and *X*2, (**B**) *X*2 and *X*3, and (**C**) *X*1 and *X*3 on the response *Y*2.

**Author Contributions:** Conceptualization, D.W. and L.L.; methodology, D.W., M.Z., J.Z.; and L.L.; formal analysis, L.L.; investigation, D.W.; writing—original draft preparation, D.W. and M.Z, writing—review and editing, D.W. and M.Z.

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

**Acknowledgments:** We are thankful to Colorcon Coating School® for technical and excipient supports.

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


**2.2 Piezoelectric pump with single chamber and a bimorph actuator**

As the name implies, the bimorph actuator is composed of two PZT disks with mechanical serial and parallel structure. **Figure 5** shows the structure of a piezoelectric pump with single chamber and a bimorph actuator (PPSCBA). The working principle of PPSCB is similar to that of PPSCSW in **Figure 1**. However, compared with single wafer piezoelectric pump, double piezoelectric actuators usually have symmetrical structures. In theory, PPSCBA can monitor the output performance by using a PZT disk as a monitor, which will make it more widely application.

In order to study the PPSCBA, the three-dimensional structure and prototype of PPSCBA have been designed, as shown in **Figures 6** and **7**. A bimorph is used as the actuator and sensor. The check valve is an umbrella valve made of rubber.

**Figure 4.** *The relationship between flowrate and driving frequency and voltage [12].*

**Figure 5.** *The structure of PPSCBA [13].*

**Figure 6.** *The three-dimensional structure of PPSCBA [13].*

*Design, Characterisation and Prospect of Piezoelectric Microfluidic Technology DOI: http://dx.doi.org/10.5772/intechopen.98559*

**Figure 7.** *The prototype of PPSCBA [13].*

#### **Figure 8.**

*The relationship between flowrate and driving frequency and voltage [13].*

The material of pump body is PMMA. The overall dimension of PPSCBA is 40 mm × 40 mm × 17 mm. When one PZT disc is used as the driver and the other as the monitor, the monitor of output flow rate is achieved.

As shown in **Figure 8**, when one piezoelectric wafer of the bimorph piezoelectric vibrator is used as the driver and the other as the sensor, the relationship between the output flow rate and the sensing voltage of PPSCBA and the driving frequency and voltage is analyzed. When the driving voltage is fixed at 150 Vpp, the peak values of sensing voltage and output flow, 6.88 Vpp and 3.12 ml/min, are obtained at 21 Hz. Therefore, the optimal frequency of PPSCBA is able to realize self-sensing. When the driving frequency is fixed at 21 Hz, the flow rate and sensing voltage increase with the increase of driving voltage, and the increasing trend is similar. When the driving voltage increases to 300 Vpp, the output flow rate and sensing voltage reach the maximum, which are 28.71 ml/min and 28.0 Vpp respectively. Therefore, PPSCBA can realize the self-sensing of output flow rate.

#### **2.3 Piezoelectric pump with single chamber and double piezoelectric actuator**

In order to improve the drive ability of pump chamber, the piezoelectric pump with single chamber and double piezoelectric actuator (PPSCDPA) is proposed. **Figure 9** shows the structure of PPSCDPA. PPSCDPA consists of an inlet, two

#### *Piezoelectric Actuators - Principles, Design, Experiments and Applications*

**Figure 9.** *The structure of PPSCDPA [14].*

#### **Figure 10.** *The 3-D structure of PPSCDPA [14].*

**Figure 11.** *The prototype of PPSCDPA [14].*

#### *Design, Characterisation and Prospect of Piezoelectric Microfluidic Technology DOI: http://dx.doi.org/10.5772/intechopen.98559*

piezoelectric diaphragm, two check valves, a pump chamber, a pump body and an outlet. The vibration of the two piezoelectric diaphragms is the same, that is, their driving voltage signals are the same. In the process of suction, two piezoelectric diaphragms bend out of the chamber at the same time; while discharging, the two piezoelectric diaphragms bend to the chamber at the same time. Combined with the unidirectional characteristic of the check valve, the PPSCDPA can realize the one-way transportation of fluid from the inlet to the outlet under the AC voltage signal drive.

The three-dimensional structure and prototype of PPSCDPA with the overall dimension of 65 mm × 40 mm × 12 mm are shown in **Figures 10** and **11**, respectively. The check valves are umbrella valve made of rubber. The material of pump body is PMMA. The performance test platform of PPSCDPA is established, and the output performance of piezoelectric pump is tested. The experimental results show that the maximum flow rate of 45.98 ml/min is achieved when the driving parameters are 15 Hz and 200 Vpp.


*<IP_ADDRESS> Two-stage horizontal spray dryer*

A horizontal spray dryer has been suggested as an alternative to the conventional vertical types [57, 58] for pharmaceutical materials and heat-sensitive food [59, 60]. The low temperatures of drying permit flavor reservation, controlled porosity and density, high solubility, as well as fine goodness agglomerated products. Essential energy provisions relative to the conventional vertical spray dryer with a lower electrical loading for a specific capacity.

A suggested design is described in **Figure 15**. The high-pressure pump is used for feed pushing to the spray nozzles. These nozzles are organized in several configurations. Then, the spray travels horizontally from the nozzles, and due to gravity, it goes down. After that, transporting of the dried powders using a conveying strap in the bottom and carried to bag filters or cyclones. A model for the horizontal spray dryer (6.0 m 3.0 m 3.0 m) has been presented by Cakaloz et al. [61].

In the single-stage horizontal spray dryer process (**Figure 16**), it has been shown that the dwell period might be overly short for allowing the droplets for drying completely. Therefore, to overcome this constraint, a new two-stage, two-dimensional horizontal spray dryer idea is suggested by Mujumdar [58] and shown in **Figure 15**,

#### **Figure 15.**

*Proposed layout of a two-stage horizontal spray dryer.*

#### **Figure 16.**

*Schematic layout of the horizontal spray dryer.*

which is prepared for permitting long drying periods. This is necessary for big droplet sizes and heat-sensitive products. The variance between single and two-stage horizontal spray driers is that in the single spray drier, there is a fluid-bed dryer, which is installed at the horizontal chamber bottom. Whereas, the two-stage horizontal spray drier is not commercialized yet. The CFD process could be utilized to emulate the horizontal spray dryer, which should be connected to a fluid-bed drying system. The droplet's surface moisture is taken away rapidly from the chamber of spraying. While the inner water occupies longer, which could be extracted in a thin layer, out of a deliberation dryer in the bottom of the room.

## *<IP_ADDRESS> Low-humidity spray drying*

On occasion, it has to choose freeze dryers instead of spray dryers, particularly for pharmaceuticals and biochemicals drying. This is because spray drying utilizes hightemperature gas like the medium of drying and the sediments on the wall cannot be averted in feasible spray dryers. The products are subject to degradation by

*Drying Technology Evolution and Global Concerns Related to Food Security… DOI: http://dx.doi.org/10.5772/intechopen.109196*

**Figure 17.**

*Schematic layout of the low-dew-point spray dryer.*

overheating [48]. Yet, the freeze dryer has high operating, and capital costs, as well as high energy consumption, compared with the spray dryer [62, 63].

To decrease the active compound degradation in the dried powders, a novel kind of spray dryer was proposed, such as low dew point (LDP) spray dryer. This type utilizes air at a nearby surrounding temperature of 80°C and quite minimum humidity (**Figure 17**). It comprises four units: the preprocessing system of drying air including heating and dehumidification; the preparation system of feed; the drying system and feed atomization including drying chamber, air disperser, and atomizer, etc., in addition to the system of product collection.

#### *<IP_ADDRESS> Spray freeze drying*

The freeze drying (FD) is a significant drying machinery for pharmaceuticals, foods, and biochemicals, which could preserve the biological activity, aroma or flavor, etc. Freeze drying is a drying method, in which the solvent in the material is solidified at low temperatures, then sublimates directly from the solid into the vapor state under vacuum. The freeze-drying process of an aqueous solution contains three phases: product freezing, ice sublimation, and removal of solvent vapor. Nevertheless, compared with spray drying, the freeze drying is an order-of-magnitude costlier drying method because of its need for vacuum, refrigeration, and long operating times [64]. In a recent combination of both drying processes, called spray freeze drying (SFD), this process was carried out as a batch method as follows: The liquid nitrogen is atomized in a cryogenic medium to spray the droplets to freeze. Frozen droplets are scuttled in a cryogenic medium and processed out and dried in the freeze-dryer under a vacuum. A model flow slab is illustrated in **Figure 18**. This method is conducted in batches instead of continuously, because of the required long freeze-drying time. In addition, it is appropriate for high-significant products with low tonnages [65].

A new method called a combined atmospheric spray and fluidized-bed freeze drying (ASFBFD) process was examined. The planned schematic flow graph is described in **Figure 19**. It mainly comprises internal fluidized bed, drying or freezing room, liquid nitrogen cooler, internal bag filter, fan, nozzles, valves, pipes, pump, etc. [65].

The process is briefly explained as follows: The feed is transported into the nozzle from the top through a pump, where it is atomized using a nozzle. The fine spray is

**Figure 18.** *A schematic flowchart of the conventional spray freeze drying.*

**Figure 19.** *Batch-type atmospheric pressure combined with spray and fluidized-bed dryer.*

connected with freeze nitrogen or air immediately. Upon the material of feed, the drying medium is frozen to about 90°C using liquid nitrogen and inflated by a fan within the fluidized-bed cribriform tray. The fluidized bed is placed at the bottom of the chamber. As the temperature of the cool air is lower enough for spray freezing, the frozen atoms conserve their main conditions and go to the bottom or to the fluidized bed through gravity. Nearly a few frozen atoms might be restricted by air or nitrogen; nonetheless, they are detached from the air using the interior bag filter. Throughout the phase, the spray freezing drying is investigated continuously until the exhaustion of feed in the batch procedure. After this phase is completed, suitable drying terms are chosen, such as liquid nitrogen, which is regulated to meet freeze-drying terms. Drying actual operation and its time terms should be specified. Many investigators have already investigated many studies on atmospheric freeze drying [66–72]. These reviews showed that the atmospheric spray and fluidized-bed freeze drying grouping is a viable new procedure that needs additional R&D. Lastly, a summary of the comparison between the four drying processes, such as SD, FD, SFD, and ASFBFD, is assumed in **Table 1**.

#### *<IP_ADDRESS> Encapsulation*

Spray drying and fluid-bed drying lead to another common food application of these technologies, that is, microencapsulation. It is defined as a process by which one material or more is entrapped within alternative material [73]. This method is generally used to prevent the core material from degradation and to control releasing or separate reactive constituents in a formulation. Because the spray dryer is generally fast, available, economical and produces good-quality products [74], it enhances the

*Drying Technology Evolution and Global Concerns Related to Food Security… DOI: http://dx.doi.org/10.5772/intechopen.109196*

#### **Table 1.**

*Drying method properties [65].*

most public means of encapsulation process. This procedure is simple and comparable with the one-phase spray drying technique. The coated substance is named active or core substance, and the coating substance is entitled a shell, while the wall substance is called a carrier or encapsulant [75]. The encapsulated active substance is scattered in the hydrocolloid transporter, such as maltodextrin, dextrin, Arabic gum, gelatin, and modified starch. Subsequently, the emulsifier is supplemented, and the mix should be homogenized to compose an oil-in-water emulsion, then it's consumed by the atomizer for the spray dry. In the drying room, the aqueous stage dries, and the active substance is captured as atoms through the protein film or hydrocolloid, discharging of the active substance from capsules under specific terms. The temperature, moisture, and pressure are the main controlling factors of its release [76].

#### *3.4.2 Energy efficiency enhancement*

Spray drying is an energy-intensive procedure. With the increased energy costs with overall production stage changes, spray dryer consumers have to look for techniques to enhance the spray dryer system's thermal efficiency. For typical single-stage drying, the best method to control energy practice is to raise the inlet temperature and keep the outlet temperature low, as well as take full benefit of the energy introduced. Nevertheless, the weakness of this procedure is the potential product degradation in food spray drying [52, 77, 78].

#### **3.5 Freeze drying**

Freeze drying is used for high-quality foodstuffs stabilization, certain biological materials, and pharmaceuticals such as proteins, vaccines, bacteria, and mammal cells. These substances are freeze-dried; thus, the quality of the dried product is retained [63, 79]. Freeze drying is a process in which the water or another solvent is sublimated by the direct transition of water from solid (ice) to vapor, thus omitting the liquid state, and then desorbing water from the "dry" layer [80–84].

As a rule, freeze drying produces the highest-quality food product obtainable by any drying method. This is due to the fact that freezing water in the material prior to lyophilization inhibits chemical and biochemical such as non-enzymatic browning, protein denaturation,, microbiological, and enzymatic reactions processes. Consequently, the content of various nutrients, smell, and taste do not change. Raw materials comprise water, ranging from 80% to 95%. The water is removed by sublimation resulting in extremely porous structure creation of the freeze-dried products, and the finalized rehydration or lyophilizing happens immediately when water is added to the substance at a later time [85, 86]. However, the water in foodstuffs could be free or bound to the solution using different powers. The free water is freezed, but the bound does not. In the freeze-drying method, the iced and some bound water should be detached. Consequently, lyophilization is an extremely complex and multistep procedure that comprises (a) freezing under atmospheric pressure phase, (b) main drying; appropriate freeze drying; ice sublimation, at decreased pressure. For example, iced water is managed, then sublimation at 0°C and absolute pressure of 4.58 mmHg could happen. Nevertheless, subsequently the water usually exists in a solution or a joint state, the substance should be cooled below 0°C to preserve water in a frozen phase. So, during the main drying step, the frozen layer temperature as showed in **Figure 20** is at 10°C or lower at absolute pressures of about 2 mmHg or less. As the ice sublimes, the sublimation interface, which began at the external surface (**Figure 20**), dried material retreats and porous shell residues. The latent heat (2840 kJ/kg ice) could be operated using the dried substance and frozen layers, as illustrated in **Figure 20**. The vaporized water is conveyed within the dried substance porous sheet. Through the main drying step, in the dried layer, a large amount of nonfrozen water might be desorbed. The desorption method might affect the amount of heat that reaches the sublimation interface, and subsequently, it might affect the velocity of the sublimation front moving. The period when there are no frozen sheets is possessed to perform the end of the primary drying phase. (c) Secondary drying phase; desorption drying; drying the product to the required final humidity, this stage starts at the end of the primary drying stage, and the desorbed water vapor is transported through the pores of the material that is dried [63, 80, 87]. During the above three phases, six main physical phenomena could be distinct, which have a significant impact on the process path, the quality of the obtained substance, and

#### **Figure 20.**

*Diagram of a material on a tray during freeze drying. The variable X denotes the position of the sublimation interface (front) between the freeze-dried layer (layer I) and the frozen material (layer II).*

#### *Drying Technology Evolution and Global Concerns Related to Food Security… DOI: http://dx.doi.org/10.5772/intechopen.109196*

its overall costs [88]. Those phenomena are as follows: the water is transitioned into ice, then the ice to a vapor phase, the water particles are desorpted from substance structures, the obtainment of sufficiently low pressure, the resublimation of water vapor removed from the substance on the condenser surface, and the removal of a layer of ice from the capacitor surface.

In this context, the conditions of the freeze-drying process should be selected in a way that does not melt the water. The presence of water during freeze drying may result in many changes in the composition, morphology, and physical properties of foods (e.g., shrinkage), then reducing product quality during storage [89]. The effect of freeze-drying conditions on the nutritional properties, antioxidant activities, and glass transition characteristics of different food materials can be found in the literature [90–95]. Despite the long processing time and high cost, it is preferred for extreme-quality products. Though some damages in bio compounds could be set after freeze drying, this method is preferable to maintain nutritional qualities, particularly when operated under a vacuum. Moreover, the quality parameters such as freeze-dried products' rehydration and porosity are favorable for manufacturing foods. Newly, the freeze-drying process condensation with innovative technologies or pretreatments permit overcoming some of these drying processing challenges [96].

#### *3.5.1 Microwave freeze drying*

The limitations on heat transfer amounts in conventionally managed freeze-drying operations have led to providing internal heat generation with the use of microwave energy [97, 98]. Hypothetically, the use of microwaves must result in an instant drying rate, because the transferring of heat does not need internal temperature slopes and the temperature of ice could be preserved near to the maximum allowable temperature for the frozen layer exclusive of the need for extreme surface temperatures. For example, if it is allowable to keep the iced layer at 12°C, after that it has been assessed that the drying time for an ideal process using microwaves for a hypothetical 1-in. slab would be 1.37 h [99]. It should be notable that this drying time compares very approvingly with 8.75 h needed for the heat input within the dry layer, while 13.5 h for heat input within the iced layer without the removal of dry layer, and even with relatively short time of 4 h of drying for continuous removal of dry layer. Examining the freeze drying of a 1-in.-thick slab beef, the actual drying time of slightly over was 2 h, compared with 15 h for traditionally dried slabs [100]. Despite these benefits, the microwave application has not been successful [99, 101–105]. Because of the following reasons: (A) Supplied energy in the microwave form is too expensive [101]. (B) The tendency to glow discharge, which could cause gases ionization in the room and food deleterious changes, and losses of useful energy. The tendency to glow discharge is larger in the pressure ranged from 0.1 to 5 mmHg and could be decreased by operating the freeze dryers at pressures under 50 mm. The operation at low pressures has a double drawback: (1) it is much expensive, because of the demand for condensers operating at a very low temperature and (2) At these low pressures, the drying rate is much slower. (C) The process control is very difficult. Meanwhile, water has an inherently greater dielectric loss factor than ice, any localized melting produces a rapid chain reaction, which results in runaway overheating. (D) The economical equipment suitable for a large continuous scale is not yet available. In spite of these constraints, microwave freeze drying is considered a potential development [101].

#### *3.5.2 Industrial freeze dryers*

The main types of industrial freeze dryers include the following:

### *<IP_ADDRESS> Tray and pharmaceutical freeze dryers*

The hugest amount of industrial freeze dryers in process are of the vacuum batch style with freeze drying of the food stuff in trays. There are two main styles, depending on the type of condenser used. The first style showed the condenser plates in the same chamber and near the tray-heater assembly. The second style is representing the condenser in a separate chamber linked to the first by a wide, in over-all, butterfly valve. This latter type of the factory is permanently used in pharmacological industries, but it can also be used for freeze-drying foods. To reduce product contamination risk, especially in the pharmaceutical industries, a new freeze-dryer plant concept has been developed. The system, as illustrated in **Figure 21,** has two doors: a little one is for charging the stuff before drying, when the full door, which is opposed to the little door, is for discharging the stuff after drying. The condenser is positioned on the floor, which is under the first floor, where the drying chamber is placed. The freeze dryer shelves are lowered to the drying chamber bottom and then lifted one by one to a location in line with the filling machine. The charging of foodstuff is prepared under a laminar flow of sterilized air; the little door is unlocked only for each plate filling and then is directly closed [87].

#### **Figure 21.**

*Industrial freeze dryer design with stoppering device (Criofarma model C300-7): 1, drying chamber; 2, inspection window; 3, automatic small door opening; 4, full door; 5, hydraulic press for stoppering the bottles after drying; 6, PTFE elbows for double sterile condition inside the stoppering plug; 7, reenforcing member and cooling coils after steam sterilization; 8, isolation butterfly valve; 9, ice condenser chamber; 10, loading device; 11, discharging device; 12, unloaded shelves.*

*Drying Technology Evolution and Global Concerns Related to Food Security… DOI: http://dx.doi.org/10.5772/intechopen.109196*

#### *<IP_ADDRESS> Multi-batch freeze dryers*

The freeze-drying system in a batch factory is usually programmed and organized to minimalize the drying time and enlarge the factory production. With a single-batch factory, the load on the several systems will be very variable during the drying rotation. The product flow and handling operations will be alternating because of the batch process specifically. This means that optimum use of supplies will not be probable in a sole-cabinet batch system. To extent this weakness, an industrial freezedrying factory is built with numerous batch cabinets instructed to operate with staggered and overlying drying rotations. Each cabinet can be filled with materials from the same system, and they are assisted by the similar central system for vacuum pumping, tray heating, and condenser refrigeration. Although, the procedure is individually organized in each cabinet from a unattached control panel. This builds probably the simultaneous different foodstuffs production, which rises the operation flexibility of the factory. With only two cabinets in operation, an important part of the batch weakness may be excluded; for example, with four cabinets, an excellent leveling of loads will be accomplished. A huge amount of industrial freeze-drying factories operate today in this style as multi-cabinet batch factories [81, 99, 106].

#### *<IP_ADDRESS> Tunnel freeze dryers*

The process in the tunnel freeze dryer (**Figure 22**) takes place in a large vacuum cabinet into which the tray-carrying trolleys are loaded at intervals through a large vacuum lock at one end of the tunnel and discharged similarly at the other end. The drying conditions are carefully controlled in a number of sections of the tunnel by temperature-pneumatic controllers [81]. The plates of vapor constriction fit closely inside the channel walls yet permitting the trolleys to passage through two locations in the tunnel main unit, and gate valves turn off the locks from the main unit. Thus, the tunnel is divided into five autonomous process zones. When trolley is not moving during the period, a tray-lifting scheme causes all trays to sit in each trolley on heaters below the top. The heaters consume flat-top surfaces and ribs under which vacuum

**Figure 22.** *Typical tunnel freeze dryer schematic diagram.*

steam passes. They are cantilevered in couples from both sides of the tunnel. Vacuum steam heating has numerous benefits, including high latent heat of condensation and temperature control operating pressure. The cooling system comprises a great aqua ammonia absorption freezer instead of a compression factory. Because of easiness with the refrigeration, load can be diverse by controlling the feed of oil to the boiler that heats the absorber.

The whole capacity of the tunnel freeze dryer can be boosted as it increases volume of business. Large business factories for cottage cheese and coffee processing have been set up in this way. The tunnel freeze dryers have the same benefits of factory capacity operation that can be attained as in multi-batch factories. On the other hand, the flexibility for simultaneous production of materials or in swapping between products is missing.

#### *<IP_ADDRESS> Vacuum spray freeze dryers*

The vacuum spray freeze dryer system is illustrated in **Figure 23**. It has been industrialized for tea infusion, coffee extract, or milk. The product is applied by spraying from a sole jet upward or downward in a cylindrical tower of about 3.7-m diameter by about 5.5-m high [81, 107]. The solutions are solidified into little particles by evaporative freezing. In the tower, a frozen helical condenser is coiled between the internal wall and central hopper, the latter accumulating the partially dry powder as it drops freely to the tower bottommost. This is in turn associated with a tunnel where the drying technique is accomplished on a stainless-steel belt migrant among radiant heaters. The dried material passes into a hopper that feeds a vacuum padlock, allowing alternating product removal for packing. The whole system operates under a vacuum of about 67 Pa. In the initial evaporation, the diameter of frozen particles obtained by spraying into a vacuum is about 150 mm and loses about 15% moisture, and there is no sticking of these particles.

**Figure 23.** *Vacuum spray freeze dryer layout.*

*Drying Technology Evolution and Global Concerns Related to Food Security… DOI: http://dx.doi.org/10.5772/intechopen.109196*

#### *<IP_ADDRESS> Continuous freeze dryers*

Continuous freeze dryers are used for products drying in trays and for agitating bulk material drying. When the product is handled in trays, then the most delicate treatment of the product is attained. The food material is placed in the tray. Hence, it is not exposed to scratch, and it falls in contact with surfaces only that completely meet required hygiene values. While agitating a crushed product, more efficient heat transfer to sole product particles can be realized. Thus, a significant decrease in the heating surface is achievable. Although, scratch of the product by agitation increased the production of water vapor per unit, the heating surface tends to bring little product particles with vapor stream away from the bulk bed of product and cause product loss. Any system problems for water vapor elimination to retrieve the product loss may more than counterbalance the advantage of the higher heater surface load [108]. The heat transfer to the product and the trays is by radiation, which is the easiest mode to safeguard a correct as well as consistently distributed heat transfer to the material during the practice. The radiant heat is formed by horizontal heater saucers that are gathered within temperature zones. Each tray remains for a fixed time in each temperature zone for drying time minimized [108].


**Appendix B. Robustness Check**

Since the dataset is from 2010 to 2014, the change in macroeconomic environment in these years may influence the decisions of the investors and the behavior of the borrowers. As China has 36 different regions, regional differences may be found in financial behavior. Thus, we added region and year dummy variables into the model to control the fixed effect of time and region.

The loan application distribution by region and year are listed in Tables A2 and A3 accordingly.

**Table A2.** Loan Application Distribution by Region.

**Table A3.** Loan Application Distribution by Year.

The regression result with region and year dummy is presented in Table A4. The results are in line with original regression, in that most of the hard information variables have opposite results in the two models while most of the soft variables have consistent results. This proves the existence of TYPE II errors in the investors' decision-making process.

**Table A4.** Robustness Test with Region and Year Dummy.

Heteroscedasticity-Robust, standard errors in parentheses. \*\*\* *p* < 0.01, \*\* *p* < 0.05, \* *p* < 0.1.

To control for multicollinearity, we analyzed the variance inflation factors (VIF) of our chosen variables. The results1 show that all the independent variables' VIFs are within 2 and with an average of 1.27. In other words, the variance of the estimated coefficients is inflated with very low factors and within a reasonable rule-of-thumb of 10. For verification, we also calculated the square root of VIF, the R square for the correlation between the given independent variable and the rest of the independent variables, and the tolerance indicators, which are computed as 1- R square. The results prove the non-existence of multicollinearity.

#### **Note**

1 We checked the variance inflation factor, the R square for the correlation between the given independent variable and the rest of the independent variables, and the tolerance indicators for each independent variable. The results show that all variables have VIFs lower than 2, R square less than 0.2, and tolerance less than 1.

#### **References**

Akerlof, George A. 1970. The market for "lemons": Quality uncertainty and the market mechanism. *The Quarterly Journal of Economics* 84: 488–500. [CrossRef]

Arrow, Kenneth J. 1964. The role of securities in the optimal allocation of risk-bearing. *The Review of Economic Studies* 31: 91–96. [CrossRef]

Barasinska, Nataliya, and Dorothea Schäfer. 2014. Is crowdfunding different? Evidence on the relation between gender and funding success from a German peer-to-peer lending platform. *German Economic Review* 15: 436–52. [CrossRef]


**4.13 Plaque assay**

Plaque-based assays are the standard method used to determine virus concentration in terms of infectious dose. Viral plaque assays determine the number of plaqueforming units (pfu) in a virus sample, which is one measure of virus quantity. This assay is based on a microbiological method conducted in Petri dishes or multi-well plates like 6 well or 24 well etc. Specifically, a confluent monolayer of host cells is infected with the virus at varying dilutions and covered with a semi-solid medium, such as agar or carboxymethyl cellulose, to prevent the virus infection from spreading indiscriminately. A viral plaque is formed when a virus infects a cell within the fixed cell monolayer [30]. Virus quantification involves counting the number of viruses in a specific volume to determine the virus concentration. In research and development (R&D) based commercial and academic laboratories, the production of viral vaccines, recombinant proteins using viral vectors, viral antigens and clone screening, multiplicity of infection (MOI) optimization, and adaptation of methods to cell culture all require virus quantification. For quantification of virus incubated after infection at 37°C in a 5% CO2 incubator at 6, 12, 24, 36, 48, 60, and 72 h post-inoculation (hpi) based on the requirement to visualize plaques in wells [31, 32]. To quantify virus there are a lot of other methods used such as Focus forming assay (FFA), Endpoint dilution assay, Protein assays, Hemagglutination assay (HA), Bicinchoninic acid assay, Single radial immunodiffusion assay, Transmission electron microscopy (TEM).


of neuronal cells, being then susceptible for non-enzymatic oxidation in the prooxidant cellular environment (**Figure 4**). Indeed we could only detect significant levels of the specific DHA-derived lipoperoxide 4-hydroxy-2-hexenal (HHE) after 36 h exposure to DHA, just before upregulation of Gpx4 expression occurred. These findings were interpreted as HHE providing the signal to trigger transcriptional regulation, likely through activation of Nrf2 transcription factor. Indeed, it is known that Nrf2 is a master factor for the regulation of antioxidant response elements (ARE) in several antioxidant and phase II detoxifying proteins in different cell types, including nerve cells (Kobayashi and Yamamoto, 2005; Zhang et al., 2013), and several reports have shown that although DHA itself does not bind Nrf2, it may stimulate transcriptional activity of AREcontaining genes through activation of Nrf2 upon generation of 4-hydroxy-2-hexenal, which indeed activates Nrf2 (Ishikado et al., 2013; Kusunoki et al., 2013; Yang et al., 2013).

The results obtained in the hippocampus of WT mice were particularly interesting, since transcriptional activation of all Gpx4 isoforms was significantly higher under the Low-DHA dietary condition compared to the High-DHA diet. Though apparently these results might contradict the results obtained in HT22 cells, the outcomes suggest that it is under a low DHA diet with limited availability of n-3 precursors, when it is especially important to activate mechanisms (like upregulation of Gpx4 isoform expression) that may ensure the preservation of DHA in brain membranes. In fact, analyses of hippocampal lipid composition in WT and APP/PS1 animals revealed a significant reduction of DHA contents in Low-DHA diet compared to the High-DHA diet, yet the amount of DHA in the Low-DHA condition was surprisingly higher than expected from the dietary supply (**Table 3**), which reflects an extremely efficient ability to preserve brain DHA even when subjected to negligible levels in the diet (Díaz et al., accepted). This exceptional capability is mainly attributable to the favorable gradient for fatty acids to cross the blood-brain barrier, either passively or via fatty acid binding proteins (FATPs), since once in the brain, unesterified fatty acids are converted to CoA thioesters by the long-chainfatty-acid-CoA synthase (ACSL) family of proteins, therefore maintaining a continuous concentration gradient (see (Bazinet and Layé, 2014) for comprehensive review).

As mentioned before, APP/PS1 animals also responded to the different diets with changes in the transcriptional levels of Gpx4 expression. However, we only observed increased expression of c-Gpx4 isoform, and in the High-DHA diet. The apparent discrepancy between WT and APP/PS1 animals is likely related to the involvement of constitutive oxidative stress associated to amyloid generation in the brain of transgenic animals (Aso et al., 2012; Fabelo et al., 2012). Indeed, it is now widely accepted that amyloid peptide induces oxidative stress, and inflammation and oxidative stress generate more Aβ, giving rise to a vicious cycle between Aβ and free radical formation/lipid peroxidation in the presence of transition metals (Behl et al., 1994; Pamplona et al., 2005; Solfrizzi et al., 2006). In line with this matter, increased levels of LCPUFA–derived lipid hydroperoxides like HHE, HNE, and

other endoperoxides like isoprostanes have been observed in the brain of transgenic models of Alzheimer's disease, including 3xTgAD and APP/PS1 animals, and more relevantly, in different areas of human brain and cerebrospinal fluid of AD patients (Marcus et al., 1998; Markesbery and Lovell, 1998; Praticò et al., 1998; Valko et al., 2007; Gwon et al., 2012). In this order of ideas, it seems plausible that in the high-DHA diet, lipid-derived hydroperoxides must be elevated in the more prooxidant environment of brain parenchyma in transgenic animals, including DHA-related HHE, which may trigger the stimulation of Nrf2 transcription factor, which eventually would favor the activation of a transcriptional program to increase Gpx4 isoforms expression, especially the cytoplasmic isoform. In agreement with our findings, recent genomic gene expression profiles by microarray analyses in a "gene dose-response" model of Nrf2-null model have revealed that graded activation of Nrf2 increased the transcription of a large number of genes involved in Nrf2-mediated oxidative stress response, glutathione metabolism, and xenobiotic metabolism, including several genes responsible for the reduction of superoxide ions using GSH, i.e., glutathione peroxidases Gpx2 and Gpx4, which augmented by 2260 and 105%, respectively, in Keap1-HKO mice compared to WT mice (Wu et al., 2011).

Alternatively, altered genetic regulation of Gpx4 expression caused by overexpression of mutated APP and PS1 in transgenic animals, may have disturbed the normal gene expression pattern, as it has been recently demonstrated to occur in the frontal cortex and hippocampus of transgenic mice carrying the 1 E9 mutant hPS1 gene (Unger et al., 2005). Likewise, recent comparative studies in sporadic (icv-STZ Mouse) and familial (3xTg-AD mouse) mice models of AD have revealed the transcriptional alteration of more than 80 genes related to synapse function, apoptosis and autophagy, AD-related protein kinases, glucose metabolism, insulin signaling, and mTOR pathway in the hippocampus and the cerebral cortex, not only when compared to WT animals, but also between both AD models (Chen et al., 2012).

In summary, we hypothesize that by upregulating Gpx4 gene expression and increasing GPx4 protein levels and enzyme activity, DHA initiates a self-protective biochemical strategy in the hippocampal neuron to control non-enzymatic peroxidation of DHA-containing phospholipids in the membrane from cellular and subcellular compartments. Given the extremely low capacity of hippocampal cells to biosynthesize DHA (reviewed in Plourde and Cunnane, 2007; Bazinet and Layé, 2014), and the essential nature of this fatty acid in the nervous system, this strategy provides an adaptive mechanism to keep the high DHA levels in hippocampal phospholipids.

# Retained Intronic Sequences in Gpx4 mRNA

In initial experimental strategy used for the detection of the different Gpx4 mRNAs, we designed a couple of primers targeted to exons E1b (forward primer) and the boundary between exons E2/E3 (reverse primer) for the specific detection of nuclear Gpx4 mRNA. Unexpectedly, in these experiments we detected the presence of two amplicons: one of the expected molecular size (157 bp) for a fully processed n-Gpx4 mRNA, and another fragment 370 bp. Once sequenced, we observed that this later amplicon contained an extra 213 bp sequence corresponding to intron I1b located between exons E1b and E2. This unexpected sequence (named here I-Gpx4) was detected in control HT22 cells (treated with vehicle), but more interestingly, that its expression level was subjected to significant upregulation by DHA treatment (near 100% compared to control cells), which was in the range observed for the changes in expression for the set of other Gpx4 mRNA variants (**Figure 2**). In absolute terms, I-Gpx4 quantification revealed that this isoform was nearly 3 times more abundant than n-Gpx4 under unstimulated conditions, and about

TABLE 2 | Oligonucleotides used as primers for quantification of different GPX4 mRNA isoforms and for mapping 5′ end of I-GPX4 mRNA in SHSY-5Y cells.

Determination of absolute mRNA levels by real-time quantitative PCR (RT-qPCR) were performed as described in Expósito-Rodríguez et al. (2011). HPRT1, Hypoxanthine guanine phosphoribosyl transferase 1; RPL32, Ribosomal protein L32.

FIGURE 4 | Schematic model depicting the hypothetical mechanism of DHA-mediated regulation of Gpx4 in HT22 cells. For details see "Defining a unified rationale for DHA-induced regulation of Gpx4 gene expression." Modified from Casañas-Sánchez et al. (2014).

Results are expressed as mole % and represent means ± SEM of four different animals under each dietary condition and genotype. DMA: Dimethyl acetals. \*p < 0.05 compared to High-DHA. #, 1: p < 0.05 and p < 0.1 compared to APP/PS1. Fatty acid extraction and determination was performed as described in Fabelo et al. (2012) and Martín et al. (2006). Data were analysed by One-Way ANOVA followed by Tukey's multiple comparison test. Comparisons between Low-DHA and High-DHA treatments were performed using Student's t-test or Mann-Whitney U-test where appropriate. Main monoenoic (oleic acid, 18:1n9), n-6 LCPUFA (arachidonic acid, 20:4n-6), and n-3 LCPUFA (DHA, 22:6n-3) are highlighted on pink background.

7 times higher after DHA treatment, pointing to a significant biological role of this intron-retaining isoform in hippocampal cells. Nonetheless, I-Gpx4 was expressed a much lower amount than c-Gpx4, even after DHA treatment.

In order to gain a deeper insight on the transcription start site for the intron-retained transcript, we designed several amplification primers directed to intron I1a (**Figure 1** and **Table 1**). While the primers targeted to E1b exon and intron I1b amplified the expected amplicons, all other primers targeting E1b exon upstream sequences were unsuccessful in these PCR assays. These results strongly indicate that a Gpx4 CIRT transcript represents an unprocessed variant of the n-Gpx4 mRNA isoform.

In order to assess for the biological relevance of this intronretaining Gpx4 isoform, we used these specific amplicons to unveil the presence in the hippocampus of C57Bl/6 mice. We observed that not only the amplicon was detected, but also that its expression was modulated by the contents of DHA in the diet. Thus, levels of I-Gpx4 isoform were significantly higher (around 2 times) in animals receiving Low-DHA diet compared to animals receiving a High-DHA diet. A similar result was obtained in transgenic APP/PS1 animals, though in this case the increase in I-Gpx4 gene expression was around 1.6 times higher in animals maintained in a Low-DHA diet (**Figure 3**). Absolute comparisons between WT and APP/PS1 animals revealed that I-Gpx4 was higher in WT animals than in the APP/PS1 genotype. Overall these data indicates that the lack of sufficient DHA or its deficient supply is accompanied by an increase in I-Gpx4. Again, these higher expression levels observed in APP/PS1 animals suggests a genotype-related transcriptional regulation, which may be related to increased hippocampal oxidative stress in transgenic animals (Aso et al., 2012).

Whether this regulated Gpx4 mRNA variant constitutes an intermediate processing stage of Gpx4 splicing or represents a different level of transcriptional regulation by intron retention remains unrevealed. However, the notion of intron retention has been gaining consistency in the last decade, as an alternative processing of conventional (nuclear) mRNA, adding a novel level of complexity in gene expression regulation. These sequences, named as CIRTs (Cytoplasmic Intron-sequence Retaining Transcripts), comprise only few introns and are limited to small fractions of introns within the gene (Buckley et al., 2014). Indeed, in recent years, a number of transcripts containing intronic sequences and subjected to cytoplasmic splicing have been unveiled across a number of cell types and species (Buckley et al., 2014), and the list keeps growing steadily. Noticeably, a recent study performed in mouse brains have revealed the broad presence of cytoplasmic intron-sequence retaining transcripts in hippocampal neurons, with 59.4% of total gene transcripts in dendrites containing intronic reads (Khaladkar et al., 2013). Examples of retained introns from individual transcripts of relevance in neuronal cells include the large-conductance calcium-dependent potassium channel (BK) encoded by KCNMA1 gene (Bell et al., 2008, 2010), γsubunit of GABA-A receptor (encoded by GABRG3) (Buckley et al., 2011), ionotropic Glutamate receptor (encoded by GRIK1 gene) and type 1 NMDA receptor (encoded by GRIN1) (Buckley et al., 2011) or the α-1 subunit of T type, voltagedependent Calcium channel (encoded by CACNA1H) (Zhong et al., 2006). Though the biological relevance of CIRTs is still largely unknown, their abundance in neuronal dendrites (and also in enucleated platelets) suggest that endogenous cytoplasmic transcripts harboring retained intronic substrates could be subject to extranuclear splicing, thereby obviating the need for shuttling molecules to and from the nucleus for

# References

Alessandri, J. M., Guesnet, P., Vancassel, S., Astorg, P., Denis, I., Langelier, B., et al. (2004). Polyunsaturated fatty acids in the central nervous system: evolution of concepts and nutritional implications throughout life. Reprod. Nutr. Dev. 44, 509–538. doi: 10.1051/rnd:20 04063

processing (Buckley et al., 2014). In this manner, dendritic splicing could represent a novel method of post-transcriptional gene regulation.

In this order of ideas, it is tempting to speculate that CIRT in Gpx4-I gene may be part of the normal endogenous posttranscriptional regulation pathway. Such regulated extranuclear splicing variants may be part of what Buckley et al. (2014) designed as "sentinel RNA hypothesis," according to which a master transcript harboring CIRTs would serve to generate transcript variants within the cytoplasm in response to specific stimulation, or under conditions in which a rapid processing is required and adaptive. A plausible scenario for this stimulusdependent cytoplasmic splicing of I-Gpx4 in hippocampal cells, is that this transcript processing in cellular domains distant from cellular nucleus, as is the case of dendrites, would enhance the antioxidant buffering capacity against the acute generation of lipid peroxides by oxidative insults, which would commit the integrity of membrane unsaturated phospholipids, especially those enriched in DHA.

# Conclusions and Hypothesis

In summary, current available data indicate that Gpx4 gene is subjected to a complex transcriptional regulation that renders nuclear, cytoplasmic, and mitochondrial isoforms, but also generate an intron-retaining variant, likely corresponding to a CIRT, all derived from the same gene. At least in hippocampal HT22 cells and in the hippocampus of wild type mice, the transcriptional control of most of these Gpx4 isoforms is modulated by DHA following an unprecedented homeostatic strategy aimed to buffer its potential oxidation and to ensure its preservation in the hippocampal neuronal membrane. The observations in transgenic APP/PS1 mice, suggest that such homeostatic mechanism is disturbed by the presence of mutated APP and/or PS1, and may be relevant in Alzheimer's disease, as suggested by the generalized observation that DHA contents are reduced in the hippocampus of AD brains.

# Acknowledgments

Supported by grants SAF2010-22114-C02-01/02 and SAF2014- 52582R from Ministerio de Economía y Competitividad (Spain). VC and DQ hold fellowships from Gobierno de Canarias and Fundación Cajacanarias, respectively. We are grateful to Biosearch Life-Puleva Biotech for continuous technical support to the project at the Laboratory of Membrane Physiology and Biophysics.

Arnér, E. S. J., and Holmgren, A. (2000). Physiological functions of thioredoxin and thioredoxin reductase. Eur. J. Biochem. 267, 6102–6109. doi: 10.1046/j.1432- 1327.2000.01701.x

Aso, E., Lomoio, S., López-Gonzalez, I., Joda, L., Carmona, M., Fernández-Yagüe, N., et al. (2012). Amyloid generation and dysfunctional immunoproteasome activation with disease progression in animal model of familial Alzheimer disease. Brain Pathol. 22, 636–653. doi: 10.1111/j.1750-3639.2011.00560.x

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2015 Casañas-Sánchez, Pérez, Fabelo, Quinto-Alemany and Díaz. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Sub-toxic Ethanol Exposure Modulates Gene Expression and Enzyme Activity of Antioxidant Systems to Provide Neuroprotection in Hippocampal HT22 Cells

Verónica Casañas-Sánchez 1, 2, José A. Pérez 1, 2, David Quinto-Alemany 3, 4 and Mario Díaz 2, 3, 4 \*

1 Departamento de Bioquímica, Microbiología, Biología Celular y Genética, Universidad de La Laguna, Tenerife, Spain, 2 Instituto Universitario de Enfermedades Tropicales y Salud Pública de Canarias, Tenerife, Spain, 3 Departamento de Biología Animal, Edafología y Geología, Universidad de La Laguna, Tenerife, Spain, 4 Unidad Asociada de Investigación ULL-CSIC, "Fisiología y Biofísica de la Membrana Celular en Patologías Neurodegenerativas y Tumorales", Tenerife, Spain

#### Edited by:

Ali Mobasheri, University of Surrey, UK

#### Reviewed by:

Alberto Passi, University of Insubria, Italy Angel Catala, National University of La Plata, Argentina

> \*Correspondence: Mario Díaz<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Membrane Physiology and Membrane Biophysics, a section of the journal Frontiers in Physiology

> Received: 19 April 2016 Accepted: 11 July 2016 Published: 27 July 2016

#### Citation:

Casañas-Sánchez V, Pérez JA, Quinto-Alemany D and Díaz M (2016) Sub-toxic Ethanol Exposure Modulates Gene Expression and Enzyme Activity of Antioxidant Systems to Provide Neuroprotection in Hippocampal HT22 Cells. Front. Physiol. 7:312. doi: 10.3389/fphys.2016.00312 Ethanol is known to cause severe systemic damage often explained as secondary to oxidative stress. Brain is particularly vulnerable to ethanol-induced reactive oxygen species (ROS) because the high amounts of lipids, and because nerve cell membranes contain high amounts of peroxidable fatty acids. Usually these effects of ethanol are associated to high and/or chronic exposure to ethanol. However, as we show in this manuscript, a low and acute dose of ethanol trigger a completely different response in hippocampal cells. Thus, we have observed that 0.1% ethanol exposure to HT22 cells, a murine hippocampal-derived cell line, increases the transcriptional expression of different genes belonging to the classical, glutathione/glutaredoxin and thioredoxin/peroxiredoxin antioxidant systems, these including Sod1, Sod2, Gpx1, Gclc, and Txnrd1. Paralleling these changes, enzyme activities of total superoxide dismutase (tSOD), catalase, total glutathione peroxidase (tGPx), glutathione-S-reductase (GSR), and total thioredoxin reductase (tTXNRD), were all increased, while the generation of thiobarbituric acid reactive substances (TBARS), as indicators of lipid peroxidation, and glutathione levels remained unaltered. Ethanol exposure did not affect cell viability or cell growing as assessed by real-time cell culture monitoring, indicating that low ethanol doses are not deleterious for hippocampal cells, but rather prevented glutamate-induced excitotoxicity. In summary, we conclude that sub-toxic exposure to ethanol may well be neuroprotective against oxidative insults in hippocampal cells.

Keywords: ethanol, antioxidant systems, superoxide dismutases, glutathione, thioredoxins, hippocampal cells, HT22 cells

# INTRODUCTION

Ethanol is known to induce neurocognitive deficits and to provoke neuronal injuries associated with neuronal degeneration (Givens et al., 2000). Although oxidative stress and mitochondrial damage are implicated in nerve tissue injury (Suh et al., 2004; Das and Vasudevan, 2007) the precise mechanisms underlying ethanol-induced neurological disorders remain unclear. Ethanol-induced oxidative stress is linked to its metabolism in both microsomal and mitochondrial systems, which is directly involved in the generation of reactive oxygen species (ROS) and reactive nitrogen species (Das and Vasudevan, 2007). It is known that high or chronic ethanol exposure results in the depletion of reduced glutathione (GSH) levels, decreases antioxidant activity, and elevates malondialdehyde and hydroxynonenal protein adducts (Das and Vasudevan, 2007). These increased levels of oxidative stress (and secondary membrane lipid peroxidation, in particular) may disrupt neuronal energy metabolism and ion homeostasis by impairing the function of membrane ion-motive ATPases and glucose and glutamate transporters. Such oxidative and metabolic disturbances may thereby render neurons vulnerable to excitotoxicity and apoptosis. Indeed, it is known that acute alcohol exposure inhibits cognitive functions, including learning, and memory in humans (Givens et al., 2000), and that ethanol-induced memory impairments are mainly due to deficits in processing new memories, rather than retrieval of consolidated memories (White et al., 2000). The hippocampus is the main locus for ethanol-induced alterations of cognitive functions. Compelling evidence have demonstrated that acute ethanol exposure inhibits long-term potentiation (LTP) in the CA1 region of the hippocampus both in vivo and in vitro (Givens and McMahon, 1995; White et al., 2000; Ramachandran et al., 2015). Apparently, the primary mechanism by which ethanol inhibits LTP is the increase in GABAergic transmission, which, in turn, inhibits the depolarization phase required for N-methyl-D-aspartate (NMDA) receptor activation (White et al., 2000; Schummers and Browning, 2001).

Apart from these deleterious effects, several lines of evidence have shown that consumption of low doses of alcohol may provide neuroprotective effects against Alzheimers disease (AD). Indeed, Belmadani et al. (2004) observed that acute administration of ethanol was able to prevent the toxic effects of amyloid beta peptides (Aβ), the main protein aggregates in Alzheimers disease, in brain slices. Pre-treatment with physiologically relevant concentrations of ethanol (0.02–0.08%) protected neurons against Aβ-induced synapse damage, and recovered levels of synaptophysin, an indicator of synapse density in cortical and hippocampal neurons (Bate and Williams, 2011). Further, it has been reported that moderate wine consumption reduced neuropathologic traits (decreased amyloid plaques and reduced spatial memory impairment), in Tg2576 transgenic mice which overexpress amyloid pre-cursor protein (Wang et al., 2006). Moreover, epidemiological studies in a large cohort of subjects (>3600) have pointed out that low or moderate alcohol consumption is associated with lower risk of incident dementia among older adults, and these individuals are less likely to develop phenotypic symptoms of Alzheimers disease (Mukamal et al., 2003). Interestingly, ethanol also protected neurons against synapse damage induced by pre-synaptic aggregates of α-synuclein, which are characteristic of Parkinson's disease and dementia with Lewy bodies (Bate and Williams, 2011).

The potential neuroprotective mechanism(s) of action of ethanol remain unknown. However, given that amyloid beta peptides and α-synuclein toxicities have been linked to increased oxidative stress (Simonian and Coyle, 1996; Mattson et al., 1999; Bossy-Wetzel et al., 2004; Mancuso et al., 2006), in the present study we have examined a possible mechanism by which moderate ethanol concentrations might exert a neuroprotective effect on hippocampal cells through modification of cellular antioxidant capabilities. We demonstrate for the first time, that sub-toxic ethanol exposure triggers the activation of cellular antioxidant systems and that its effects may be unraveled both at transcriptional and enzymatic levels. Our results demonstrate that, at low doses, ethanol exerts a role as an "Indirect Antioxidant," at least in hippocampal cells.

# MATERIALS AND METHODS

# Cell Culture Conditions, Ethanol Supplementation and Preparation of Cell Extracts

The immortalized mouse hippocampal cell line, HT22, was cultured in standard Dulbecco's modified Eagle's medium (DMEM) as described in Martín et al. (2006). Culture medium was changed every 2 days and, when reached 90% confluence, cells were subcultured after treatment with 0.25% trypsin-EDTA mixture, at a density of 4 × 105 /ml on T25 flasks (for gene expression analyses) and T75 flasks (for enzyme activity studies).

After allowing 24 h for attachment, culture media were replaced with standard medium supplemented with ethanol in a 0.1% final concentration or vehicle phosphate buffered saline (PBS). Every 24 h of incubation the medium was replaced with fresh medium containing ethanol in PBS or PBS. Cell cultures were collected at 6, 24, 30, and 48 h, and immediately processed for either total RNA extraction or preparation of total extracts required for determination of enzyme activities, and glutathione and TBARs levels.

Cell extracts were prepared by homogenization in cold hypotonic buffer (Tris-HCl 20 mM, pH = 7.6) containing 1X protease inhibitors cocktail (Roche Diagnostics, Barcelona, Spain), then centrifuged at 900 g for 10 min, and the supernatants collected and stored at −80◦C until analyses. For glutathione measurements, cell extracts were homogenized in cold hypotonic buffer containing 5% TCA and centrifuged at 10,000 g for 10 min at 4◦C to isolate the post-mitochondrial supernatant. Supernatants were stored in 150 µl aliquots at −80◦C. Protein determination was carried out using the Bradford assay (Bradford, 1976).

# RNA Purification, cDNA Synthesis and Relative Quantification of Gene Expression

Total RNA was purified from 3 × 106 HT22 cells using a commercial kit and following the manufacturer's recommendations (RNeasy R Protect Minikit, Qiagen), and on-colum DNase I digestion to remove genomic DNA (gDNA). The integrity of purified RNA was estimated through the 3′ :5′ assay (Nolan et al., 2006).

cDNA samples for real-time reverse transcription quantitative PCR (RT-qPCR) experiments were obtained with the Transcriptor First Strand cDNA Synthesis Kit (Roche), using 6µg total RNA as template and anchored oligo(dT)18 primers. A mixture of the 33 diluted cDNA samples was used for the selection of the optimal concentration of each PCR primer pair (see **Table 1**), based on the lowest quantification cycle (Cq) values. Resulting amplicons for each primer pair from the cDNA pool were checked by electrophoresis on 3% agarose gels and sequenced. The possibility of gDNA contamination in the RT-qPCR assays was controlled in several ways. First, amplification primers were targeted to different exons, often spanning an exon/exon boundary (see **Table 1**). Next, each primer pair was tested by real-time qPCR using 1 ng genomic DNA as template. The level of gDNA contamination in each of the 32 RNA samples was assessed, assaying a quantity equivalent to the cDNA used in the amplification reactions (i.e., 40 ng of total RNA) was amplified by real-time qPCR using primers targeted to alpha-tubulin sequences.

Real-time amplifications were run in triplicate using SYBR Green detection on a LightCycler 480 platform (Roche). Relative quantities of the targeted mRNAs were calculated from Cq data following an efficiency-correction model implemented in the REST software (Pfaffl, 2001). The normalization factor for each cDNA sample was calculated as the geometric mean of the expression values of reference genes Hprt1, Polr2f, and Tbp genes.

# Antioxidant Enzyme Activities and Levels of Glutathione and Thiobarbituric Acid Reacting Substances (TBARS)

Superoxide dismutase (SOD) activity was measured using the pyrogallol method following Marklund and Marklund (1974). One unit was defined as 50% inhibition of the rate of autoxidation of pyrogallol. The activity of SOD is expressed as units/mg protein. Catalase (CAT) activity was determined as described previously (Sani et al., 2006), by following the rate of decomposition of H2O2 in 10 mM potassium phosphate buffer at 240 nm. One CAT unit was defined as the decomposition of 1 mmol H2O2/min, and was expressed as units/mg protein.

Total glutathione peroxidase (GPX) and phospholipidhydroperoxide glutathione peroxidase (GPX4) activities were measured using the glutathione reductase-NADPH methods described by Lawrence and Burk (1976) and Scheerer et al. (2007), respectively, by monitoring the rate of decrease in the concentration of NADPH as recorded at 340 nm. Glutathione reductase (GSR) was analyzed by determining the reduction of oxidized glutathione (GSSG) at the expenses of NADPH oxidation and monitored at 340 nm, according to the method of Carlberg and Mannervik (1985). GPX, GPX4 and GSR activities were expressed as nmol NADPH oxidized/min.mg protein.

Glutathione-S-transferase (GST) activity was determined following the conjugation of GSH with CDNB (1-chloro-2,4 dinitrobenzene) at 340 nm (Habig et al., 1974) and expressed as nmol GS-DNB/min.mg protein.

Thioredoxin reductase (TXNR) activity was measured using the DTNB-NADPH assay described by Arnér et al. (1999), in which the generation of thionitrobenzoate ion (TNB) catalyzed by TXNR upon oxidation of NADPH is monitored at 412 nm. TXNR activity was expressed as nmol TNB/min.mg protein.

Glutathione levels were determined fluorimetrically using excitation/emission wavelengths of 355 nm/420 nm according to Hissin and Hilf (1976). GSH, GSSG, and total glutathione (GSH+2GSSG) levels were determined against proper calibration curves and expressed as nmol/mg protein. Lipid peroxidation was determined by the thiobarbituric acid reacting substances (TBARs) method (Ohkawa et al., 1979), using TMP (1,1,3,3-tetramethoxypropane) as standard for calibration curves. TBARS were measured fluorimetrically with 485 nm (excitation)/ 535 nm (emission) wavelengths. TBARS contents were expressed as nmol/mg protein.

# Real-time Cell Proliferation Assays

Real-time cell proliferation studies were performed using the xCELLigence biosensor technology (Roche), an electrical impedance-based system that allows for the measurement of real-time cell proliferation (Ke et al., 2011). Briefly, HT22 cells were trypsinized, and seeded at a density of 3.5 × 103 cells/well into 3 independent E16—xCELLigence plates. After an initial stabilization period, the impedance was recorded at 15 min intervals along the experiment, and the values converted to Cell Index (CI), a measure of the degree of cellular adhesion to the multi-electrode array. Generally, cell number directly correlates with output CI reading until confluency is achieved (CImax). 48 h after seeding, 10 µl of vehicle (PBS), or ethanol (final concentration 0.1%) were added to each well, and incubated for additional 24 h before replacing the incubating solutions with either DMEM, DMEM+0.1% EtOH, or DMEM+1% EtOH (see **Figure 5A**). In some experiments, glutamate (20 mM final concentration) was added during the last medium replacement. The concentration of glutamate chosen for these experiments was calculated as the IC50 obtained in the dose-response analyses performed using this same device (**Figure 5B**). For this purpose 24 h after initial attachment, cells were exposed to single doses of glutamate, ranging from 3 to 30 mM, while CI was continuously monitored. 24 h after exposure CImax was obtained and used for logistic analyses.

# Statistics

Gene expression data were processed following an efficiencycorrected model for relative quantification (Pfaffl, 2001) and normalization with multiple internal controls as implemented in the qBASE software (Hellemans et al., 2007). Four genes showing high expression stability in relative expression analysis were tested as potential reference genes according to qBASE normalization tools, three of which were finally selected. A relative expression software tool (REST 2008, Pfaffl et al., 2002) was used to obtain the corresponding significance levels for each individual change in gene expression. Comparisons of gene expression levels between PBS and ethanol and expressed as fold values. P-values below 0.05 were considered statistically different.

Dose-response curves for glutamate were obtained from realtime cell proliferation assays. Data were fitted to a four-parameter logistic equation to obtain the IC50 value, using the software implemented in XCELLigence device.

Data from enzyme activity assays and glutathione/TBARs levels are expressed as mean ± SEM, and were analyzed by

TABLE

1

Gene

names,

cellular

locations,

and

oligonucleotides

used

as

primers

in

amplification

reactions.

IS, internal standard for normalization; RIA, RNA integrity assessment by 3 ′:5 ′ assay. Subcellular localization of protein encoded by the detected\*\*N/A: the primer pair does not amplify with gDNA as template, at least with a short extension time amplification reactions.

 mRNA variant.

\* one-way ANOVA followed by Tukey's multiple comparison test. Comparisons between PBS and ethanol treatments at each time were performed using Student's t-test or Mann-Whitney U-test where appropriate.

# RESULTS

# Effects of Sub-toxic Exposure to Ethanol on the Transcriptional Activity of Genes Encoding for Antioxidant Systems

First, we analyzed the time-course of the effects of subtoxic ethanol exposure (0.1%) on the expression of different genes encoding for antioxidant enzymes with relevant activity in neuronal cells. We explored key enzymes from the three major antioxidant systems, namely classical, thioredoxin/peroxyredoxin, and glutathione/glutaredoxin systems. Target genes analyzed are detailed in **Table 1**, together with the information on the primers used for PCR amplification.

Results in **Table 2** show that superoxide dismutase encoding genes were up-regulated yet with different time-courses. Thus, while Sod1 expression was stimulated just 6 h after each exposure to ethanol (0 and 24 h), Sod2 was up-regulated after 30 h exposure to ethanol and remained so until the end of the experiment. The other gene relevant in the classical system, Cat, encoding for catalase, remained unaltered, or was down-regulated (at 24 h). Within the thioredoxin-peroxiredoxin system, Txnrd1 gene, encoding for cytosolic thioredoxin reductase was soon up-regulated and its expression kept stimulated throughout the experiment. Paradoxically, Txnrd2, and Txnrd3 genes, encoding for thioredoxin reductases from mitochondrial and endoplasmic reticulum isoforms, respectively, were downregulated throughout the experiment, although only significantly for Txnrd3. A similar set of changes were observed for genes encoding for peroxyredoxins 2–5, all of which were significantly down-regulated yet with different time patterns. Finally, Txnip gene, encoding for the inhibitory thioredoxin interacting protein, was highly down-regulated, particularly 24 h after each addition of ethanol. No changes in the expression patterns were observed for either thioredoxin-encoding genes.

Lastly, within the glutathione/glutaredoxin system, all affected genes were up-regulated. The most important changes were observed for Gpx1, encoding for cytosolic glutathione peroxidase 1, which remained stimulated throughout the experiment, and

TABLE 2 | Gene expression of antioxidant systems in HT22 cells exposed to ethanol (0.1%) or PBS.

Results correspond to the mean of four different experiments. PBS, phosphate buffer saline; p, probability value; \*Significant differences. Bold values indicate those genes whose expression was altered at some stage in the experiments.

reached the maximal transcriptional stimulation amongst all genes studied at 48 h. These changes were specific for Gpx1, and were not observed for Gpx4 gene which encodes the membraneassociated isoform. Also, significant changes were detected for Gclc gene, which encodes for the catalytic subunit of glutathionecysteine ligase.

# Effects of Sub-toxic Exposure to Ethanol on Key Antioxidant Enzyme Activities and Antioxidant Metabolites

Next, we assessed the activities of most antioxidant enzymes encoded by genes that were affected by ethanol treatment and following the same time-course used to explore changes in transcriptional activity. Results shown in **Figure 1A** shows that total superoxide dismutase activity was increased by ethanol treatment from 24 h after exposure, being the maximal activity reached at 48 h. Surprisingly, catalase activity remained similar to vehicle (PBS) until the end of the experiment, where at 48 h a significant increase (40%) was observed (**Figure 1B**).

Within the thioredoxin system, we explored the time-course of total thioredoxin reductase (tTXNRD) activity (**Figure 2**). We observed that, in line with the results of Txnrd1 up-regulation, TXNRD activity was stimulated from 6 h, and that maximal activation was observed at 48 h.

With regards to the glutathione/glutaredoxin system, we assessed the enzyme activities of total glutathione peroxidase, glutathione peroxidase 4, glutathione-S-reductase, and glutathione-S-transferase (**Figure 3**). When compared to PBS, total glutathione peroxidase (tGPx) was found to be significantly increased from the begin of the experiment (**Figure 3A**), reaching nearly a 200% increase at 48 h (similar to what was observed for Gpx1 gene expression). This increase is likely attributable to stimulation of the GPX1 isoform, since GPX4 activity remained unaltered all along the experiment (**Figure 3B**). Glutahione-S-reductase (**Figure 3C**) was only affected at 48 h, which is compatible with the significant increase in Gsr gene expression detected at 30 h, and agrees with the expected delay in Gsr mRNA translation. On the other hand, glutathione-S-transferase was completely unaffected by ethanol treatment (**Figure 3D**).

We also determined cellular levels of total glutathione, reduced glutathione, oxidized glutathione (**Figure 4**). None of these oligopeptides appeared to be affected by ethanol treatment throughout the experiment, when compared to PBStreated cells. These observations on glutathione species are in contrast to the expression levels of Gclc gene, which were significantly increased by the end of the experiment (**Table 2**). Several possible explanations are that even at 48 h, (1) Gclc mRNAs has not been fully translated, (2) that newly-synthetized GCLC protein might not be physiologically active or, (3) that expression of the regulatory subunit (GCLM) might be limiting, as demonstrated for HepG2/C3A cells and murine embryonic fibroblasts cultured in cysteine-deficient medium (Sikalidis et al., 2014). Regarding TBARs, it is noticeable that their levels did not increase at any point of the experiment, indicating that potential lipid peroxidation induced by ethanol is buffered by concerted activation of antioxidant systems. Indeed, although not

significantly, there appear to occur a time-dependent reduction of TBARs levels in ethanol-treated cells (**Figure 4D**).

# Effects of Ethanol on the Time-course of HT22 Cell Proliferation and Resistance to Excitotoxicity

We finally analyzed the effects of different doses of ethanol treatment on HT22 cells proliferation. Results summarized in **Figure 5** shows that HT22 cells reach the maximal cell index (CImax) after 120–160 h of seeding in the conditions of the present experiments (**Figure 5A**). In the presence of 0.1% ethanol, maximal CI values were similar between PBSand ethanol-treated cells. However, when challenged with a 10 times higher dose of ethanol, a significant reduction of cell proliferation was observed, indicating an important degree of toxicity by this dose of ethanol (**Figure 5A**), likely caused by an excessive oxidative stress, which overcame the cytoprotective effects of endogenous antioxidant defense induced by low ethanol exposure.

In order to assess whether changes in antioxidant gene expression might confer resistance to toxic oxidative insults, we used glutamate as it has been reported to cause excitotoxicity in HT22 cells (He et al., 2013) and oxidative stress secondary to cysteine depletion (Li et al., 1998). First, using non-linear regression to a four parameter logistic equation, we determined the IC50 value for glutamate toxicity against cell index, and we found a value of 19.5 mM (**Figure 5B**). Then, we assayed

a glutamate concentration of 20 mM in ethanol (0.1%) treated cells, while monitoring real time cell proliferation. We observed that pre-exposure to 0.1% ethanol significantly prevented glutamate-induced cell death to about 80% of ethanoltreated cells, compared to around 90% cell death in the absence of EtOH. Therefore, we concluded that ethanol efficiently reduced cell death caused by glutamate-induced excitotoxicity.

# DISCUSSION

Our present results demonstrate that treatment of hippocampal HT22 cells with sub-toxic doses of ethanol modifies the expression of specific genes within the classical, thioredoxin, and glutathione systems. We also show that these transcriptional changes are accompanied by consistent modifications in enzyme activities of the three systems and modulation of cellular antioxidant status. First, we observed that ethanol exposure stimulated gene expression of both superoxide dismutase genes (Sod1 and Sod2), and soon increased total SOD activity. The effects of ethanol exposure on the activity of superoxide dismutase are controversial, with reports of an increase (Somani et al., 1996; Enache et al., 2008), no change (Gonenc et al., 2005), or a decrease (Ledig et al., 1981), depending on the brain region, the dose and the duration of ethanol exposure. However, our results are in agreement with previous results showing significant increases in superoxide dismutase and catalase activities in the hippocampus of rats receiving acute intraperitoneal injections

of ethanol (1.5 g/kg) (Enache et al., 2008). Similar results were observed in the rat cortex in response to acute ethanol administration (Somani et al., 1996). Interestingly, in our present study, although Cat gene expression was not altered by ethanol, a significant increase in enzyme activity was observed by the end of the experiment, which suggests a post-translational modulation of catalase, perhaps associated to increase H2O2 as a result of SOD activation. The existence of factors modulating catalase activity in response to oxidative stress has been demonstrated in different cell lines (Uenoyama and Ono, 1973; Cao et al., 2003). Several studies have shown that catalase interacts with c-Abl and Arg non-receptor tyrosine kinases, upon activation by H2O2through a mechanism dependent on protein kinase Cδ (Sun et al., 2000; Cao et al., 2003). The functional significance of these interactions are supported by the demonstration that cells deficient in both c-Abl and Arg exhibit substantial increases in H2O2 levels and a marked increase in H2O2-induced apoptosis (Cao et al., 2003).

Within the thioredoxin system we observed that the expression of the gene encoding for cytoplasmic thioredoxin reductase (Txnrd1) was significantly increased by ethanol from 6 h, while thioredoxin reductases of mitochondrial and endoplasmic reticulum origins were unaffected or down-regulated. Paralleling these observations, an equivalent increase in tTXNRD activity was observed shortly after the upregulation of Txnrd1 expression was observed. The finding that tTXNRD activity was augmented is consistent with the fact that cytosolic thioredoxin reductase (encoded by Txnrd1 gene) is the most abundant isoform in nerve cells (Arnér and Holmgren, 2000; Turanov et al., 2010). Another interesting observation was the dramatic down-regulation of Txnip gene expression in response to ethanol. It is known that Txnip gene encodes for the thioredoxin interacting protein, a 55 kDa protein that stabilizes reduced thioredoxin and keeps it inactive, thereby functioning as an endogenous inhibitor (Yoshihara et al., 2014). Despite ethanol does not alter thioredoxin genes (Txn1 and Txn2) expression in HT22 cells, reduction in the amount of TXNIP would obviously increase free thioredoxin (Trx) proteins, which would enhance its ROS buffering capacity. This effect is physiologically relevant since mammalian TRXRD reduces oxidized substrates, such as Trx and H2O2, but also other non-disulfide-containing molecules, such lipid hydroperoxides and other hydroperoxides even independently of Trx, but coupled to selenocysteine or selenodiglutathione reduction (Björnstedt et al., 1995; Arnér and Holmgren, 2000), which notably increases the antioxidant spectrum of TRXR.

A family of proteins related to Trx and regulated by ethanol is peroxiredoxins. Peroxiredoxins contain two conserved cysteines in their active site and utilize Trx as reductant (Rhee et al., 2005), therefore, tightly linked to Trx oxidative status (Hawkes

data from three different experiments and are indicated as mean ± SEM. (C) Neuroprotective effects of 0.1% ethanol. Illustrated correspond to a representative experiment of glutamate excitotoxicity in the presence or absence of ethanol. Arrows indicate the time where cells were exposed to either DMEM, EtOH 0.1, or EtOH 0.1% + glutamate in 1, or EtOH 0.1%, EtOH 0.1% + glutamate, or glutamate alone in 2. Three different replicates were performed for experiments illustrated in (A,C).

et al., 2013).  We have observed that ethanol treatment brings about a generalized and significant down-regulation (18–39%) of all peroxyredoxin genes. These transcriptional alterations occurred soon after ethanol exposure (from 6 h) and lasted until 48 h. Noticeably, Srxn1 gene expression (which encodes for sulfiredoxin, the main protein responsible for reactivation of oxidized peroxiredoxins) remained unaffected in response to ethanol. At present, we have no explanation for this concerted reduction of peroxiredoxins but a plausible hypothesis is that by down-regulating their expression (at the same time Txnrd1 expression increases and Txnip expression decreases), reduction of Trx is mostly under the control of thioredoxin reductase activity, with lower amounts of reduced thioredoxin being used to reduce peroxiredoxins. As mentioned before, peroxiredoxins become oxidized upon ROS attack, and their reduction is coupled to oxidation of thioredoxin (Rhee et al., 2005). Holistically, such a mechanism will help to maintain higher cytosolic levels of reduced thioredoxin. Additional experiments, including determination of cytosolic levels of oxidized and reduced thioredoxin in response to ethanol, will help to unravel the physiological significance of these findings.

The last antioxidant system modulated by ethanol in HT22 cells is glutathione/glutaredoxin system. We observed that soon after ethanol exposure, expression levels of Gpx1 increased, and remained higher than in PBS throughout the experiment. Paralleling these findings, tGPx activity was higher in ethanoltreated cells from 6 h, therefore, correlating changes in expression levels. The increase in tGPx activity was likely attributable to GPx1 activity, since GPx4 activity (and gene expression) remained unaffected. Gclc gene expression also exhibited a significant increase in ethanol-treated cells, though in this case the response was delayed compared to Gpx1 gene, and was observed only after 30 h ethanol treatment. Clearly, this modulation of the glutathione/glutaredoxin system enhances the cellular antioxidant potential attributable to glutathione in neuronal cells, at the same time that provides an efficient strategy to ensure the reduction of oxidized glutathione. Indeed, in the present study, we have observed that levels of reduced (and also oxidized) glutathione remained unchanged in spite of the two ethanol challenges and also that TBARs levels were not different from those observed in PBS-treated cells throughout the experiment. These observations pinpoint to an efficient ROSbuffering capacity in HT22 cells in response to sub-toxic ethanol exposure. Interestingly, in agreement with our results, in vivo studies performed in the rat cerebral cortex and corpus striatum have shown that ethanol (1.6 g/kg) significantly increases total GPx (as well as SOD) activity (Somani et al., 1996).

It is well-known that an important effect of ethanol is to increase the generation of ROS, including superoxide and the hydroxyethyl radical (Das and Vasudevan, 2007). Generation of ethanol-derived ROS is expected to interact with cellular targets, particularly with membrane lipids, giving rise to lipid hydroperoxides, which, in turn, initiate a self-propagating oxidative damage (Niki et al., 2005). Therefore, it is expected that levels of lipid-related oxidative species progressively augment as time progresses. Although ROS are generally considered to exert deleterious effects, it is becoming increasingly evident that ROS may serve second messengers implicated in signaling processes, and participate in a number of normal physiological phenomena (Dröge, 2002). Thus, it is likely that ethanol-derived oxidized metabolites may be responsible for triggering transcriptional signals to boost the expression of components of cellular antioxidant systems, as we have previously observed for lipoperoxides derived from docosahexaenoic acid (Casañas-Sánchez et al., 2014). Indeed, It is known that some of the genes studied here, and upregulated by ethanol, contain "antioxidant response elements" (ARE) in their promoter regions (Kaspar et al., 2009; Hawkes et al., 2013). In this sense, recent studies have demonstrated that NF-E2-related factor 2 (Nrf2) is the master transcription factor for the regulation of ARE in different tissues, including the brain (Kobayashi and Yamamoto, 2005; Singh et al., 2010; Zhang et al., 2013).

Overall, we may conclude that sub-toxic ethanol exposure enhances global antioxidant capacity of hippocampal neurons by at least three mechanisms: (1) by enhancing the expression and activity of the generic system through up-regulating superoxide dismutase expression (2) by increasing thioredoxin reductase 1 expression, the most abundant isoform in neuronal tissue, at the same time that down-regulates Txnip and peroxiredoxins expression, and (3) by upregulating glutathione peroxidase 1 and glutathione-S-reductase genes expression. Taken together these observations led us to envisage that hippocampal cells become more resistant to oxidative insults after being exposed to acute sub-lethal ethanol. Indeed, recent in vivo evidence have shown that ethanol pre-conditioning render brain tissue more resistant to oxidative damage in animal models such as the ischemia-reperfusion injury in gerbil and rat models (Liao et al., 2003; Wang et al., 2007), pro-inflammatory lipopolysaccharideinjected rats (Singh et al., 2007), or even mice models of Alzheimers disease (Wang et al., 2006).

 Effects of alcohol pre-conditioning on inflammatory protein (gp120IIIB)-induced neurotoxicity have also been explored in organotypic slices of rat hippocampus-entorhinal cortex, two brain regions significantly impacted in Alzheimers disease and other dementias (Collins et al., 2000, 2010). More recently, Muñoz and coworkers have shown that low concentrations of ethanol protect against synaptotoxicity induced by Aβ in hippocampal neurons (Muñoz et al., 2015). In line with this, we show here that ethanol (0.1%) can prevent excitotoxicity induced by glutamate exposure, which is in consonance with the results reported for NMDA exposure in rat primary cultured cells (Chandler et al., 1993) or in rat cerebellar granular cells (Cebere and Liljequist, 2003) in a similar range of concentrations and time-course as used here.

Different mechanisms have been proposed to underlie the neuroprotective effects of sub-lethal ethanol exposure. Overall, an emerging hypothesis is that alcohol pre-conditioning-induced neuronal survival mechanisms involve induction of heat-shock proteins (HSP27 and/or HSP70) upon ROS generation, and that HSP-dependent protection is intimately associated to selective protein kinase C (PKCα and PKCδ) and FAK (focal adhesion kinase) activation, NOS (nitric oxide synthase), the focal adhesion complex, and stabilization of the cytoskeleton (Collins et al., 2009). However, the present study is the first demonstrating that ethanol provides resistance to oxidative insults through mechanisms directly linked to transcriptional modulation of specific components within the set of antioxidant systems. Therefore, under this paradigm, ethanol may be considered an "Indirect Antioxidant," as it has been coined for molecules which although lacking antioxidant activity per se, are capable to potentiate cellular antioxidant capacity by enhancing gene expression (Jung and Kwak, 2010). This newly identified neuroprotective (and perhaps anti-excitotoxic) effect of ethanol in vitro is clearly hormetic and might be clinically relevant (Rattan, 2004), but certainly it requires further studies before its significance and window of application is completely understood.

# AUTHOR CONTRIBUTIONS

VC and JP performed genetic analyses. DQ and VC performed analyses of enzyme activities. MD designed the study, analyzed the data and drafted the manuscript.

# REFERENCES

# ACKNOWLEDGMENTS

Supported by grants SAF2010-22114-C02-01 and SAF2014- 52582-R from Ministerio de Economía y Competitividad (Spain). VC and DQ held fellowships from Gobierno de Canarias and Fundación Cajacanarias, respectively. We thank Dr. Juan Carlos Sánchez-Hernández (Universidad Castilla-La Mancha, Spain) for advice and support on antioxidant enzymology.

Proc. Natl. Acad. Sci. U.S.A. 95, 7748–7753. doi: 10.1073/pnas.95. 13.7748

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2016 Casañas-Sánchez, Pérez, Quinto-Alemany and Díaz. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.


**11. GSK3 and NPC migration and polarization**

Correct positioning of newly generated neurons is crucial during development and for healthy function of the adult brain. Neuronal migration is tightly regulated by extracellular cues, including Wnts and growth factors. Accordingly, there is emerging evidence that their common downstream target, GSK3, may be involved in this process, primarily via regulation of cytoskeleton-associated proteins. Migration requires cellular polarization and extension at the leading edge of the cell. This involves dynamic reorganization of microtubules by a variety of microtubule-binding proteins. Several of these are directly phosphorylated and regulated by GSK3, including Tau, CRMP2, MAP1B, MAP2C, CLASP2, pVHL and APC (for reviews, see [145, 146]). Phosphorylation of many of these substrates by GSK3 reduces their ability to bind microtubules, thus making them less stable. Interestingly, GSK3 activity is typically low at the leading edge of migrating cells or at the tips of growing neurites in neuronal polarization [147-149]. This would promote substrate binding and stabilization of microtubules, facilitating forward movement of the cell's leading edge or growing neurite. Similarly, local inhibition of GSK3 activity is essential for polarization of newly generated neurons and growth of the nascent axon [150, 151]. Meanwhile, global inhibition of GSK3 induces formation of multiple axons [150, 152]. Several upstream inhibitors of GSK3 have been implicated in this process, including Cdc42 [149], ILK [153], LKB1 [147] and Akt [150]. Apart from the latter, these signaling proteins are not established regulators of GSK3 activity, so the precise molecular mechanisms by which they inhibit GSK3 activity await clarification.

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Author details

Jerry L. Hatfield\* and Christian Dold

\*Address all correspondence to<EMAIL_ADDRESS>
USDA-ARS National Laboratory for Agriculture and the Environment, Ames, Iowa, USA