Roy Schwartz and Zohar Habot-Wilner

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/60484

#### **Abstract**

Determining the cause of intraocular inflammation has important implications both for the treatment and prognosis of uveitic diseases. This chapter describes ocular di‐ agnostic procedures and their indications while mainly focusing on diagnostic vi‐ trectomy. The chapter discusses the history of elective diagnostic procedures; main indications for invasive procedures in the diagnosis of uveitic disease; surgical prin‐ ciples and techniques for each of the diagnostic procedures; descriptions of the vari‐ ous laboratory techniques being used; and selected examples of conditions that may require the use of such techniques.

**Keywords:** Uveitis, diagnostic vitrectomy, tap

### **1. Introduction**

The term uveitis refers to a large and varied group of disease entities, each with its own set of manifestations. While some may fit textbook characteristics, others may present in a way that baffles us as clinicians and leaves us with a wide differential diagnosis. Determining the cause of intraocular inflammation has important implications both for treatment and for prognosis of the disease. That is where the field of invasive diagnostic procedures comes into place. Through the use of different laboratory techniques, this diagnostic modality adds to the battery of other methods available to the clinician in order to reach the final diagnosis and provide proper management.

This chapter covers ocular diagnostic procedures, while focusing mainly on diagnostic vitrectomy.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


**4. Applications of POF grating devices**

*Application of Optical Fiber in Engineering*

uniform phase mask under strain performance [54]. The spectral reflected power of a 10 mm bandwidth with a chirp of ~0.26 nm/mm under 1.6% strain, and the strain and temperature sensitivity obtained with 0.71 ± 0.02 pm/με and 56.7 pm/°C. Then, Chirped POFBGs have been also obtained by hot water-assisted gradient thermal annealing, where one grating device with around 1.1 nm/mm chirp performance was obtained as **Figure 6** shown [55]. The simplicity of this method is one of the main advantages since no special phase mask or additional etching is needed, and enables easy control of the chirp characteristics and the central wavelength.

Finally, regarding LPG in POF, the extensive literatures with different methods and mechanisms appeared in the last years. Recently, the researcher in Spain [56]

*The spectrum and the bandwidth varies of the chirped POF FBG fabrication with strong gradient annealing.*

**34**

**Figure 6.**

**Figure 5.**

*The end face and the reflected spectrum of three types mPOF.*

POF FBG already goes to industry measurement such as used for water content detection in aviation fuel as shown in the **Figure 7**, the water content in Jet-A1 was measured by using POF FBG sensing technology which calibrated with both coulometric titration and environmental chamber. The results indicate a better performance compare with coulometric titration [58].

POF FBG can be also used to monitor the strain of human arteries with pulse wave signals. A variety of different vital signs including blood pressure can be derived from the signals, which show a higher signal to noise than silica FBG [59], the experimental measurement as **Figure 8** shown.

POF FBG can be used as health equipment for dynamic monitoring of gait. Five FBGs inscribed in CYTOP POF was embedded in a cork insole, as shown in **Figure 9**. The advantages of POF such as higher flexibility and robustness enabled monitoring patients with higher body mass, compared the results obtained with similar systems based on silica fiber, a mean sensitivity of ~8.14 PM/kPa was obtained, which is much higher compared with FBGs in silica optical fiber [60].

Consider the special POF grating applications, due to polymer special characteristics, strain sensing is the most attractive and reliable applications. There is a lot of literature reported POF FBG for strain sensing [61–63]. However, strain sensing under variable humidity and temperature conditions is always an issue for POF sensing technology go to real applications. The researchers in Spain demonstrated

**Figure 7.** *Schematic of the bench test rig [58].*

#### **Figure 8.**

*Experimental measurement of blood pressure uses silica FBG and POF FBG [59].*

#### **Figure 9.**

*Experimental measurement of blood pressure uses silica FBG and POF FBG. (a) Foot plantar area designation ans sensing point. (b) Polymer optical fiber Bragg grating embedded cork insole [60].*

one method use the effective bandwidth of the tunable chirped POFBG, which is highly dependent on the strain and remains practically constant with temperature and humidity changes, can be implemented combine with wavelength measurement, for strain sensors under temperature and humidity variable environments, the spectrum varies under strain condition as **Figure 10** shown [64].

Due to the polymer characteristics, POF grating devices are attracting attention for biomedical applications. An essential feature of these systems is the possibility to detect temperature spatial distributions, which also name as thermal maps. A linearly chirped POF FBG reported as a semi-distributed temperature sensor for monitor the temperature profile along the grating length as **Figure 11** shown [65]. The grating device has been placed close to the radiofrequency applicator, which have one tip inserted in situ of the target. The reflection spectrum of the chirped

**37**

applications.

**Figure 11.**

*Schematic of thermal ablation experiment [65].*

**Figure 10.**

*strain; (c) Bandwidth vs strain [64].*

**Figure 12**.

*Fabrication and Application of Polymer Optical Fiber Grating Devices*

POF FBG was detected by LUNA OBR 4600 and the temperature gradient was estimated use the Gaussian model method. The results indicate that chirped POF FBG can provide significant improvement in thermal detecting for biomedical

*Chirped POF FBG spectrum varies with strain. (a) Spectral reflected power vs strain; (b) Wavelength shift vs*

Also, a high spatial resolution distributed strain sensing approach based on Chirped POF FBG was reported by researchers in China [66], through spatial wavelength encoded characteristic of Chirped POF FBG, a fully distributed high resolution strain measurement can be achieved by optical frequency domain reflectometry method, which is a promising approach for short-range fully distributed sensing systems, schematic of the experiment setup shown in

*DOI: http://dx.doi.org/10.5772/intechopen.94351*

*Fabrication and Application of Polymer Optical Fiber Grating Devices DOI: http://dx.doi.org/10.5772/intechopen.94351*

#### **Figure 10.**

*Application of Optical Fiber in Engineering*

one method use the effective bandwidth of the tunable chirped POFBG, which is highly dependent on the strain and remains practically constant with temperature and humidity changes, can be implemented combine with wavelength measurement, for strain sensors under temperature and humidity variable environments,

*Experimental measurement of blood pressure uses silica FBG and POF FBG. (a) Foot plantar area designation*

Due to the polymer characteristics, POF grating devices are attracting attention for biomedical applications. An essential feature of these systems is the possibility to detect temperature spatial distributions, which also name as thermal maps. A linearly chirped POF FBG reported as a semi-distributed temperature sensor for monitor the temperature profile along the grating length as **Figure 11** shown [65]. The grating device has been placed close to the radiofrequency applicator, which have one tip inserted in situ of the target. The reflection spectrum of the chirped

the spectrum varies under strain condition as **Figure 10** shown [64].

*ans sensing point. (b) Polymer optical fiber Bragg grating embedded cork insole [60].*

*Experimental measurement of blood pressure uses silica FBG and POF FBG [59].*

**36**

**Figure 9.**

**Figure 8.**

*Chirped POF FBG spectrum varies with strain. (a) Spectral reflected power vs strain; (b) Wavelength shift vs strain; (c) Bandwidth vs strain [64].*

#### **Figure 11.**

*Schematic of thermal ablation experiment [65].*

POF FBG was detected by LUNA OBR 4600 and the temperature gradient was estimated use the Gaussian model method. The results indicate that chirped POF FBG can provide significant improvement in thermal detecting for biomedical applications.

Also, a high spatial resolution distributed strain sensing approach based on Chirped POF FBG was reported by researchers in China [66], through spatial wavelength encoded characteristic of Chirped POF FBG, a fully distributed high resolution strain measurement can be achieved by optical frequency domain reflectometry method, which is a promising approach for short-range fully distributed sensing systems, schematic of the experiment setup shown in **Figure 12**.

**Figure 12.** *Schematic of the experiment setup for fully distributed strain measurement [66].*


*2.5. E*ff*ect of AT1R Deficiency on the Severity of Inflammatory Cell Infiltration in TNFtg Mice*

Since an excess of exogenous Ang II accelerated inflammatory bone destruction (Figure 2A), we investigated whether endogenous Ang II could play a role in bone destruction using AT1R-deficient arthritic mice generated by crossing TNFtg mice with AT1R-knockout (AT1R−/−) mice. AT1R deficiency did not significantly alter body weight (Figure 4A). We found that the severity of clinical arthritis (Figure 4B,C) and the extent of inflammatory cell infiltration (Figure 4D,E) were comparable between TNFtg and TNFtg/AT1R−/− mice. In addition, the severity of cartilage damage was not affected by AT1R deficiency (Figure 4D,F).

**Figure 4.** Effect of angiotensin II type 1 receptor (AT1R) deficiency on the severity of inflammatory cell infiltration in tumor necrosis factor-transgenic (TNFtg) mice. TNFtg mice were crossed with AT1R-deficient (AT1R−/−) mice. WT (*n* = 9), AT1R−/− (*n* = 7), TNFtg (*n* = 12), and TNFtg AT1R−/− (*n* = 8) male mice were analyzed at the age of 16 weeks. (**A**) Body weight at the age of 16 weeks. (**B**) Arthritis score at the age of 16 weeks. (**C**) The number of arthritic paws with an arthritis score of 2 or higher. (**D**) Representative images of stained sections around the talus bones of indicated mice; sections were stained with hematoxylin and eosin (H&E) and Safranin O; original magnification ×40. (**E**,**F**) Histological scores of inflammation (**E**) and cartilage damage (**F**). Values are the mean ± SEM. n.s., not significant. \*, *p* < 0.05.


*4.2. Energy Production*

Among the nutrient management options, gross and net energy output was affected significantly by the rate of N and P application, Zn fertilization and microbial inoculation. The rate of N and P application had the highest contribution to variation in energy production, while Zn fertilization had the lowest contribution to energy production. The highest gross energy in RDN + Zn was the outcome of highest yield, while the highest net energy production in 75% RDN + Zn + MC1 or MC2 was due to reduction in cost of cultivation on 25% of N and P fertilizer. The difference in energy input across CEMs had a higher contribution to the variation in net energy production than gross energy production. The variation in energy input across CEM was 6.53 × 103 to 15.47 × 103 MJ ha−1 for rice, 3.95 to 12.19 MJ ha−1 for wheat and 10.49 to 27.66 MJ ha−1 for RWCS, while variation in gross energy production was 125.0–157.1, 125.7–153.1 and 250.9–309.2 MJ ha−1 for rice, wheat and RWCS, respectively.

The nutrient application through chemical fertilizers is the single most important source of nutrients. Its importance has increased over the years due to increasing nutrient deficiency [55,56], response to fertilization and use of high-yielding nutrient responsive varieties. In terms of energy, fertilizer contributes 59–64% to total energy input in RWCS and the cost of chemical fertilizer is also going to increase in future on account of the increasing cost of fertilizer production, depletion in natural reserves and increasing demand. The rice and wheat together contribute 61% (17.67 million tonnes) to total fertilizer consumption in India. Considering this, complimentary options such as use of microbial inoculations with partial replacement of chemical fertilizers will help in making the RWCS more energy-efficient.


*Polymer-Based Membranes for C3+ Hydrocarbon Removal from Natural Gas DOI: http://dx.doi.org/10.5772/intechopen.103903*

#### **Figure 5.**

*Schematic of the NGL recovery process: (A) without membrane; (B) utilizing rubbery membrane to divert NAG stream to feed LRU.*

**Figure 6.** *Schematic of the cascaded-refrigeration process to recover C2+ NGL from natural gas.*

gas leaving the third cooling stage is recycled back to exchange heat with gas feeding each stage and leaving the first stage at around 450 psi (referred to as high pressure (HP) residue gas). The liquids collected in the three stages are fed to the demethanizer, which is operating at lower pressure. The C2+ NGL product is obtained as the bottom product, while the top gas product from the demethanizer (low pressure (LP) residue gas) is consolidated with HP residue stream and sent to SG network. This LRU process is ideally designed to process AG stream, which is C2 <sup>+</sup> rich and free of or very lean in nitrogen. However, under certain situations of higher demand for C2+ NGL, the LRU is designed or operated with feed that is composed partially of NAG. As noted in **Table 4**, the N2 content of NAG stream is higher, and the C2+ content is considerably lower, than in AG stream.

**Table 5** compares the performance of the NGL process when 140 MMSCFD of the NAG stream is diverted to mix with the 500 MMSCFD AG stream. In reference (base case), **Table 5** provides the performance of the NGL process when only AG stream is fed to the LRU. When portion of NAG is let down directly to mix with AG, the condensability of the feed stream to LRU is reduced, as indicated by the increase in N2 content and reduction in the C2+ content. Nevertheless, the C3+ NGL liquids production has increased as a direct result of the increase in feed to LRU, while liquid C2 production has decreased in almost similar rate rendering the cascaded-refrigeration NGL recovery process not sensitive to the increase in the flow of C2+ to LRU. On the other hand, diverting part of the NAG selectively through membrane has resulted in increasing the condensability of the feed to LRU. As a result of enhancing the


Meet the editor

Yves Rybarczyk received a PhD degree in Robotics from the University of Evry, France, in 2004. His teaching and research activities focus on human–machine interaction and artificial intelligence. He was assistant professor at the Nova University of Lisbon between 2007 and 2015. Then, he moved to South America where he held the position of Associate Professor and Head of the Intelligent and Interactive Systems Laboratory at

the Universidad de Las Américas until 2019. Currently, he is an associate professor at the University of Skövde, Sweden. He has participated in several projects on the modeling and development of complex and interactive systems. He is the author of over 80 scientific publications. Prof. Rybarczyk is a member of the steering committee of the International Summer Workshops on Multimodal Interfaces and a member of the editorial board of *IEEE Latin America Transactions*.

Contents

**Section 1**

**Section 2**

and Rehabilitation Systems

Recovery in Post-Hip Arthroplasty Patients

*by Yves Rybarczyk*

*and Sophia Ortiz*

Systems

Control

**Section 3**

*by Keiko Tsujioka*

Study—ePHoRt Project

**Preface III**

Introduction **1**

**Chapter 1 3**

State of the Art **9**

**Chapter 2 11**

**Chapter 3 31**

**Chapter 4 51**

Rehabilitation Systems **71**

**Chapter 5 73**

Technical Contributions to the Quality of Telerehabilitation Platforms: Case

*by Patricia Acosta-Vargas, Janio Jadán-Guerrero, Cesar Guevara,*

*Sandra Sanchez-Gordon and Tania Calle-Jimenez*

Introductory Chapter: Transdisciplinary Considerations on Assistive

Toward a Design of a Telerehabilitation Program for the Functional

*by Wilmer Esparza, Arian Ramón Aladro-Gonzalvo, Jonathan Baldeon*

A Systematic Review of Usability and Accessibility in Tele-Rehabilitation

*by Jorge Luis Pérez Medina, Patricia Acosta-Vargas and Yves Rybarczyk*

Dual Loop Theory: Eidetic Feedback Control and Predictive Feedback


**3.1 TNF- gene**

416 Amyotrophic Lateral Sclerosis

parameters and infiltrating T cells (Holmoy et al., 2008). The majority of T cells characterized in the infiltration were CD8+ cytotoxic T cells, but a substantial number of T CD4+ cells

Alterations of total lymphocyte count (Provinciali et al., 1988; Tavolato et al., 1975) and T subset distribution in peripheral system of ALS patients (Westall et al., 1983) have been reported. Low T cells numbers and decreased proliferative capacity in T cells are found in

As concerned CD8+ and natural killer T cells, they were found increased in ALS patients

Interestingly, ALS patients showed a reduction of CD4+/CD25+ regulatory T cells that are known to interact with the local microglia, reinforcing the hypothesis of the involvement of the adaptive immune system associated with neuroinflammatory process in ALS (Mantovani et al., 2009). Beers and colleagues (Beers et al., 2011) observed that regulatory T cells as CD4+/CD25+/FoxP3 correlated with disease progression; in fact, the number of T

Animal studies showed that in ALS model T cells deficiency decreases microglia reactivity and accelerates ALS disease progression; specific and progressive accumulation of monocytes/macrophages was observed along the length of degenerating nerve fibers and activated microglia was detected in spinal cord of ALS model mice (Chiu et al., 2009). No infiltrating B-cells have been found even if a role of B lymphocytes in the pathogenesis of ALS has been hypothesized, as secreted autoantibodies by B cells identified in CSF and

As concern antibodies, since the eighties the presence of IgG in serum or tissues of ALS patients has been documented (anti-ganglioside GM1, anti-sulfoglucuronyl paragloboside, anti-neurofilaments and anti-Fas) (Sengun & Appel, 2003; Yi et al., 2000). Indeed, IgG deposits have been demonstrated in motor cortex, spinal cord and in motor neurons from ALS patients (Donnenfeld et al., 1984; Engelhardt & Appel, 1990; Fishman & Drachman, 1995). Serum immunoglobulins from ALS patients showed enhanced binding to rat spinal cord cells *in vitro* (Digby et al., 1985), demonstrating cytotoxic effects when they were added to a motor neuron cell cultures (Alexianu et al., 1994; Demestre et al., 2005) and that the presence of an immune response to spinal cord cell membrane components in patients with

IgG from ALS patients reacts with the skeletal muscle DHP (bisognerebbe spiegare cosa è) sensitive Ca2+ channels reducing the peak of the Ca2+ current and the charge movement in single cut fibres from the rat extensor muscle (Delbono et al., 1991). About 60% of ALS sera contained different monoclonal immunoglobulins: in particular IgG (72.7%) and IgM

Different interactions have been found between innate and adaptive immunity in ALS, as concern cytokines involvement. Cytokines have an effect on the expression of other inflammatory factors and on each other, and these functional relationships are non-linear: the causal relationships of cytokines and disease are complex and difficult to prove

Several studies regarding immune system changes in sporadic ALS reveal that there are increased levels of circulating monocytes and macrophages, producing cytokines as IL-1

were also present (Beers et al., 2008).

the blood of ALS patients (Holmoy et al., 2008).

compared to control cohort (Rentzos et al., 2011).

serum from ALS patients (Naor et al., 2009).

motor neuron disease was a damaging event.

(27.3%) have been found (Duarte et al., 1991).

**2.3 Cytokines**

(Marklund et al., 1992).

cells were found inversely correlated with disease progression rate.

The gene coding for TNF- is located on chromosome 6 within the region encoding the Major Histocompatibility Complex (MCH), HLA in human, between the HLA-DR class II and HLA-B class I genes (Fig. 3). Its location and strict linkage disequilibrium present between some alleles of class I and class II genes has permitted to hypothesize associations between TNF- alleles and some diseases. The gene for TNF- include about 3 Kb and contains four exons (almost 80% of the protein is codified by the exon four) and three introns.

The Role of TNF-Alpha in ALS: New Hypotheses for Future Therapeutic Approaches 419

As concerned -238 polymorphism, has been described a direct effect on gene expression, although studies suggest that this region contains a strong repressor site (-280 to -172). -238 TNFG/A allele genotype may be in linkage disequilibrium with a functional polymorphism

Several of these polymorphisms have been studied extensively in some diseases, mainly -

The mutated allele -308A is a marker of susceptibility to several autoimmune and inflammatory diseases such as lupus erythematosus, celiac disease and Alzheimer's disease (Candor et al., 2002 and 2004), but so far it has been published in a very large number of

Most SNPs (-863, -238, -1031) do not affect the levels of expression but their pathologic

As concerned SNPs in coding region, some polymorphisms are described (http://www.ncbi.nlm.nih.gov/SNP) but they are not correlated with any diseases or

The functions of TNF- are biologically dependent on the amount of cytokine produced. If it is present in small amounts, TNF- acts locally as autocrine and paracrine mediator of inflammation in leukocytes and endothelial cells, determines the expression of surface receptors for leukocyte migration, acting as an angiogenic factor, such as fibroblast growth factor and induces apoptosis in certain cell types. If it is present in high amounts, however, is distributed in the systemic circulation where it stimulates the production of IL-1 and IL-6 by leukocytes and the synthesis of acute phase proteins in the liver (such as fibrinogen) and

TNF- exerts many of its effects by binding, as a trimer, the cell membrane receptor TNF-R1 of 55 kDa (p55) or TNF-R2 of 75 kDa (p75) belonging to the superfamily of TNF receptors,

TNF-R1 is expressed in almost all tissues and may be activated by trimeric soluble form and also by membrane-associated form of TNF-; TNF-R2 is expressed only by cells of the immune system and it is activated only by trimeric soluble form of the TNF-. Differently from TNF-R1, TNF-R2 do not own a death domain (DD), and its activation may only induce

Instead, the binding of TNF- to the TNF-R1 may cause both cell death or survival depending on which pathway is activated and it also depends on the second signal involved (Hsu et al., 1996; Darnay et al.,1997). Following the binding of TNF-, TNF-R1 produces a conformational change that determines the separation of the intracellular death domain (DD). This dissociation allows the adapter protein TRADD to bind the domain of death (DD) to induce apoptosis or form a platform for the subsequent binding proteins to make cell survival. If TRADD directly binds FADD, which in turn recruits caspase-8, the apoptosis way is activated. High concentrations of caspase-8 induce its proteolytic activation and subsequent cleavage of downstream caspases, leading to cell apoptosis. Cell death induced by TNF- plays a minor role compared to the role of this cytokine in inflammation. Its ability to induce apoptosis is in fact modest when compared to that of other members of the

works in order to reach the correct conclusions on the role of these polymorphisms.

involvement is related to the variation of allelic or genotypic frequencies.

that impacts TNF production (Liu et al., 2008).

activates the intravascular thrombus formation.

which also includes FAS, CD40, CD27 and RANK.

family as Fas and often masked by the anti-apoptotic effects.

308 and -857 SNPs.

functions.

**3.2 TNF- protein function**

the survival pathway (Fig. 4).

Fig. 3. Localization of TNF-α gene on chromosome 6 (6p21.3)

TNF- gene codifies for a protein of 233 amino acids with a molecular weight of 25.6 kDa. TNF- is, at the beginning, a transmembrane protein of 212 amino acids associated to homotrimers; the N-terminal portion loses 76 amino acid by cleavage of TNF- converting enzyme (TACE or ADAM-17), producing a soluble monomeric TNF- form (17 kD) and next, the trimetric form of 51 kD (Bazzoni & Beutler, 1996; Reddy et al., 2000).

The trimer is the biological active soluble form because of its ability to bind its receptors. However, the trimeric soluble form spontaneously tends to dissociate into a monomeric inactive form, that is a physiological process that allows to limit the deleterious effects of excessive concentration of TNF- (Smith et al., 1987).

TNF- response to a variety of extracellular signals is very rapid and transient and includes a transcriptional component as well as posttranscriptional events. Its transcriptional control occurs predominantly at the level of transcriptional initiation.

The approximately 1000 base pairs of the TNF- gene's 5' flanking region contains a number of important regulatory elements that affect TNF- transcription in response to various stimuli. The basic promoter region is defined by TATA box sequence, located about 20 bp upstream from the transcription site and about 200 bp form the translation start codon. Multiple potential regulatory sites, including consensus sequences for the AP-1 (Activator Protein-1) and AP-2 (Activator Protein-2) sites, the cAMP-responsive element, and sequences similar to the NF-kB, (Nuclear Factor kappa-light-chain-enhancer of activated B cells) sequences found in immunoglobulin and cytokine regulatory elements are present in 5' flanking region. This sequence has been demonstrated to be responsive to LPS (lipolysaccharide) and TNF- stimulation. The 3' untranslated region contains a sequence element affecting posttranslational control of TNF- through mRNA stability and translation efficiency.

Many polymorphisms have been described in TNF- promoter region (-308, -857, -863, -238, -1031) defining its correlation with TNF- mRNA amounts: wild-308G allele is responsible for a higher transcription gene (Helmig et al., 2011), as the A mutated allele at position -857 results in a high production of TNF- (McCusker et al., 2001).

As concerned -238 polymorphism, has been described a direct effect on gene expression, although studies suggest that this region contains a strong repressor site (-280 to -172). -238 TNFG/A allele genotype may be in linkage disequilibrium with a functional polymorphism that impacts TNF production (Liu et al., 2008).

Several of these polymorphisms have been studied extensively in some diseases, mainly - 308 and -857 SNPs.

The mutated allele -308A is a marker of susceptibility to several autoimmune and inflammatory diseases such as lupus erythematosus, celiac disease and Alzheimer's disease (Candor et al., 2002 and 2004), but so far it has been published in a very large number of works in order to reach the correct conclusions on the role of these polymorphisms.

Most SNPs (-863, -238, -1031) do not affect the levels of expression but their pathologic involvement is related to the variation of allelic or genotypic frequencies.

As concerned SNPs in coding region, some polymorphisms are described (http://www.ncbi.nlm.nih.gov/SNP) but they are not correlated with any diseases or functions.

### **3.2 TNF- protein function**

418 Amyotrophic Lateral Sclerosis

TNF- gene codifies for a protein of 233 amino acids with a molecular weight of 25.6 kDa. TNF- is, at the beginning, a transmembrane protein of 212 amino acids associated to homotrimers; the N-terminal portion loses 76 amino acid by cleavage of TNF- converting enzyme (TACE or ADAM-17), producing a soluble monomeric TNF- form (17 kD) and

The trimer is the biological active soluble form because of its ability to bind its receptors. However, the trimeric soluble form spontaneously tends to dissociate into a monomeric inactive form, that is a physiological process that allows to limit the deleterious effects of

TNF- response to a variety of extracellular signals is very rapid and transient and includes a transcriptional component as well as posttranscriptional events. Its transcriptional control

The approximately 1000 base pairs of the TNF- gene's 5' flanking region contains a number of important regulatory elements that affect TNF- transcription in response to various stimuli. The basic promoter region is defined by TATA box sequence, located about 20 bp upstream from the transcription site and about 200 bp form the translation start codon. Multiple potential regulatory sites, including consensus sequences for the AP-1 (Activator Protein-1) and AP-2 (Activator Protein-2) sites, the cAMP-responsive element, and sequences similar to the NF-kB, (Nuclear Factor kappa-light-chain-enhancer of activated B cells) sequences found in immunoglobulin and cytokine regulatory elements are present in 5' flanking region. This sequence has been demonstrated to be responsive to LPS (lipolysaccharide) and TNF- stimulation. The 3' untranslated region contains a sequence element affecting posttranslational control of TNF- through mRNA stability and

Many polymorphisms have been described in TNF- promoter region (-308, -857, -863, -238, -1031) defining its correlation with TNF- mRNA amounts: wild-308G allele is responsible for a higher transcription gene (Helmig et al., 2011), as the A mutated allele at position -857

next, the trimetric form of 51 kD (Bazzoni & Beutler, 1996; Reddy et al., 2000).

Fig. 3. Localization of TNF-α gene on chromosome 6 (6p21.3)

excessive concentration of TNF- (Smith et al., 1987).

translation efficiency.

occurs predominantly at the level of transcriptional initiation.

results in a high production of TNF- (McCusker et al., 2001).

The functions of TNF- are biologically dependent on the amount of cytokine produced. If it is present in small amounts, TNF- acts locally as autocrine and paracrine mediator of inflammation in leukocytes and endothelial cells, determines the expression of surface receptors for leukocyte migration, acting as an angiogenic factor, such as fibroblast growth factor and induces apoptosis in certain cell types. If it is present in high amounts, however, is distributed in the systemic circulation where it stimulates the production of IL-1 and IL-6 by leukocytes and the synthesis of acute phase proteins in the liver (such as fibrinogen) and activates the intravascular thrombus formation.

TNF- exerts many of its effects by binding, as a trimer, the cell membrane receptor TNF-R1 of 55 kDa (p55) or TNF-R2 of 75 kDa (p75) belonging to the superfamily of TNF receptors, which also includes FAS, CD40, CD27 and RANK.

TNF-R1 is expressed in almost all tissues and may be activated by trimeric soluble form and also by membrane-associated form of TNF-; TNF-R2 is expressed only by cells of the immune system and it is activated only by trimeric soluble form of the TNF-. Differently from TNF-R1, TNF-R2 do not own a death domain (DD), and its activation may only induce the survival pathway (Fig. 4).

Instead, the binding of TNF- to the TNF-R1 may cause both cell death or survival depending on which pathway is activated and it also depends on the second signal involved (Hsu et al., 1996; Darnay et al.,1997). Following the binding of TNF-, TNF-R1 produces a conformational change that determines the separation of the intracellular death domain (DD). This dissociation allows the adapter protein TRADD to bind the domain of death (DD) to induce apoptosis or form a platform for the subsequent binding proteins to make cell survival. If TRADD directly binds FADD, which in turn recruits caspase-8, the apoptosis way is activated. High concentrations of caspase-8 induce its proteolytic activation and subsequent cleavage of downstream caspases, leading to cell apoptosis. Cell death induced by TNF- plays a minor role compared to the role of this cytokine in inflammation. Its ability to induce apoptosis is in fact modest when compared to that of other members of the family as Fas and often masked by the anti-apoptotic effects.

The Role of TNF-Alpha in ALS: New Hypotheses for Future Therapeutic Approaches 421

As concerns the activation of MAP kinase (MAPK), TNF- induces a strong activation of JNK (c-Jun N-terminal Kinase), one of three major cascades of MAPK, evokes a moderate response of the p38-MAPK, and is responsible of a minimal activation of ERK. TRAF2 actives MEKK1 and ASK1 directly or indirectly, which phosphorylate MKK7, which in turn activates JNK. The latter AP-1 translocates into the nucleus and activates transcription factors such as c-Jun and AFT2. The way of JNK is involved in differentiation, cell

In the central nervous system TNF- is produced by astrocytes, microglia and neurons in response to several stimuli both intra and extracellular, and seems to play a central role in the genesis and perpetuation of neuroinflammatory signal. An alteration in the regulatory mechanisms of TNF- was found in a wide variety of disorders such as depression, carcinogenesis, and Alzheimer's disease. In the nervous system different TNF- activities were defined, as inducer or inhibitor of neuronal apoptosis underlining how complex TNF-

Moreover, electrophysiological experiments have shown a negative effect determined by TNF- on neuronal function. Studies of hippocampal sections showed that the addition of pro-inflammatory cytokines decreased the long-term potentiation (Long-term potentiation, LTP), a correlate of learning and memory processes (Tancredi et al., 1992). The mechanism by which this occurs is still under investigation, but it has been suggested that activation of p38 plays a major role in reducing the early phase of LTP in response to TNF-, while the

However, other studies have shown that TNF- alone is not able to initiate apoptosis in the absence of a second signal and may actually prevent apoptosis in response to certain types of cell damage (Badiola et al., 2009). In fact, although apoptosis is primarily triggered by the TRADD, TNF- may also be associated with cell survival signals, because it is also able to facilitate the binding of other molecules such as JNK and NF-kB, which were instead important in cells endurance, indicating how complex are the signalling pathways of TNF-. In fact, TNF- facilitates axonal regeneration, induces neuronal survival through the antiapoptotic pathway mediated by NF-kB, limits the demyelination in experimental autoimmune encephalomyelitis, but these effects appear to be highly dose-dependent and

protein expression changes would play a role in the late phase (Butler et al., 2004).

related to the exposure time and interaction with other factors (Schwartz et al., 1991).

presence/absence of secondary signals from endogenous or exogenous stimuli.

glial cells were detected in CSN from PD patients (Imamura et al., 2003).

The protective effect of the cascade triggered by TNF- has been documented experimentally: mice lacking the receptor for TNF- were subjected to cerebral ischemia. This result was attributed to an increase of reactive oxygen species, suggesting that TNF- could induce an antioxidant protection due to ischemic events (Bruce et al., 1996). Similarly, in a model of glutamatergic excitotoxicity, stimulation of TNF-R2 with TNF- led to protection against this toxicity (Marchetti et al., 2004). In conclusion, these data suggest that TNF- could affect neuronal viability in different ways depending on the receptor subtype involved and the

In neurodegenerative disease, as Parkinson's (PD) and Alzheimer's (AD), neuroinflammatory processes appear to play key roles in neuronal dysfunction and death (Hakansson et al., 2005). TNF- was found increased in striatum and CSF from PD patients compared to controls (Nagatsu et al., 2000), and a large number of TNF- immunoreactive

proliferation and apoptosis.

pathway may be.

**3.3 TNF- and nervous system**

**Copyright:** The Brazilian Journal of Medical and Biological Research (BJMBR) applies the Creative Commons Attribution License (CCAL) to all works published (read the human-readable summary or the full license legal code). Under the CCAL, authors retain ownership of the copyright for their article and can allow anyone to download, reuse, reprint, modify, distribute, and/or copy articles published in the BJMBR, as long as the original authors and source are cited.

Fig. 4. TNF- and TNFR pathway (Rosenquist M., 2003)

Both TNF- receptors activate different survival intracellular signalling pathways, especially because of the IkB kinase (IKK) and the cascade of MAP kinases (MAPKs) that control the gene expression through NF-kB, and AP-1 respectively. In detail the term NF-kB refers to a family of five structurally-related transcription factors (p50, p52, RelA/ p65, c-Rel and RelB), all containing the Rel homology domain (RHD) within the N-terminus and acting as homoand heterodimeric DNA binding complexes (O'Dea et al., 2010). Several studies showed that NF-kB activity is induced in most cell types in response to a broad variety of stimuli, ranging from cytokines, radiation and reactive oxygen species (ROS) (such as exposure to H2O2), with major roles in coordinating innate and adaptative immunity, cell activation and proliferation, survival, development and apoptosis (Ghosh et al., 2008; Vallabhapurapu et al., 2009).

TRADD recruits TRAF2 (TNF receptor-associated factor 2) and RIP (receptor interacting protein). TRAF2 in turn recruits the protein kinase IKK (inhibitor of nuclear factor kappa-B kinase), which is activated by RIP. The IKBα inhibitory protein that normally binds the NFkB and inhibits its translocation, is phosphorylated by IKK and then is degraded, releasing NF-kB. The latter NF-kB is a transcription factor that translocates into the nucleus and mediates the transcription of a wide variety of genes and theirs products involved in survival and cell proliferation, in the inflammatory response and anti-apoptotic factors.

As concerns the activation of MAP kinase (MAPK), TNF- induces a strong activation of JNK (c-Jun N-terminal Kinase), one of three major cascades of MAPK, evokes a moderate response of the p38-MAPK, and is responsible of a minimal activation of ERK. TRAF2 actives MEKK1 and ASK1 directly or indirectly, which phosphorylate MKK7, which in turn activates JNK. The latter AP-1 translocates into the nucleus and activates transcription factors such as c-Jun and AFT2. The way of JNK is involved in differentiation, cell proliferation and apoptosis.

### **3.3 TNF- and nervous system**

420 Amyotrophic Lateral Sclerosis

**Copyright:** The Brazilian Journal of Medical and Biological Research (BJMBR) applies the Creative Commons Attribution License (CCAL) to all works published (read the human-readable summary or the full license legal code). Under the CCAL, authors retain ownership of the copyright for their article and can allow anyone to download, reuse, reprint, modify, distribute, and/or copy articles published in

Both TNF- receptors activate different survival intracellular signalling pathways, especially because of the IkB kinase (IKK) and the cascade of MAP kinases (MAPKs) that control the gene expression through NF-kB, and AP-1 respectively. In detail the term NF-kB refers to a family of five structurally-related transcription factors (p50, p52, RelA/ p65, c-Rel and RelB), all containing the Rel homology domain (RHD) within the N-terminus and acting as homoand heterodimeric DNA binding complexes (O'Dea et al., 2010). Several studies showed that NF-kB activity is induced in most cell types in response to a broad variety of stimuli, ranging from cytokines, radiation and reactive oxygen species (ROS) (such as exposure to H2O2), with major roles in coordinating innate and adaptative immunity, cell activation and proliferation,

survival, development and apoptosis (Ghosh et al., 2008; Vallabhapurapu et al., 2009).

TRADD recruits TRAF2 (TNF receptor-associated factor 2) and RIP (receptor interacting protein). TRAF2 in turn recruits the protein kinase IKK (inhibitor of nuclear factor kappa-B kinase), which is activated by RIP. The IKBα inhibitory protein that normally binds the NFkB and inhibits its translocation, is phosphorylated by IKK and then is degraded, releasing NF-kB. The latter NF-kB is a transcription factor that translocates into the nucleus and mediates the transcription of a wide variety of genes and theirs products involved in survival and cell proliferation, in the inflammatory response and anti-apoptotic factors.

the BJMBR, as long as the original authors and source are cited. Fig. 4. TNF- and TNFR pathway (Rosenquist M., 2003)

In the central nervous system TNF- is produced by astrocytes, microglia and neurons in response to several stimuli both intra and extracellular, and seems to play a central role in the genesis and perpetuation of neuroinflammatory signal. An alteration in the regulatory mechanisms of TNF- was found in a wide variety of disorders such as depression, carcinogenesis, and Alzheimer's disease. In the nervous system different TNF- activities were defined, as inducer or inhibitor of neuronal apoptosis underlining how complex TNF pathway may be.

Moreover, electrophysiological experiments have shown a negative effect determined by TNF- on neuronal function. Studies of hippocampal sections showed that the addition of pro-inflammatory cytokines decreased the long-term potentiation (Long-term potentiation, LTP), a correlate of learning and memory processes (Tancredi et al., 1992). The mechanism by which this occurs is still under investigation, but it has been suggested that activation of p38 plays a major role in reducing the early phase of LTP in response to TNF-, while the protein expression changes would play a role in the late phase (Butler et al., 2004).

However, other studies have shown that TNF- alone is not able to initiate apoptosis in the absence of a second signal and may actually prevent apoptosis in response to certain types of cell damage (Badiola et al., 2009). In fact, although apoptosis is primarily triggered by the TRADD, TNF- may also be associated with cell survival signals, because it is also able to facilitate the binding of other molecules such as JNK and NF-kB, which were instead important in cells endurance, indicating how complex are the signalling pathways of TNF-. In fact, TNF- facilitates axonal regeneration, induces neuronal survival through the antiapoptotic pathway mediated by NF-kB, limits the demyelination in experimental autoimmune encephalomyelitis, but these effects appear to be highly dose-dependent and related to the exposure time and interaction with other factors (Schwartz et al., 1991).

The protective effect of the cascade triggered by TNF- has been documented experimentally: mice lacking the receptor for TNF- were subjected to cerebral ischemia. This result was attributed to an increase of reactive oxygen species, suggesting that TNF- could induce an antioxidant protection due to ischemic events (Bruce et al., 1996). Similarly, in a model of glutamatergic excitotoxicity, stimulation of TNF-R2 with TNF- led to protection against this toxicity (Marchetti et al., 2004). In conclusion, these data suggest that TNF- could affect neuronal viability in different ways depending on the receptor subtype involved and the presence/absence of secondary signals from endogenous or exogenous stimuli.

In neurodegenerative disease, as Parkinson's (PD) and Alzheimer's (AD), neuroinflammatory processes appear to play key roles in neuronal dysfunction and death (Hakansson et al., 2005). TNF- was found increased in striatum and CSF from PD patients compared to controls (Nagatsu et al., 2000), and a large number of TNF- immunoreactive glial cells were detected in CSN from PD patients (Imamura et al., 2003).

The Role of TNF-Alpha in ALS: New Hypotheses for Future Therapeutic Approaches 423

TNF- and its soluble receptors, sTNFRs, were already found significantly higher in plasma of ALS patients than in those of healthy controls (Poloni et al., 2000). They found a significant correlation between levels of TNF- and sTNF-R1 and sTNF-R2, confirming that a general activation of the TNF- system occurred in ALS patients. Activation of the TNF- system however did not correlate neither with the disease duration nor with the disease severity. Even after dividing the patients in two subgroups, with high and low TNF- levels, they did not

Our research group analyzed the possible implication of TNF- pathway in ALS pathogenesis (Cereda et al., 2008). We assayed both the levels of TNF- and its soluble receptors in plasma from ALS patients overtime during disease progression. We assayed the concentrations of TNF- and its soluble receptors in plasma of 88 patients with sporadic ALS and 40 healthy controls; blood sample from each patient was taken since two months after diagnosis up to death, or along 80 months. We found that circulating levels of TNF- and its two soluble receptors were significantly increased in the plasma of patients with sporadic ALS. Our data show that TNF- high plasma concentration is present already at disease onset in the majority of ALS patients and remains over the normal range during the whole disease progression

We hypothesised that in the majority of ALS patients TNF- plasma concentration has already reached its peak at disease onset, remains high during all disease duration and starts to decrease at the end of the disease. This finding suggests that TNF- pathway could be activated in the first stage of the disease and it decreases its effect with the progression of

Preliminary genetic analysis are documented in ALS only about (-308, -857) TNF- promoter polymorphisms and they do not show statistically significant differences in allelic and genotypic frequencies (Cereda et al., 2008). In 2008 over 100 sporadic ALS patients' DNA samples collected at the Neurological Institute "C. Mondino" (Pavia, Italy) and DNA sample from 228 healthy controls were used to study polymorphisms of TNF-, TNF-R1 and TNF-

In our work we studied -308 G/A and -857 A/G. Moreover, we investigated MspaI polymorphism in exon 1 TNF-R1 gene, a SNP at +36 A/G positions, and Nla III polymorphism in exon 6 TNF-R2 that identified a SNP (T/G) at 196 codon, which leads to an amino acid substitution (Met/Arg). We found no statistically significant differences in allele and genotype frequencies between patients and controls for polymorphisms considered. In our recent work, we performed a molecular study of polymorphisms of many cytokines (IL-1, IL-1β, IL-1R, IL-1R, IL-4Rα, IL-12, IFN-, TGF-β1, IL-2, IL-4, IL-6, IL-10) including -238 TNF- on 70 ALS patients (unpublished data). Although no difference was found in allele frequencies of this polymorphism, we observed a statistical significance in AA genotype of the TNF- -238 SNPs comparing ALS patients respect to healthy control. The most common G allele of -238 polymorphism of TNF- gene is associated with high production of TNF- (Huizinga et al., 1991; Wilson et al., 1997) but no data is available on the relationship between the minor allele and transcriptional level. Indeed the results obtained from studies on multiple sclerosis, a chronic inflammatory disease, suggest a

find any difference in terms of clinical parameters of the disease (Poloni et al., 2000).

time, even though it slightly decreases during disease progression.

**4.2 Polymorphisms and TNF- transcription gene**

possible protective effect of A allele in -238 position.

the disease (Cereda et al., 2008).

R2 genes by RFLP.

**4.1 TNF- level in peripheral blood of ALS patients**

As concern AD, TNF- was found upregulated in both CSF and serum, and its levels correlate with disease severity (Dickson, 1997; Fillit et al., 1991; Paganelli et al., 2002); examination of post-mortem AD brains reveals that TNF- increased and co-localizes with Aβ plaques (Montgomery et al., 2011). AD patients showed elevated levels of TNF- in the brain (Tarkowski et al., 2000) and in vitro studies have shown that TNF- induces the production of Aβ peptides through the regulation of the gamma secretase complex (Blasko et al., 1999).

### **4. TNF- system and ALS**

Starting from TNF- literature in ALS disease, the data often come from ALS animal model: TNF- mRNA was found in the spinal cord of G93A mice in the early stages of the disease (4 months of age) in correlation with the astroglia activation (Hensley et al., 2003) . Moreover gene expression in mice increases with age up to a peak at the final stages of the disease (7-8 months). Although transcripts of both receptors, TNF-R1 and TNF-R2, have been identified in the spinal cord of the G93A rat (Elliot et al., 2001; Hensley et al., 2003). Yoshihara et al. (2002) have shown that the expression of TNF- in the marrow of G93A mice overlaps with the activation of microglia already in a pre-symptomatic stage. Then, using immunohistochemistry approach authors found that TNF- was located mainly at the level of motor neurons and microglia. Some genes involved in apoptosis showed the same pattern of TNF- gene expression, suggesting a correlation between the inflammatory reaction and the apoptotic pathway (Yoshihara et al., 2002). To this regard, Hensley et al. (2003) have characterized the relationship between the inflammatory genes, oxidative stress and apoptotic events in the G93A mice. In the spinal cord of mice at the presymptomatic stadium expression of FADD and TNF-R1 and many members of the caspase apoptotic cascade were found increased; however, they were expressed at the highest level only in the early stage of the disease, during which an increased protein oxidation was also observed.

Kiaei et al. (2006) have seen an increase in immunoreactivity for TNF- in sections of the spinal cord of G93A mice and familial or sporadic ALS patients; in G93A mice treatment with thalidomide and lenalidomide, drugs capable of inhibiting the expression of TNF-, attenuates disease progression was. This result is a further confirmation of the hypothesis that TNF- plays an important role in the pathogenesis of ALS, probably giving rise to an apoptotic pathway (Kiaei et al., 2006).

Veglianese et al. (2006), showed p38MAPK activation in the G93A mice in the presymptomatic stage at the level of motor neurons, and in later phase also in astrocytes and microglia. It has also been demonstrated to be involved in the activation of kinases upstream of MAPK pathway. An increased expression of both receptors of TNF- is also observed in the presymptomatic stage, confirming the activation, mediated by TNF-, of the signalling cascade that leads to MAPK. The MAPK seems to be implicated in the development and disease progression in G93A mice, as already said. Once activated it is able to phosphorylate neurofilaments, causing their accumulation within motor neurons, which is considered one of the pathogenetic features of ALS. In addition, p38MAPK is able to stimulate nitric oxide synthase in neurons and in glia, leading to the formation of peroxynitrite (Wengenack et al., 2004). The generation of SOD1 knock-out mice for TNF-, however, has shown that the absence of TNF- has no effect on axonal degeneration but influences onset, severity and progression of the disease in G93A mice; these data suggest that TNF-, despite its high expression during disease, is not the only factor involved in the degeneration caused by mutations in SOD1 motor neurons in animal models (Gowing et al., 2006).

### **4.1 TNF- level in peripheral blood of ALS patients**

422 Amyotrophic Lateral Sclerosis

As concern AD, TNF- was found upregulated in both CSF and serum, and its levels correlate with disease severity (Dickson, 1997; Fillit et al., 1991; Paganelli et al., 2002); examination of post-mortem AD brains reveals that TNF- increased and co-localizes with Aβ plaques (Montgomery et al., 2011). AD patients showed elevated levels of TNF- in the brain (Tarkowski et al., 2000) and in vitro studies have shown that TNF- induces the production of Aβ peptides through the regulation of the gamma secretase complex (Blasko et al., 1999).

Starting from TNF- literature in ALS disease, the data often come from ALS animal model: TNF- mRNA was found in the spinal cord of G93A mice in the early stages of the disease (4 months of age) in correlation with the astroglia activation (Hensley et al., 2003) . Moreover gene expression in mice increases with age up to a peak at the final stages of the disease (7-8 months). Although transcripts of both receptors, TNF-R1 and TNF-R2, have been identified in the spinal cord of the G93A rat (Elliot et al., 2001; Hensley et al., 2003). Yoshihara et al. (2002) have shown that the expression of TNF- in the marrow of G93A mice overlaps with the activation of microglia already in a pre-symptomatic stage. Then, using immunohistochemistry approach authors found that TNF- was located mainly at the level of motor neurons and microglia. Some genes involved in apoptosis showed the same pattern of TNF- gene expression, suggesting a correlation between the inflammatory reaction and the apoptotic pathway (Yoshihara et al., 2002). To this regard, Hensley et al. (2003) have characterized the relationship between the inflammatory genes, oxidative stress and apoptotic events in the G93A mice. In the spinal cord of mice at the presymptomatic stadium expression of FADD and TNF-R1 and many members of the caspase apoptotic cascade were found increased; however, they were expressed at the highest level only in the early stage of the disease, during which an increased protein oxidation was also observed. Kiaei et al. (2006) have seen an increase in immunoreactivity for TNF- in sections of the spinal cord of G93A mice and familial or sporadic ALS patients; in G93A mice treatment with thalidomide and lenalidomide, drugs capable of inhibiting the expression of TNF-, attenuates disease progression was. This result is a further confirmation of the hypothesis that TNF- plays an important role in the pathogenesis of ALS, probably giving rise to an

Veglianese et al. (2006), showed p38MAPK activation in the G93A mice in the presymptomatic stage at the level of motor neurons, and in later phase also in astrocytes and microglia. It has also been demonstrated to be involved in the activation of kinases upstream of MAPK pathway. An increased expression of both receptors of TNF- is also observed in the presymptomatic stage, confirming the activation, mediated by TNF-, of the signalling cascade that leads to MAPK. The MAPK seems to be implicated in the development and disease progression in G93A mice, as already said. Once activated it is able to phosphorylate neurofilaments, causing their accumulation within motor neurons, which is considered one of the pathogenetic features of ALS. In addition, p38MAPK is able to stimulate nitric oxide synthase in neurons and in glia, leading to the formation of peroxynitrite (Wengenack et al., 2004). The generation of SOD1 knock-out mice for TNF-, however, has shown that the absence of TNF- has no effect on axonal degeneration but influences onset, severity and progression of the disease in G93A mice; these data suggest that TNF-, despite its high expression during disease, is not the only factor involved in the degeneration caused by

mutations in SOD1 motor neurons in animal models (Gowing et al., 2006).

**4. TNF- system and ALS**

apoptotic pathway (Kiaei et al., 2006).

TNF- and its soluble receptors, sTNFRs, were already found significantly higher in plasma of ALS patients than in those of healthy controls (Poloni et al., 2000). They found a significant correlation between levels of TNF- and sTNF-R1 and sTNF-R2, confirming that a general activation of the TNF- system occurred in ALS patients. Activation of the TNF- system however did not correlate neither with the disease duration nor with the disease severity. Even after dividing the patients in two subgroups, with high and low TNF- levels, they did not find any difference in terms of clinical parameters of the disease (Poloni et al., 2000).

Our research group analyzed the possible implication of TNF- pathway in ALS pathogenesis (Cereda et al., 2008). We assayed both the levels of TNF- and its soluble receptors in plasma from ALS patients overtime during disease progression. We assayed the concentrations of TNF- and its soluble receptors in plasma of 88 patients with sporadic ALS and 40 healthy controls; blood sample from each patient was taken since two months after diagnosis up to death, or along 80 months. We found that circulating levels of TNF- and its two soluble receptors were significantly increased in the plasma of patients with sporadic ALS. Our data show that TNF- high plasma concentration is present already at disease onset in the majority of ALS patients and remains over the normal range during the whole disease progression time, even though it slightly decreases during disease progression.

We hypothesised that in the majority of ALS patients TNF- plasma concentration has already reached its peak at disease onset, remains high during all disease duration and starts to decrease at the end of the disease. This finding suggests that TNF- pathway could be activated in the first stage of the disease and it decreases its effect with the progression of the disease (Cereda et al., 2008).

### **4.2 Polymorphisms and TNF- transcription gene**

Preliminary genetic analysis are documented in ALS only about (-308, -857) TNF- promoter polymorphisms and they do not show statistically significant differences in allelic and genotypic frequencies (Cereda et al., 2008). In 2008 over 100 sporadic ALS patients' DNA samples collected at the Neurological Institute "C. Mondino" (Pavia, Italy) and DNA sample from 228 healthy controls were used to study polymorphisms of TNF-, TNF-R1 and TNF-R2 genes by RFLP.

In our work we studied -308 G/A and -857 A/G. Moreover, we investigated MspaI polymorphism in exon 1 TNF-R1 gene, a SNP at +36 A/G positions, and Nla III polymorphism in exon 6 TNF-R2 that identified a SNP (T/G) at 196 codon, which leads to an amino acid substitution (Met/Arg). We found no statistically significant differences in allele and genotype frequencies between patients and controls for polymorphisms considered. In our recent work, we performed a molecular study of polymorphisms of many cytokines (IL-1, IL-1β, IL-1R, IL-1R, IL-4Rα, IL-12, IFN-, TGF-β1, IL-2, IL-4, IL-6, IL-10) including -238 TNF- on 70 ALS patients (unpublished data). Although no difference was found in allele frequencies of this polymorphism, we observed a statistical significance in AA genotype of the TNF- -238 SNPs comparing ALS patients respect to healthy control. The most common G allele of -238 polymorphism of TNF- gene is associated with high production of TNF- (Huizinga et al., 1991; Wilson et al., 1997) but no data is available on the relationship between the minor allele and transcriptional level. Indeed the results obtained from studies on multiple sclerosis, a chronic inflammatory disease, suggest a possible protective effect of A allele in -238 position.

The Role of TNF-Alpha in ALS: New Hypotheses for Future Therapeutic Approaches 425

In fact, hydrogen peroxide serves as a messenger mediating directly or indirectly the activation of transcription factors such as NF-kB that mediates the induction of various

Regarding NF-kB, its pathway is also involved in SOD1, NF-κB was one of the first transcription factor shown to be redox-regulated. Rojo and colleagues (Rojo et al., 2004) showed that cell treatment with H2O2 initiates the PI3K/Akt cascades, which participates in NF-κB activation and in subsequent SOD1 transcriptional induction. Indeed, NF-κB binding site was identified in the human SOD1 promoter (GGTAAGTCCC) demonstrating that Aktactivated NF-κB presents increased binding to this sequence, mediating the up-regulation of

About ALS disease, we have already underlined the importance of NF-kB role in SOD1 activity, which altered expression and mutations are implicated in ALS disease. Our laboratory studied SOD1 mRNA expression (Gagliardi et al., 2010), we demonstrated that SOD1 mRNA level were altered in ALS patients. In fact we found that the SOD1 gene expression was increased in ALS patients than in controls population. Our unpubblished data show that TNF- mRNA level is higher in patients' lymphocytes than controls as mRNA SOD1 gene. Unfortunately only few data are available about NF-kB and ALS, that may help to understand the relationship between SOD1 and ALS, so far NF-kB have been studied in ALS mouse model but the inhibition of NF-kB pathway has not effect on the

Several trials both controlled and uncontrolled using immunomodulating agents have been conduced in patients with ALS. These have included plasma exchange, steroids, azathioprine, cyclophosphamide, recombinant human IFN, cyclosporine, immunoglobulin,

High-dose therapy with intravenous immunoglobulins was used in ALS, the rationale was strengthened by observations that Ig was effective in improving the muscle strength of patients with a paraproteinemic or conduction block polyneuropathy and also in other autoimmune neuromuscular disease. The authors (Meucci et al. 1996, Dalakas et al. 1994) concluded that IVIg had no apparent therapeutic role in improving the symptoms or arresting the progression in ALS patients (Meucci et al. 1996). Meucci et al included in the study seven patients with a diagnosis of definite or probable ALS according to El Escorial criteria. All patients were treated with intravenous infusions of IVIg 0,4 g/kg/die for 5 consecutive days, followed by monthly, two day infusions at the same daily dosage for 4 to 13 months. All patients were concomitantly treated with oral cyclophosphamide, 1-2 mg/kg/die, as this therapy is effective delaying the frequency of IVIg maintenance infusions in other diseases. The response to treatment was assessed by the Medical Research Council rating scale for muscle strength on ten muscles per limb, a clinical bulbar function scale, a modified Rankin disability scale. The effect of treatment on the progression of the disease was evaluated by comparing the monthly rate of progression of upper and lower limb muscle weakness before and during treatment. All patients continued to deteriorate during treatment, reflected by the worsening of scores after treatment compared with the scores before therapy. The monthly rate of progression of limb weakness during therapy

proinflammatory genes (Schreck et al.,1991).

progression of the disease (Crosio et al., 2011).

**5. Therapeutic strategies**

glatiramer acetate, minocycline.

**5.1 Classic immunotherapy in ALS**

SOD1 expression.

Our study shows that mRNAs of TNF- were expressed at higher level in lymphocytes of sporadic ALS patients than in controls but there was not relationship with site of disease onset (spinal or bulbar), disease duration at the time of blood sample withdrawal or disease severity. We suppose that mRNA level increase may be due to an ALS disease's point common, as oxidative stress involvement, in all patients analyzed (Carrì et al., 1997; Ciriolo et al., 2002). Interaction between TNF-, oxidative stress and SOD1 will be better described in the next paragraph.

### **4.3 TNF- and SOD1 pathways**

Several data support the hypothesis that TNF- and SOD1 may not only take part to a common cellular pathway but they may also regulate directly and indirectly their self. In fact, an inverse correlation between the expression of SOD1 and TNF- has been described: cytotoxic effects of TNF- can be reduced by increasing levels of SOD1 as inhibition of SOD1 mRNA and protein may result in a decrease in the protective effect of SOD1 against inflammation (Meier et al.,1989; Wong & Goeddel ,1988).

In 2006 Afonso and collaborators demonstrated that, in U937 cells, TNF- down-regulated SOD1 protein expression in a time-dependent manner (Alfonso et al., 2006). Afonso and colleagues performed different experiments treating U937 cells with TNF- (10 ng/ml) for 1, 4 , 24 hours and their data showed a decline of SOD1 mRNA at 1 hour (22%), maximal suppressor at 4 h (54%) and lesser at 24h (38%).

Although SOD1 activity is modified in some specific situations, the direct effect of the proinflammatory cytokine TNF- on SOD1 promoter has not been reported. Variable results were reported regarding SOD1 regulation by TNF- (Chovolou et al.,2003), confirmed by gene expression studies that show the same tendency of SOD1 and TNF- and suggest that these two genes may have a common system, or, at least, they may take part to the same one.

As concern SOD1 and TNF-, it is well documented that both TNF- and SOD1 pathways are regulated by reactive oxygen species (ROS) concentration and we suppose that oxidative stress is a common regulation point thought NF-kB activation.

The important role of ROS is reported in TNF- signalling although it is unclear whether the TNF- action may be producer or reducer of ROS concentration. In fact, TNF- has been reported to increase ROS production from electron transport in mitochondria, plasma membrane NADPH oxidase and cytosolic phospholipase A2-linked cascade through signal transduction pathways triggered by TNFR-related proteins (Chandel et al., 2001; Micheau et al., 2003; Woo et al., 2000). Multimerization of TNFRs may lead to recruitment of TRAFs (TNFR-associated factors) by the receptors resulting in activation of kinases and transcription factors, such as c-Jun and NF-kB (Chandel et al., 2001).

About the reducer role, in a mice model it was demonstrated that TNF- stimulation in mice deficient in TNF receptor-associated factor 2 (TRAF2) or p65 NF-kB subunit did not induce ROS accumulation, indicating that TRAF-mediated NF-kB activation normally suppresses the TNF-induced ROS accumulation (Sakon et al., 2003). ROS in lower concentrations may function as second messengers in mediating TNF- activated signal transduction pathways that regulate the NF-kB system (Grisham et al.,1998; Janssen-Heininger et al.,2000).

As concern ROS role, Scott and collaborators demonstrated that ROS may up-regulate TACE activity and consequently, this increased activity may change TNF- cleavage by TACE (Scott et al., 2011).

In fact, hydrogen peroxide serves as a messenger mediating directly or indirectly the activation of transcription factors such as NF-kB that mediates the induction of various proinflammatory genes (Schreck et al.,1991).

Regarding NF-kB, its pathway is also involved in SOD1, NF-κB was one of the first transcription factor shown to be redox-regulated. Rojo and colleagues (Rojo et al., 2004) showed that cell treatment with H2O2 initiates the PI3K/Akt cascades, which participates in NF-κB activation and in subsequent SOD1 transcriptional induction. Indeed, NF-κB binding site was identified in the human SOD1 promoter (GGTAAGTCCC) demonstrating that Aktactivated NF-κB presents increased binding to this sequence, mediating the up-regulation of SOD1 expression.

About ALS disease, we have already underlined the importance of NF-kB role in SOD1 activity, which altered expression and mutations are implicated in ALS disease. Our laboratory studied SOD1 mRNA expression (Gagliardi et al., 2010), we demonstrated that SOD1 mRNA level were altered in ALS patients. In fact we found that the SOD1 gene expression was increased in ALS patients than in controls population. Our unpubblished data show that TNF- mRNA level is higher in patients' lymphocytes than controls as mRNA SOD1 gene. Unfortunately only few data are available about NF-kB and ALS, that may help to understand the relationship between SOD1 and ALS, so far NF-kB have been studied in ALS mouse model but the inhibition of NF-kB pathway has not effect on the progression of the disease (Crosio et al., 2011).


**3. Conclusion**

Consumption of AFB1-contaminated food is the current major cause of HCC in many countries. Many studies aim to lower AFB1-induced toxicity particularly the utilization of edible plants as protective foods. This review proposed the edible plants which could alleviate AFB1-induced toxicity and concluded the possible mitigation of AFB1 toxicities through several related pathways (**Table 1** and **Figure 1**). Although the detoxification mechanism of AFB1 activated by various plants has been investigated in a pre-clinical study for a decade, clinical trial is still rarely clarified. Further investigation on a risk reduction of AFB1 still needs to be carried out especially in the clinical study.

**273**

**Plants** Cruciferous vegetables

[10] [14] [15] [16] [17] [20] [21]

> Green tea

[25] [27] [28] [29] [30] [31]\*

> Purple rice

[38] [39] [40]

> Turmeric

[43] [44] [45] [46] [47] [48] [49]

/

/

/

> /

/ / / / /

/ /

/

/

/

/

/

/

/

/

/

/

/

/

/

/ / / /

/ / /

**Reference**

**Protective effects**

**Inhibit AFB1 biosynthesis**

**Inhibit AFB1**

**Anti- oxidant**

**Anti- genotoxicity**

**Reduce cytotoxicity**

**Modulate metabolism enzymes**

/

**Inhibit hepatotoxicity**

**Decrease liver cancer**

/ / /

**absorption** / /

*Modulation of Edible Plants on Hepatocellular Carcinoma Induced by Aflatoxin B1*

/ /

/

*DOI: http://dx.doi.org/10.5772/intechopen.87296*

#### *Modulation of Edible Plants on Hepatocellular Carcinoma Induced by Aflatoxin B1 DOI: http://dx.doi.org/10.5772/intechopen.87296*

*Phytochemicals in Human Health*

ized these parameters [83].

ity by administration of quercetin AFB1.

**2.10 Rosemary plant (***Rosmarinus officinalis L***.)**

the cellular defensive mechanism against AFB1 [89].

detoxification [85].

AFB1-contaminated diet in rat resulted in a decrease of total proteins and RNA content and fatty acid synthase (Fas) and tumor necrosis factor (TNF) gene expression in the liver tissue caused by AFB1 while co-administration with quercetin normal-

Even though numerous studies revealed the hepatoprotective effects of quercetin against xenobiotic-induced cellular toxicity, low bioavailability of quercetin absorbed into circulation is the remarkable barrier [84]. One of the supreme strategies widely used is nanoformulation. Quercetin nanoparticles not only demonstrated a noteworthy reduction of AFB1-induced cell death, but it also suppressed the liver toxicity caused by AFB1 including ROS formation, lipid peroxidation, mitochondrial membrane potential collapse, and GSH depletion. In addition, both quercetin and quercetin nanoparticles significantly enhanced the function of hepatic enzymes (AST, ALT, and ALP) and hepatic antioxidant enzymes (SOD, CAT, and GPx) (p < 0.05). Interestingly, quercetin nanoparticles showed higher effects than quercetin [84]. These result reflexes an inhibiting ability of AFB1 toxic-

AFB1 also caused increase of cytotoxicity in a bovine mammary epithelial cell line. The pre-incubation with quercetin affected to increase cell viability, AFM1 biosynthesis (low toxic metabolite of AFB1), GSH content, and mRNA level of glutathione S-transferase alpha 1 (GSTA1) which are important for AFB1

Rosemary plant (*Rosmarinus officinalis L*.), naturally found in the western Mediterranean region, has been widely used as a food additive. As it contains high polyphenolic contents, it shows many pharmacological properties such as antioxidant activity and antimicrobial and antimycotic properties, etc. [86]. Previous study proved that the growth of *Aspergillus flavus and A. parasiticus* were significantly inhibited by 4% commercial rosemary essential oil from 28.2 to 59.5% and 41.5 to 52.4%, respectively [87]. Apart from antimycotic properties, dose-dependent exposure of carnosic acid—major polyphenolic compound in rosemary plants—clearly decreased cell death caused by 10 μM AFB1. Pre-treatment to carnosic acid also reduced the production of ROS and the concentration of 8-OH-deoxyguanine, clearly confirming an involvement of carnosic acid in the protection of cytotoxicity induced by AFB1 [88]. Furthermore, both rosemary extract and its active components (carnosol and carnosic acid) exhibited a potent inhibition of DNA adduct formation. They not only inhibit phase I metabolizing enzymes but also induce phase II metabolizing enzymes such as GST that promote

Consumption of AFB1-contaminated food is the current major cause of HCC in many countries. Many studies aim to lower AFB1-induced toxicity particularly the utilization of edible plants as protective foods. This review proposed the edible plants which could alleviate AFB1-induced toxicity and concluded the possible mitigation of AFB1 toxicities through several related pathways (**Table 1** and **Figure 1**). Although the detoxification mechanism of AFB1 activated by various plants has been investigated in a pre-clinical study for a decade, clinical trial is still rarely clarified. Further investigation on a risk reduction of AFB1 still needs to be carried out

**272**

**3. Conclusion**

especially in the clinical study.

**275**

**Plants** Rosemary

[87] [88] [89] *Alleviate serum cytokine and procollagen III, NO.*

*\*\*Alleviate content of nucleic acid of liver tissue.*

**Table 1.** *The protective effects of edible plants against AFB1-induced toxicity.*

*\**

/ /

/

/

/

**Reference**

**Protective effects**

**Inhibit AFB1 biosynthesis**

**Inhibit AFB1**

**Anti- oxidant**

**Anti- genotoxicity**

**Reduce cytotoxicity**

**Modulate metabolism enzymes**

**Inhibit hepatotoxicity**

**Decrease liver cancer**

**absorption**

*Modulation of Edible Plants on Hepatocellular Carcinoma Induced by Aflatoxin B1*

*DOI: http://dx.doi.org/10.5772/intechopen.87296*

*Phytochemicals in Human Health*

**274**

**Plants**

**Reference**

**Protective effects**

**Inhibit AFB1**

**Inhibit AFB1**

**Antioxidant**

**Anti- genotoxicity**

**Reduce cytotoxicity**

**Modulate metabolism enzymes**

**Inhibit hepatotoxicity**

**Decrease liver cancer**

/ / /

/

/

/

**absorption**

/

/

/ / /

/ / /

/

/ /

/

/

/

/

/

**biosynthesis**

Green

[52]

vegetables

[53] [54] [55] [56]

> Ginger

*Dialium*

[62]

*guineense*

*Parkia*

[64]

*biglobosa*

Carotenoidrich fruits and

[69] [70] [71] [72]

Allii Fistulosi

[76] [78] [79] [80] [81] [82]\* [83]\*\*

[84] [85]

/ /

> /

/ / /

/ /

/

/

/

/

/

/

/

/

/

Bulbus

/ /

/

/

/

/

vegetables

[57]

#### **Table 1.**

*The protective effects of edible plants against AFB1-induced toxicity.*


**1. Introduction**

Pavement design is essentially and usually a structural long-term evaluation process which is needed to ensure that traffic loads are efficiently distributed at all levels of the total road structure. The total road structure consists of a top layer, one or more base layers, subbase and subgrade. The stresses and strains developed at those levels should be within the capabilities of the materials used. The objective of the pavement design is to produce an engineering structure that will distribute traffic loads efficiently within the selected burden parameters by minimizing the whole-life cost of the pavement including the work costs (materials, construction, maintenance and residual value); the user costs (traffic delays, accidents at roadworks, skidding accidents, fuel consumption/tire wear and residual allowance); environmental impact and so forth. Mechanical pavement design usually involves the selection of the materials/mixtures for the different layers of the pavement structure and the calculation of the required thickness of the designed pavement. Stiffness is a fundamental

and important parameter that must be fully understood in the selection of the materials. Usually pavement layers have a higher stiffness while the layers underneath such as base and subbase have a greater thickness with a lower stiffness to get the same pressure on the underside. The thickness of the pavement structure layers can be reduced by increasing the stiffness of the layers and introducing multilayered pavement structures which are commonly used in, for example, Belgium, France and The Netherlands. One of the important facets of material behavior is the consideration of the situation where the layers are susceptible to moisture. If the area becomes saturated, the stiffness is reduced (normally imposed stresses taken by the dry material cause the layer to fail). For sure it can be avoided if drainage is installed in such a way that groundwater never reaches the pavement layers. Since moisture may affect the subgrade and the sub-base (and, also the base if it is unbound). Another one is a temperature monitoring, as temperature affects the bitumen-bound layers. It is essential that the design process takes the climatic conditions into account [1,2]. Also, the pavement compaction quality is a very important facet.

In the last 50 years, pavement research and related pavement techniques have grown. These theories, principles, and/or procedures which were based on the knowledge and research achievements at that time, have helped the pavement professionals to make specific analysis, design, construction and maintenance on pavements. Therefore, they have created a far-reaching influence on later pavement technology [3]. On the other hand, during the same period, a lot of new pavement materials were invented and widely used. The properties of these new materials are considerably different compared to the conventional materials. Neither traditional pavement analysis methods nor existing design principles can provide a direct way to consider these differences. This leads to the necessity and the difficulty of modifying the pavement design methods used so far. Currently, there are several important challenges to pavement research related to asphalt pavement analysis and design that include: how to deal with more and more common heavy traffic loads (the allowed max. weight for 5 axles to be 44 tons in Belgium and France and 50 tons in The Netherlands) [4], or even overloads and with increasing traffic volumes; how to consider emerging pavement materials; how to incorporate new materials, new techniques and new design concepts into pavement analysis and design; and how to consider ageing and healing effects.

The assessment of pavement mechanical state and service life is very important for design evaluation and road maintenance. However, this job seems to be a mission impossible, because it is unimaginable to learn how exactly a pavement works inside [5]. Conventional monitoring and investigation methods adopted by researchers are core drilling, pavement cutting, Benkelman beam, falling weight deflector (FWD), automatic deflectometer, surface-curvature apparatus and so forth. They are either destructive or with low-precision or low-frequency and most important is that all these methods are discontinuous and short-term. A pavement which is exposed for a long time to a natural environment, is deteriorating at the coupled effect of load, temperature, water and ultraviolet light. It is very difficult to completely understand the mechanical response of a pavement structure in an actual environment by regular methods [5]. In fact, pavement monitoring is a rather complicated process and each pavement is another case to survey. Furthermore, it cannot be monitored only for a short-term period to get a complete analysis of the pavement behavior.

The past two decades some major technological breakthroughs produced by the fusion of different disciplines have been witnessed. This trend is likely to develop in the future due to the recent significant advancements of fiber optics communications, photonics, biomedical and nanotechnologies worldwide. In parallel with the communications and information technology revolution, fiber and waveguide optics sensor and imaging technologies have enjoyed an unseen technological maturity and revealed enormous potentials for a broad variety of new applications [6].

Asphalt material is often considered to behave in a linearly viscoelastic-plastic manner; thus, its mechanical response is a continuous function of time and temperature. Considering the stiffness of the material, its behavior at lower temperatures is equivalent to a higher strain rate, such as the strain on pavement due to fast moving traffic. In the case of high stiffness, the strain on asphalt

should ideally be measured directly for the greatest accuracy; however, instruments capable of making such measurements are not generally available. Electrical strain gauges are often assumed to have negligible stiffness and the stress transferred from the asphalt to an embedded sensor decreases drastically, thereby reducing the sensitivity of the sensor reading [7]. The sensors used for pavement instrumentation must be as much compatible with the heterogeneous nature and mechanical properties of pavement materials. First, the sensors should be as small as possible so that they are not too intrusive in the bituminous layers. Secondly for strain measurements, the stiffness of the sensors has to match that of the asphalt mixture in order to correctly measure the mechanical properties of the pavement. More-over, the embedded sensors must withstand the highest stresses experienced during the pavement construction process (high temperature and compression). After that, if a long-term monitoring is considered, the sensor should be resistant to corrosion and to thermo-mechanical fatigue conditions [8].

Several fiber optic sensor technologies (fiber Bragg grating (FBG) and Fabry-Perot (FP) interferometry) have already been used for the experimental investigation of pavement behavior and pavement monitoring with positive results. FBG is a small portion of an optical fiber several millimeters long in which a diffraction grating is written by ultraviolet (UV) exposure. The optical property of this grating is to reflect a narrow optical band (around a center wavelength called Bragg wavelength) of the incident spectrum. FBG have the intrinsic quality to be very sensitive to thermal and mechanical stimuli. The Bragg wavelength is proportional to the temperature and/or strain variation. Since, this sensor is very brittle, it needs to be packaged. The fiber FP sensor is essentially an optical cavity that is defined by two semi-reflecting parallel mirrors. The FP cavity (at least in a bulk optic form) has highly reflecting mirrors of reflectivity such that the device has a high finesse and consequently its reflection/transmission is spectrally selective and serves as an interference filter element. In its use with optical fibers, the cavity is formed in a short length (1–30 mm) of optical fiber that has partially reflecting coated ends which is then fusion-spliced onto the end of the connecting fiber [9]. Those technologies allow to perform dynamic measurements at a sampling rate of at least of 0.5–1 kHz (for the standard interrogators), they are investigated particularly for the development of traffic classification and weigh-in-motion systems. In particular, FBG sensors have been widely applied in different sensing fields, where they are used as strain and temperature sensors [10–13], for deformation investigation [14–16], rutting performance [17,18], response of asphalt concrete [19,20] and weigh-in-motion [21]. Compared with conventional sensors, FBG sensors are the most promising candidates to effectively replace conventional strain gauges for long-term real-time monitoring applications in a harsh environment. They exhibit several advantages: flexibility, embeddability, high frequency, electromagnetic interference immunity and so forth. FBG and FP technologies deliver a strain local measurement like an electrical strain gauge. Despite their high sensitivity and accuracy, they are not suitable for detection of cracks or damage. Due to their relatively small dimensions compared to those of a pavement, a crack can be detected only if it propagates in the vicinity of the sensor, by means of fiber optic sensing techniques based on the Brillouin scattering or the Rayleigh scattering [8].

The FBG monitoring system prototype installed at UAntwerp—a three-layered pavement test track (width—4 m and length—96 m), the CyPaTs bicycle path—in September 2017, proved that the FBG technique can be successfully applicable for in-situ strain and temperature measurements under real heavy loaded traffic (e.g., truck, paver, roller) in the asphalt pavement structures during the asphalt paving process [22–26]. It also proved that an FBG monitoring system can be functional for long-term pavement monitoring as it is still operational. Nowadays, the determination of strain at the bottom of asphalt pavement layers through non-destructive tests is of a great interest. Therefore, the application of FBG in an asphalt pavement structure can be considered as an advanced research method for a long-term and real-time process.

The main goals of this review are to compile an overview of the recent developments worldwide in the application of fiber optics sensors (FOS) in asphalt pavement monitoring systems; to find out if those systems provide repeatable and suitable results for a long-term monitoring; if there are certain solutions to validate an inverse modelling approach based on the results of FWD and FOS. Section two gives an overview of the standardized FWD methodology compared to several test cases using fiber optics sensors in asphalt pavements. The results of the inverse modeling approach performed by several researchers using FOS and FWD are described in section three. A concluding summary on the application of FOS to determine tensile strain at the bottom of an asphalt layer is given in Section 4.

#### **2. Testing Methods and Technologies**

This part of the review describes the standardized FWD methodology and several cases of application of FOS in asphalt pavement as an alternative method to FWD to determine tensile strains at the bottom of the asphalt layer.

#### *2.1. Falling Weight Deflectometer*

FWD is by now one of the most common non-destructive testing methods to assess bearing capacity on major road infrastructures by using deflection data generated from a loading device to quantify the response of a pavement structure to known load drops [27,28]. This non-destructive technique allows measuring the deflection response of the pavement at several positions under a given load (Figure 1). The surface deflections obtained from FWD testing are used to back-calculate in situ material properties using software which has been developed in the eighties [29]. These properties are considered representative for the pavement response to a load and can be used to assess stresses and strains that are valid for pavement structural and fatigue analysis and design. However, the accuracy of the results (stresses and strains at critical locations in the pavement) depends upon the assumptions used for the analysis [30].

**Figure 1.** Schematics of the falling weight deflectometer (FWD) test (figure is reproduced from Bilodeau & Doré [31]).

#### *2.2. Fiber Optics Sensors*

Pavement monitoring is an essential part of pavement research and plays an important role in pavement management systems [32]. It is known that it is very hard to devise an efficient method to determine realistic mechanical properties of the asphalt pavement. Several gauges were developed to instrument the pavement for its monitoring but considering their high sensitivity to a lot of parameters, not many succeeded to obtain real-time strain data [33] as an alternative method for traditional FWD. FOS gained popularity in the last two decades when an attempt was made to instrument FOS inside the pavement for monitoring purposes. FOS is not commonly used in asphalt technology due to its application restrictions during rough construction processes, which require the sensors to

endure high temperatures, moisture, high compaction force, repeated heavy loading and so forth. The accurate measurement of the pavement responses (strain and stress) distributions in pavement structure, combined with temperature, is critical for the understanding of pavement behavior and the modeling of pavement failure [32]. It was noted by Papavasiliou & Loizos [28] that implementing a FOS system is a time consuming and delicate procedure but it has proved to be a useful and promising tool for in-situ strain measurements under real traffic loading; however, due to the deviations between calculated strains (pavement design, FWD data analysis) and FOS signal records (measured strains) more experimental work was needed.

Below several cases of application of FOS in asphalt pavements are discussed in further detail.

## 2.2.1. Fiber-Optic Strain Gauge (2007) (Laval University, Canada)

The proposed gauge/instrumented core relates to horizontal strain measurements at the bottom of bound surfacing layers of pavements [34,35]. The gauge is designed to be retrofitted in existing pavement surfacing layers (asphalt concrete, Portland cement concrete or other bound material) through a small diameter core hole to minimize perturbation to the pavement layer to be instrumented. Fiber optic strain sensors are imbedded in the polymeric proof body. Even though electric strain gauges can be used for the proposed application, fiber optic sensors are preferred due to their insensitivity to water, frost action and electric fields. Figure 2 shows the schematic design of OpSens interferometric fiber optic strain transducer.

**Figure 2.** Strain sensor based on the Fabry-Perot (FP) interferometer (figure is reproduced from OpSens Solutions [36]).

The sensor is made of two optical fibers that are precisely aligned inside a microcapillary tube to form an optical FP sensing interferometer. This makes the strain gauge completely immune to any electromagnetic interference and completely insensitive to transverse strains and temperature, as opposed to fiber optic Bragg gratings sensors.

Figure 3 shows a schematic illustration of an instrumented core which includes two measurement levels. Inside each polymeric proof body, two orthogonal fiber optic sensors are inserted (dash lines) for measuring the strain along the longitudinal and the transverse directions of the road. The polymeric proof body is made of a plastic composite having an elastic modulus and a thermal coefficient of contraction like asphalt concrete, allowing both materials to be mechanically compatible. The material is sufficiently robust to protect the gauge when the core is subjected to heavy traffic loads and severe climatic conditions.

**Figure 3.** Laval University fiber-optic strain gauge (figure is reproduced from Bilodeau & Doré [31]).


**5. Effects of past climate change**

The last glaciation has implied large variations in sea level, in the order of 125 m. Several areas that were under the sea level in preglacial time were drained from the sea during the Last Glacial Maximum (LGM).

This could enhance the recharge with fresh water. A Tertiary aquifer on the Kerala coast in SW India has groundwater dates varying between 23 and 34 kA BP (kA BP = kilo-annum, thousands of years before present) [8] and should thus have been recharged just before LGM. This is reasonable as the head for recharge was large, 60–80 m. However, just before LGM, the recharge has obviously been interrupted. In this case, the paleoclimatic conditions seem to have promoted the recharge during a wet SW monsoon which just before LGM was interrupted by a dry monsoon (**Figure 6**).

Recharge and Turnover of Groundwater in Coastal Aquifers with Emphasis on Hydrochemistry… http://dx.doi.org/10.5772/intechopen.73301 83

**Figure 6.** Sea water levels during the last glacial period. The SW monsoon had, as per the abundant paleoclimatological data published, a wet period before LGM followed by a dry period after the LGM.

**4. Measures to halt sea water intrusion**

water near the shoreline [16].

copper mine waste in the catchment.

82 Aquifers - Matrix and Fluids

Glacial Maximum (LGM).

**5. Effects of past climate change**

rupted by a dry monsoon (**Figure 6**).

Where sea water intrusion is already a fact, there are measures to halt it. First of all, groundwater extraction should be diminished. While there are no visible signs of sea water intrusion in the Kerala Tertiary aquifers, there is a gradual development of surface water utilization for the larger towns in the coastal plain, which is a good measure to protect the coastal aquifers. On the Indian east coast, this is more difficult as the river flows are lesser there. Ballykraya and Ravi [13] describe the creation of a barrier by artificial recharge near the coast. A similar measure has been taken in the Salalah plain by well recharge of treated waste water along the coast line [14, 15]. A new approach, which could be more efficient, is when fresh water recharge into the nonsaline part of the coastal aquifer is combined with pumping of saline

**Figure 5.** Staple diagram with δ34S ratios in groundwater indicating recharge from the river Mati with sulfate having sulfide origin from oxidation of waste rock from copper mines [12]. The high δ34S ratios in sea near wells are above the sea water ratio (21‰), which indicates sulfate reduction in intercalated clay layers. The black bars are samples from

The last glaciation has implied large variations in sea level, in the order of 125 m. Several areas that were under the sea level in preglacial time were drained from the sea during the Last

This could enhance the recharge with fresh water. A Tertiary aquifer on the Kerala coast in SW India has groundwater dates varying between 23 and 34 kA BP (kA BP = kilo-annum, thousands of years before present) [8] and should thus have been recharged just before LGM. This is reasonable as the head for recharge was large, 60–80 m. However, just before LGM, the recharge has obviously been interrupted. In this case, the paleoclimatic conditions seem to have promoted the recharge during a wet SW monsoon which just before LGM was interOther aquifers on the Indian east coast has groundwater of a similar age and the same period and mechanisms of recharge may apply [9, 17]. The paleoclimatic data from India are abundant from both marine sediments as well as local pockets on current land illustrating the variations in the strength of the SW monsoon [18–23].

The past climate change has, in several aquifers near sea, formed a sequence of Ca-HCO3 → Na-HCO3 → Na-Ca-Cl types of groundwater formed by flushing of an initially formed saline/ brackish aquifer by fresh water [24, 25]. This could be seen laterally in Kerala (**Figure 1**). The direction of the recharge flow is from SE toward NW directed by the topography of the underlying Precambrian, which is likely to be intersected by faults in the same direction. The same zonation is seen depth-wise for instance in the Mekong river delta and in the Red River delta in Vietnam [26].

A secondary effect of flushing is that the softening effect of the process creates Na-HCO3 type of groundwater (**Figure 7**) which tends to mobilize fluoride [27–30]. Where the recharge is less of a "piston flow" process, there will be more mixed forms of groundwater types. This is often the case on the Indian SE coast where the sedimentology is more intricate [10, 11].

Another effect of the last glaciation is the common occurrence of arsenic in groundwater in Holocene sediment, for instance not only in the Bengal delta [31–36] but also in other coastal plains in S and SE Asia [37–39] like in the Mekong river delta, the Red River delta, and the Irrawaddy river delta. When the sea level was lowered before Last Glacial Maximum (LGM) at around 18 kA before present, the sediments were subject to erosion and redeposition and this lowered the organic matter content [39]. Contrarily, after LGM, the sea level rose and created abundant wetlands rich in organic matter [40]. These sediments become easily anoxic with reduction of ferric oxyhydroxides and mobilization of arsenic into groundwater [41].

**Figure 7.** Na/Cl ratios versus chloride, showing the mobilization of sodium during the late stage of fresh water flushing. When the fresh water stage is approached, there is mobilization of sodium creating a Na-HCO3 type of water often with elevated levels of fluoride.

appearance of specific water such as NaHCO3

portion of the aquifer with elevated chloride levels.

18O 18O/16O for oxygen isotopes

ka BP Thousands of years before present

ESLR Effective sea level rise

LGM Last Glacial Maximum

SW monsoon Southwest monsoon

SMOW Surface mean ocean water

δ34S 34S/32S ratio for sulfur isotopes

\*Address all correspondence to<EMAIL_ADDRESS>
δD Isotopic ratio for hydrogen isotopes (2

\* and Satheesachandran Thambi2

1 Royal Institute of Technology (KTH), Stockholm, Sweden

2 Central Groundwater Board of India, Thiruvananthapuram, Kerala, India

**Abbreviations**

**Author details**

Gunnar Jacks1

formed by ion exchange. At the end of the flush-

http://dx.doi.org/10.5772/intechopen.73301

85

H/H or deuterium/common hydrogen)

ing period, there is a pronounced increase in the Na/Cl ratio formed by the uptake of calcium in the fresh water recharge and release of sodium at adsorption sites from the saline period.

**Figure 8.** Age of groundwater in the Mati plain aquifer related to the chloride levels [12]. The slow turnover rate has left

Recharge and Turnover of Groundwater in Coastal Aquifers with Emphasis on Hydrochemistry…


**Abstract**

Optic nerve can be affected by various etiologies of optic neuropathies, and it can appear swollen or pale depending on etiology and duration of the disease. These etiologies are inflammation, ischemia, malignancy, idiopathic intracranial hypertension, toxins, and nutritional deficiency. Peripapillary optical coherence tomography (OCT) is widely performed to detect these diseases and monitor them based on the changes in peripapillary retinal nerve fiber layer (RNFL) thickness. Therefore, nowadays this modality of imaging has become a routine test in follow-up of optic nerve diseases. In this chapter, clinical examinations and main findings of peripapillary OCT in common optic neuropathies are discussed.

**Keywords:** optic nerve head, optic disc, optic neuropathies, peripapillary, optical coherence tomography, retinal nerve fiber layer