Biochemical markers have a central position in the diagnosis and management of patients in clinical medicine, and also in clinical research and drug development, also for brain disorders, such as Alzheimer’s disease. The enzyme-linked immunosorbent assay (ELISA) is frequently used for measurement of low-abundance biomarkers. However, the quality of ELISA methods varies, which may introduce both systematic and random errors. This urges the need for more rigorous control of assay performance, regardless of its use in a research setting, in clinical routine, or drug development. The aim of a method validation is to present objective evidence that a method fulfills the requirements for its intended use. Although much has been published on which parameters to investigate in a method validation, less is available on a detailed level on how to perform the corresponding experiments. To remedy this, standard operating procedures (SOPs) with step-by-step instructions for a number of different validation parameters is included in the present work together with a validation report template, which allow for a well-ordered presentation of the results. Even though the SOPs were developed with the intended use for immunochemical methods and to be used for multicenter evaluations, most of them are generic and can be used for other technologies as well.

Exposure to ambient air pollution is a serious and common public health concern associated with growing morbidity and mortality worldwide. In the last decades, the adverse effects of air pollution on the pulmonary and cardiovascular systems have been well established in a series of major epidemiological and observational studies. In the recent past, air pollution has also been associated with diseases of the central nervous system (CNS), including stroke, Alzheimer's disease, Parkinson's disease, and neurodevelopmental disorders. It has been demonstrated that various components of air pollution, such as nanosized particles, can easily translocate to the CNS where they can activate innate immune responses. Furthermore, systemic inflammation arising from the pulmonary or cardiovascular system can affect CNS health. Despite intense studies on the health effects of ambient air pollution, the underlying molecular mechanisms of susceptibility and disease remain largely elusive. However, emerging evidence suggests that air pollution-induced neuroinflammation, oxidative stress, microglial activation, cerebrovascular dysfunction, and alterations in the blood-brain barrier contribute to CNS pathology. A better understanding of the mediators and mechanisms will enable the development of new strategies to protect individuals at risk and to reduce detrimental effects of air pollution on the nervous system and mental health.

The cytokine erythropoietin (EPO) possesses potent neuroprotective activity against a variety of potential brain injuries, including transient ischemia and reperfusion. It is currently unknown whether EPO will also ameliorate spinal cord injury. Immunocytochemistry performed using human spinal cord sections showed abundant EPO receptor immunoreactivity of capillaries, especially in white matter, and motor neurons within the ventral horn. We used a transient global spinal ischemia model in rabbits to test whether exogenous EPO can cross the blood-spinal cord barrier and protect these motor neurons. Spinal cord ischemia was produced in rabbits by occlusion of the abdominal aorta for 20 min, followed by saline or recombinant human (rHu)-EPO (350, 800, or 1,000 units/kg of body weight) administered intravenously immediately after the onset of reperfusion. The functional neurological status of animals was better for rHu-EPO-treated animals 1 h after recovery from anesthesia, and improved dramatically over the next 48 h. In contrast, saline-treated animals exhibited a poorer neurological score at 1 h and did not significantly improve. Histopathological examination of the affected spinal cord revealed widespread motor neuron injury associated with positive terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling in control but not in rHu-EPO-treated animals. These observations suggest both an acute as well as a delayed beneficial action of rHu-EPO in ischemic spinal cord injury. Because rHu-EPO is currently used widely with an excellent safety profile, clinical trials evaluating its potential to prevent motor neuron apoptosis and the neurological deficits that occur as a consequence of ischemic injury are warranted.

Inflammation is a crucial component of various stress-induced responses that contributes to the pathogenesis of major depressive disorder (MDD). Depressive-like behavior (DLB) is characterized by decreased mobility and depressive behavior that occurs in systemic infection induced by Lipopolysaccharide (LPS) in experimental animals and is considered as a model of exacerbation of MDD. We assessed the effects of melatonin on behavioral changes and inflammatory cytokine expression in hippocampus of mice in LPS-induced DLB, as well as its effects on NLR Family Pyrin Domain Containing 3 (NLRP3) inflammasome activation, oxidative stress and pyroptotic cell death in murine microglia in vitro . Intraperitoneal 5 mg/kg dose of LPS was used to mimic depressive-like behaviors and melatonin was given at a dose of 500 mg/kg for 4 times with 6 h intervals, starting at 2 h before LPS administration. Behavioral assessment was carried out at 24 h post-LPS injection by tail suspension and forced swimming tests. Additionally, hippocampal cytokine and NLRP3 protein levels were estimated. Melatonin increased mobility time of LPS-induced DLB mice and suppressed NLRP3 expression and interleukin-1β (IL-1β) cleavage in the hippocampus. Immunofluorescence staining of hippocampal tissue showed that NLRP3 is mainly expressed in ionized calcium-binding adapter molecule 1 (Iba1) -positive microglia. Our results show that melatonin prevents LPS and Adenosine triphosphate (ATP) induced NLRP3 inflammasome activation in murine microglia in vitro , evidenced by inhibition of NLRP3 expression, Apoptosis-associated speck-like protein containing a CARD (ASC) speck formation, caspase-1 cleavage and interleukin-1β (IL-1β) maturation and secretion. Additionally, melatonin inhibits pyroptosis, production of mitochondrial and cytosolic reactive oxygen species (ROS) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling. The beneficial effects of melatonin on NLRP3 inflammasome activation were associated with nuclear factor erythroid 2–related factor 2 (Nrf2) and Silent information regulator 2 homolog 1 (SIRT1) activation, which were reversed by Nrf2 siRNA and SIRT1 inhibitor treatment.

About 20 years have passed since the discovery of the first microRNA (miRNA) and by now microRNAs are implicated in a variety of physiological and pathological processes. Since the discovery of the powerful effect miRNAs have on biological processes, it has been suggested that mutations affecting miRNA function may play a role in the pathogenesis of human diseases. Over the past several years microRNAs have been found to play a major role in various human diseases. In addition, many studies aim to apply miRNAs for diagnostic and therapeutic applications in human diseases. In this chapter, we summarize the role of miRNAs in pathological processes and discuss how miRNAs could be used as disease biomarkers.