**2. Material and methods**

#### **2.1. Material**

**1. Introduction**

76 Hypothalamus in Health and Diseases

of the disease [9].

ered, as probable predisposing factors for AD [21].

Alzheimer's disease (AD) is a progressive, devastating, irreversible neurodegenerative disorder of the central nervous system, which has been recognized as the most common cause of serious cognitive decline in elderly people, resulting in profound dementia [1, 2] with no effective therapeutic intervention [3]. It is reasonable that AD induces a huge social burden and has a serious economic impact, since it starts frequently as mild cognitive impairment, resulting eventually in

The pathogenesis of AD involves a considerable number of cellular and molecular underlying mechanisms, as well as many genetic or acquired overlapping risk factors [8], such as diabetes, obesity, and psychosocial stress, which although are among the modifiable factors, may contribute substantially in the rapid mental deterioration, aggravating the clinical phenomenology

A substantial number of clinical observations and laboratory investigations plead in favor of brain injury [8], stress [10–12], or stress-related psychiatric disorders [13, 14], type 2 diabetes [15, 16], insulin resistance [17, 18], inflammation [19] and depression [20] which may be consid-

The neuropathological findings in AD are numerous. Among them, the amyloid containing neuritic plaques, the neurofibrillary tangles, which consist of intraneuronal aggregation of highly phosphorylated tau proteins, the morphological alterations of dendrites and spines, the synaptic pathology, and the increased neuronal loss in limbic structures and the cortex of the brain hemispheres are considered as hallmarks of the disease [22–24]. The gradual accumulation of Aβ peptide in the brain may induce inflammatory reactions, in which activated microglial cells are mostly involved [24]. It is important that the aggregation of Aβ amyloid peptide may promote selective degeneration of neurons, which are particularly vulnerable to age-related procedures, to oxidative stress, and any other type of energy deficiency [25]. The disruption of the blood brain barrier and the pathology of capillaries play a substantial role in shaping the neuropathological pattern of AD [26, 27], since they can facilitate the infiltration

of immune cells promoting the exacerbation of inflammatory reactions in the brain.

The initial clinical manifestations of AD are subtle. However, as the time advances, progressive memory and learning impairment [28]; language disturbances; visuospatial disorientation; ideomotor apraxia; behavioral disturbances; depressive symptoms [29–32]; personality changes [33–35]; and a multitude of non-cognitive symptoms, such as sleep disruption, circadian dysrhythmia, changes in body weight, and autonomic dysfunction, are progressively established as dominant deficits in AD [36]. Sleep disturbances, on the other hand, might have a negative impact on the amyloid burden and the cognitive capacity of the patients, though the entire pathogenetic mechanism in sporadic cases remains unclear and is only approached by various hypotheses. The study of familial cases of AD, on the other hand, advocates in favor of the heterogeneity of the disease, and suggests that the morphological alterations in AD follow an eventual common pathway with many other degenerative conditions of the CNS [37, 38]. Oxidative stress seems to contribute substantially in the pathogenesis of AD [39, 40]. In addition, electron microscopy revealed serious morphological alterations of mitochondria in nerve

dementia, as the time advances [4, 5], affecting over 26 million people worldwide [6, 7].

Our morphological observations are based on the study of 14 brains of patients, aged 54–82 years, who suffered from AD. The brains were excised at autopsy, performed between 4 and 8 hours post mortem at a room temperature of 4°C. All of the patients fulfilled the clinical, neurological, neuropsychological, and neuropsychiatric criteria of AD. All of them died 24–46 months following the clinical diagnosis of the disease (**Table 1**).

and twice in propylene oxide. Thin sections were cut in a Reichert ultratome, which were contrasted with uranyl acetate and lead citrate and studied in a Zeiss 9aS electron microscope.

The Hypothalamus in Alzheimer's Disease http://dx.doi.org/10.5772/intechopen.81475 79

The hypothalamus was processed for silver impregnation techniques, according to rapid Golgi method and Golgi-Nissl method. After a 4-week fixation in solution of 10% fresh formalin, the specimens were immersed in potassium dichromate (7 g potassium dichromate in 300 mL water) for 10 days at room temperature. Then, they were immersed in a solution of 1% silver nitrate for 10 days in a dark environment at a temperature of 16°C. Following rapid dehydration in graded alcohol solutions, the specimens were embedded in paraffin and cut, some of them at 100 μ and some at 25 μ, alternatively. Many sections of 25 μ were stained also with methylene blue, according to Golgi-Nissl technique [57, 58]. Then, the sections were mounted in Entellan (Merck-Millipore, Darmstadt, Germany), between two cover slips and studied in a Zeiss Axiolab Photomicroscope, equipped with digital camera and computer.

We studied extensively, mostly, the suprachiasmatic (SCN), the supraoptic (SON), and the paraventricular nuclei (PVN) of the hypothalamus [45]. For the calculation of the volume of the nuclei, we applied the Cavalieri principle [59, 60]. We estimated the dendritic arborization as a whole and subsequently we described the morphology and calculated the number of the dendritic branches. We studied, in a detailed way, the morphology of the dendritic spines in light microscope, on sections stained according to rapid Golgi and Golgi-Nissl methods.

Morphometric studies were performed with an image analyzer (Image J program). The surface of the neurons and the dendritic arbors of the hypothalamic nuclei were calculated in

The morphology and the morphometry of the neurons, the dendrites, and the dendritic spines were estimated, according to Jacobs et al. [62] principles, which concern: (a) the quality of silver impregnation of neurons and dendrites and (b) the sufficient contrast between stained

Dendritic arbores were quantitatively estimated in a centrifugal way, according to Uylings et al. [63]. The diameter of the neurons was precisely measured, as well as the total length of the apical and basal dendrites. The number of dendritic bifurcations was enumerated as well as the length and number of dendritic segments per dendritic order, and the density of spines on each one of dendritic segments. The dendrites that arise from the neuronal body up to their first symmetrical bifurcation are considered as first-order branches. Subsequently, the dentritic branches, which are located distantly, are considered as second-order segments, third-order segments, and so on. For the morphometric analysis, we applied Image J program after a calibration for the specific types of microscope (Carl Zeiss Axiolab Photomicroscope) and we counted the number and estimated the order of the dendritic branches according to Sholl's

specimens stained with silver nitrate, according to rapid Golgi method [61].

*2.2.2. Light microscope*

*2.2.3. Morphometry*

neurons and neuropile space.

*2.2.2.1. Silver impregnation techniques*

Twelve additional macroscopically intact brains of apparently healthy individuals, aged 50–80 years, who died accidentally, were used as normal controls. The definite diagnosis of AD was based on NINCDS-ADRDA criteria [54].

#### **2.2. Methods**

Samples from the hypothalamus were excised and processed for electron microscopy and silver impregnation techniques, including rapid Golgi's method, Golgi-Nissl method, and Rio Hortega and Bodian techniques [55, 56].

#### *2.2.1. Electron microscopy*

For proceeding to electron microscopy, the specimens were immediately immersed in Sotelo's fixing solution, composed of 1% paraformaldehyde, 2.5% glutaraldehyde in 0.1 M cacodylate buffer adjusted at pH 7.35. Then, they were post fixed in 1% osmium tetroxide for 30 minutes at room temperature. After fixation, the specimens were dehydrated in graded alcohol solutions


The hypothalamus was excised and studied from 1974 to 2011.

AD, Alzheimer's disease; F, female; M, male. Fixation for silver impregnation techniques.

**Table 1.** List of the AD brains.

and twice in propylene oxide. Thin sections were cut in a Reichert ultratome, which were contrasted with uranyl acetate and lead citrate and studied in a Zeiss 9aS electron microscope.

#### *2.2.2. Light microscope*

4 and 8 hours post mortem at a room temperature of 4°C. All of the patients fulfilled the clinical, neurological, neuropsychological, and neuropsychiatric criteria of AD. All of them died

Twelve additional macroscopically intact brains of apparently healthy individuals, aged 50–80 years, who died accidentally, were used as normal controls. The definite diagnosis of

Samples from the hypothalamus were excised and processed for electron microscopy and silver impregnation techniques, including rapid Golgi's method, Golgi-Nissl method, and Rio

For proceeding to electron microscopy, the specimens were immediately immersed in Sotelo's fixing solution, composed of 1% paraformaldehyde, 2.5% glutaraldehyde in 0.1 M cacodylate buffer adjusted at pH 7.35. Then, they were post fixed in 1% osmium tetroxide for 30 minutes at room temperature. After fixation, the specimens were dehydrated in graded alcohol solutions

**Length of brain fixation** 

**Braak and braak stage**

**in months**

24–46 months following the clinical diagnosis of the disease (**Table 1**).

**Duration of the disease**

M 55 3 years 1 II/III F 62 28 months 1 II/III M 63 37 months 1 II F 66 40 months 1 II/III M 72 3 years 1 III M 74 38 months 1 II/III F 75 42 months 1 II/III F 76 46 months 1 III M 78 42 months 1 II/III F 80 2 years 1 II/III M 78 42 months 1 II/III F 76 36 months 1 III M 54 2 years 1 III M 65 37 months 1 II/III

AD was based on NINCDS-ADRDA criteria [54].

The hypothalamus was excised and studied from 1974 to 2011.

AD, Alzheimer's disease; F, female; M, male. Fixation for silver impregnation techniques.

Hortega and Bodian techniques [55, 56].

**2.2. Methods**

*2.2.1. Electron microscopy*

78 Hypothalamus in Health and Diseases

**Gender Age at death (years)**

**Table 1.** List of the AD brains.

#### *2.2.2.1. Silver impregnation techniques*

The hypothalamus was processed for silver impregnation techniques, according to rapid Golgi method and Golgi-Nissl method. After a 4-week fixation in solution of 10% fresh formalin, the specimens were immersed in potassium dichromate (7 g potassium dichromate in 300 mL water) for 10 days at room temperature. Then, they were immersed in a solution of 1% silver nitrate for 10 days in a dark environment at a temperature of 16°C. Following rapid dehydration in graded alcohol solutions, the specimens were embedded in paraffin and cut, some of them at 100 μ and some at 25 μ, alternatively. Many sections of 25 μ were stained also with methylene blue, according to Golgi-Nissl technique [57, 58]. Then, the sections were mounted in Entellan (Merck-Millipore, Darmstadt, Germany), between two cover slips and studied in a Zeiss Axiolab Photomicroscope, equipped with digital camera and computer.

We studied extensively, mostly, the suprachiasmatic (SCN), the supraoptic (SON), and the paraventricular nuclei (PVN) of the hypothalamus [45]. For the calculation of the volume of the nuclei, we applied the Cavalieri principle [59, 60]. We estimated the dendritic arborization as a whole and subsequently we described the morphology and calculated the number of the dendritic branches. We studied, in a detailed way, the morphology of the dendritic spines in light microscope, on sections stained according to rapid Golgi and Golgi-Nissl methods.

#### *2.2.3. Morphometry*

Morphometric studies were performed with an image analyzer (Image J program). The surface of the neurons and the dendritic arbors of the hypothalamic nuclei were calculated in specimens stained with silver nitrate, according to rapid Golgi method [61].

The morphology and the morphometry of the neurons, the dendrites, and the dendritic spines were estimated, according to Jacobs et al. [62] principles, which concern: (a) the quality of silver impregnation of neurons and dendrites and (b) the sufficient contrast between stained neurons and neuropile space.

Dendritic arbores were quantitatively estimated in a centrifugal way, according to Uylings et al. [63]. The diameter of the neurons was precisely measured, as well as the total length of the apical and basal dendrites. The number of dendritic bifurcations was enumerated as well as the length and number of dendritic segments per dendritic order, and the density of spines on each one of dendritic segments. The dendrites that arise from the neuronal body up to their first symmetrical bifurcation are considered as first-order branches. Subsequently, the dentritic branches, which are located distantly, are considered as second-order segments, third-order segments, and so on. For the morphometric analysis, we applied Image J program after a calibration for the specific types of microscope (Carl Zeiss Axiolab Photomicroscope) and we counted the number and estimated the order of the dendritic branches according to Sholl's method of concentric circles [64], which were drawn, at intervals of 15 μm, centered on the soma of the neuron. The dendritic spines were counted on three segments of the dendritic field. Thus, we calculated those, which were located: (a) on primary dendrite, 20–30 μm in length; (b) on the secondary dendrite, 20–30 μm in length; and (c) on the tertiary dendrite, 40–50 μm in length.

In electron microscopy, we performed stereological analysis following the Nyengaard [65] and West [66, 67] principles. The number, the length, the total surface area, the volume, the circulatory ratio, and the spatial distribution of mitochondria [68] were precisely counted and estimated as well as the cisternae and vesicles of the Golgi apparatus [69].

We also estimated the mean nuclear area, the dendritic profiles [70], the total number of the dendritic spines per dendritic segment, the pre- and post-synaptic components [71–73], and the number of synaptic vesicles per presynaptic terminal [73].

The statistical analysis of the data was evaluated by Student t tests. p-Values below 0.05 were considered statistically significant, and those below 0.01 were considered as highly significant.
