Material and Methods
Sixty hippocampi were examined, the average age of the brains being 57 years (range, 19 – 85 years). To simplify this account, the hemisphere studied (right or left) and the sex of the subject it was taken from were omitted; previous studies have shown no dif- ferences stemming from the particular techniques used (Mani et al. 1986).
The brains were removed between 5 and 12 h after death and were fixed in a 10% formalin solution.
Most had already received an intravascular injection of India ink. In the following, the specific methods used in each chapter are described.
In Chaps. 3 and 4 on the structure and anatomy of the hippocampus, two methods were used:
1. Dissection of the hippocampus. With the help of an operating microscope, the hippocampus was ex- amined after ablation of blood vessels and lepto- meninges. The temporal horn was opened, and the choroid plexuses removed to inspect the ven- tricular aspect of the hippocampus.
2. Bodian’s method of silver impregnation. After fix- ation in formalin, the hippocampus was cut into sections of 10 – 20 µm and then impregnated by the silver proteinate method. This method is ap- plicable after intravascular injection, and a corre- lation can thus be established between the neural architecture and the vascular network. Compared with the usual staining by cresyl violet, Bodian’s technique has the advantage of showing not only neuronal somata, but also their processes. Con- versely, and due to density of impregnation, pho- tographs of large areas show insufficient contrast and are of little use.
In Chap. 5 on the hippocampal vascularization, two methods were applied:
1. Intravascular injection of India ink. Once the brain had been removed, 400 cc India ink solution diluted by distilled water to 50% was injected into the carotid and vertebral arteries. Gelatin (5% – 10%) was added to the mixture. In our opin-
ion, this method of total vascular injection of the brain through the main arterial trunks is the best way to obtain a good view not only of the arteries, but also of the capillaries and, in particular, the veins. In the literature, the cerebral veins have al- ways been studied by specific retrograde venous injections. Using this last method, however, it is rarely possible to obtain good filling of the venous tree. The method of intravascular India ink injec- tion permits the vascular anatomy to be observed in three different ways:
a) Observation of the superficial (leptomeningeal) vessels. The superficial vessels were dissected with the help of an operating microscope. Arter- ies and veins were identified, and the point of penetration of arteries and emergence of veins noted (16 hemispheres were used for this study).
The preparations were then photographed while immersed in distilled water to prevent reflection and bubbles.
b) Identification of the deep path of intrahippocam- pal arteries and veins. Blocks 3 – 4 mm thick were cut and cleared using the Spalteholz technique (a mixture of methyl salicylate and benzyl benzo- ate). The superficial arteries and veins identified during the preceding step were followed into the hippocampal tissue. By this method, it is possible to recognize the intrahippocampal arteries and veins and to follow them along their entire course.
c) Thick sections. After fixation, the brains were cut into coronal, sagittal, or axial sections in relation to the bicommissural plane linking the middle part of the anterior and posterior commissures.
Sections (500 µm thick) were made after dehydra- tion, immersion in toluene, and embedding in paraffin. To obtain thick sections without cracks, the block was heated between each cut. Sections were then mounted on slides in gelatin and dried under pressure. This method provides a picture of the total cerebral vascular pattern (Chap. 6) and allows the intrahippocampal vascular net- work to be studied. However, the possible defor-
CHAPTER 2
mation of the nervous tissue due to considerable shrinkage following immersion in alcohols and toluene must be taken in account. Thick sections facilitate the identification of cerebral structures according to differences in the vascular density.
White and grey substances are clearly differenti- ated. In addition, the grey matter nuclei do not have a vascular network of equal density. This technique has already been used to study the vascularization of circumventricular organs and brain stem, cerebral, cerebellar cortices and pi- neal gland (Duvernoy 1972, 1975, 1978, 1995a, 1999a,b; Duvernoy and Koritke 1964, 1965; Du- vernoy et al. 1969, 1971, 1972, 1981, 1983, 2000).
2. Intravascular injection with low-viscosity resin (Mercox). This technique was used to complete the observation of the vessels carried out by intra- vascular injection of India ink. After total vascu- lar injection of the brain with the resin (Mercox), the tissues were destroyed, and the cast of the vas- cular network was examined with a scanning electron microscope. Thus accurate observations of the fine vascular network and its spatial orga- nization became possible.
In Chap. 7 on sectional anatomy, each plate includes several figures:
1. A three-dimensional section of the hippocampus.
The principle of this method is to gradually erode the hemisphere by sectioning it from anterior to posterior for coronal sections, from medial to lat- eral for sagittal sections, and from superior to in- ferior for axial sections. Eroded tissue is dis- carded, and the newly exposed surface of the hemisphere is observed for the purpose of ana- tomical analysis.
2. A head section
3. One or several MRI views
The bicommissural plane acted as a reference for all these sections. The 3T MRI views were provided by Prof. U. Salvolini and Prof. T. Scarabino. Prof. T. Nai- dich and Dr. G. Fatterpekar supplied us with the 9.4T views obtained from anatomical preparation.
4 Chapter 2 Material and Methods
