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ORIGINAL RESEARCH ARTICLE

Front. Neuroanat., 25 September 2020 | https://doi.org/10.3389/fnana.2020.584266
  • 1Faculty of Medicine, Iuliu Haţieganu University of Medicine and Pharmacy, Cluj-Napoca, Romania
  • 2Department of Morphological Sciences – Anatomy and Embryology, Iuliu Haţieganu University of Medicine and Pharmacy, Cluj-Napoca, Romania
  • 3Department of Obstetrics and Gynecology, Imogen Research Center, Cluj-Napoca, Romania
  • 4Department of Morphological Sciences – Histology, Iuliu Haţieganu University of Medicine and Pharmacy, Cluj-Napoca, Romania
  • 5Department of Neurosurgery, Iuliu Haţieganu University of Medicine and Pharmacy, Cluj-Napoca, Romania
  • 6Department of Neurosurgery, Emergency County Hospital, Cluj-Napoca, Romania
  • 7Department of Pathology and Neuropathology, Imogen Research Center, Cluj-Napoca, Romania

Neuroplasticity is a complex process of structural and functional reorganization of brain tissue. In the fetal period, neuroplasticity plays an important role in the emergence and development of white matter tracts. Here, we aimed to study the architecture of normal fetal brains by way of Klingler’s dissection. Ten normal brains were collected from in utero deceased fetuses aged between 13 and 35 gestational weeks (GW). During this period, we observed modifications in volume, shape, and sulci configuration. Our findings indicate that the major white matter tracts follow four waves of development. The first wave (13 GW) involves the corpus callosum, the fornix, the anterior commissure, and the uncinate fasciculus. In the second one (14 GW), the superior and inferior longitudinal fasciculi and the cingulum could be identified. The third wave (17 GW) concerns the internal capsule and in the fourth wave (20 GW) all the major tracts, including the inferior-occipital fasciculus, were depicted. Our results suggest an earlier development of the white matter tracts than estimated by DTI tractography studies. Correlating anatomical dissection with tractography data is of great interest for further research in the field of fetal brain mapping.

Introduction

From ontogeny to adulthood, the brain remains the most plastic structure of the body. The formation of its cortical areas and white matter fibers is characterized by neuroplasticity. This process can be defined as the ability of the nervous system to change its activity in response to intrinsic or extrinsic stimuli by reorganizing its structure, functions, or connections. Since all the brain complexity stems from the neural tube, the fetal period corresponds to the most elaborate process of neuroplasticity, which is well regulated by genetic and molecular mechanisms.

The white matter tracts have recently come to the attention of researchers looking for a better understanding of brain functionality. The connectivity of the brain still remains a mostly uncharted area. The bundles of fibers which form the white matter can be classified in commissural (corpus callosum, anterior and posterior commissures, hippocampal commissure, and habenular commissure), projection (internal capsule), and association fibers (cingulum, superior and inferior longitudinal fasciculi, uncinate fasciculus, and inferior fronto-occipital fasciculus). Their development begins in the embryonic period and is consolidated postnatally, when myelination is still ongoing. Structural refinements in the architecture of the white matter were observed even in adolescence (Asato et al., 2010).

Recent studies have shown some of the signaling pathways involved in the development of white matter. The tracts form along the path traced by the “pioneer axons” which are guided by various molecular cues (Chédotal and Richards, 2010). This is a highly precise phenomenon, during which any disturbance can have serious consequences in postnatal life. Nonetheless, architectural reorganization and compensation may occur in pathologies like the complete or partial agenesis of the corpus callosum.

The development of fiber tracts has been studied using diffusion tensor imaging (DTI) tractography and increasingly complex imaging techniques. Used in the context of fetal MRI, tractography has the potential to diagnose anomalies in the development of the white matter tracts in utero. However, the major drawback of these techniques is the high number of false positive fiber tracts returned (Vasung et al., 2017), which makes its use very challenging in this particular situation, where a diagnosis can influence the decision to keep or terminate a pregnancy.

To improve the further research on the white matter, we used the classic dissection approach described by Klingler et al. (1935). This implies freezing formalin-fixed brains followed by thawing. It has been already used to describe connections in the human adult brain (Klingler and Gloor, 1960). Recently, the mechanism by which the technique allows the separation of the tracts has been studied at ultrastructural level, confirming its usefulness in separating the tracts without altering their structure (Zemmoura et al., 2016). Old anatomical techniques have paradoxically enjoyed renewed interest due to the development of newer imaging techniques (Dini et al., 2013). While no longer necessarily being viewed as a ground truth, the fiber dissection technique is nevertheless useful in the confirmation of DTI tractography and for the development of novel techniques (Dini et al., 2013Zemmoura et al., 2014De Benedictis et al., 2018). Although sometimes questioned, its usefulness in the field of neuroscience is confirmed by the fact that it is used in numerous recent studies (Di Carlo et al., 2019Flores-Justa et al., 2019Komaitis et al., 2019).

The present study is the first one to use Klingler’s dissection technique on fetal brains with the purpose of evaluating the dynamic evolution of the white matter tracts. Our approach can be further developed by association with other imagistic and molecular techniques.

Materials and Methods

Fetal Brains

The study was conducted on 17 normal brain hemispheres aged between 13 and 35 gestational weeks (GW). The ages of the brains were: 13, 14, 17, 20, 23, 32, 34, and 35 GW. All the brains were collected from spontaneous intrauterine death cases, caused by non-cerebral pathologies. Fetal necropsies were performed by specialized pathologists following standard protocols for intrauterine death diagnostics. The gestational age was determined based on the day of the last menstruation of the mother and on fetal ultrasonographic evaluations performed by obstetricians during the pregnancy. The brains were collected in the first 1 to 6 h after the extraction of the fetuses and they were preserved in 4% formaldehyde solution by way of intravascular perfusion and post fixation.

Some specimens included both hemispheres of the brain but some of them consisted of only one hemisphere, the other half was being utilized to histopathological examination. The study protocol was approved by the Ethics Committee of our University.

White Matter Dissection

For the tridimensional white matter dissection, the brains were prepared according to the Klingler method (Ludwig and Klingler, 1956Pascalau et al., 2016). They were immersed in plastic recipients filled with 10% formaldehyde solution and frozen at −20°C for 14 days. Afterward, they were thawed gradually at room temperature or under running warm water for several hours. As a result, the gray matter structures became friable and the white matter fibers where easy to separate from one another. The fiber dissection technique implies exposing the most accessible portions of the white matter tracts by cortex removal, following the fibers to their terminations and extracting each tract to reveal the one underneath. In adult human brains and in some mammalian brains, it is usually performed with blunt wooden instruments made from tongue depressors (Türe et al., 2000Pascalau and Szabo, 2017). However, these instruments where too gross for the fetal brains, so Wheeler spatulas were used instead. Serial photographs were taken at each dissection step, maintaining the same distance and angle relative to the specimens.

Since no cerebral pathology was expected based on in utero imaging investigations, the latero-medial and medio-lateral stepwise fiber dissection protocols used previously by our group in the adult human brain (Pascalau et al., 2018) and in some other mammalian brains (Ludwig and Klingler, 1956Türe et al., 2000Pascalau et al., 2018) were employed, with some changes. The steps of the two protocols where intermingled (Table 1 and Supplementary Video 1) in order for them to be performed on the same hemisphere without making it too fragile. As the existence and development level of the tracts could not be anticipated, the usual landmarks could not be used, so the dissection had to be performed blindly (gray matter and short association fibers where carefully removed until some parallel fibers where encountered, and the latter where followed to their terminations, which gave away the identity of the tracts).

TABLE 1

www.frontiersin.orgTable 1. Stepwise dissection protocol.

The protocol began with the separation of the brain hemispheres with a sagittal section along the midline (Step 1). The cortex on the medial surface of the hemisphere was removed first and the fibers of the cingulum where exposed along their trajectory (Step 2). Then, the cingulum was removed and the underlying corpus callosum was dissected (Step 3). At this stage, the hemisphere was switched to its lateral surface (Step 4) and the cortex on this surface was removed (Step 5). The long association fascicles that were present at the particular gestational age were exposed and removed one by one in a latero-medial order. Depending on the degree of development, the insular cortex or the sylvian fissure were used as principal landmarks for the dissection of the most superficial tracts, namely the uncinate fasciculus on the ventral part and the superior longitudinal fasciculus on the dorsal side of this region (Step 6). The inferior longitudinal fasciculus was identified as being located between the previous tracts (Step 7). The protocol continued with the removal of the layer formed by these fascicles and with the dissection of the successive white matter/gray matter layers underlying the insular cortex (extreme capsule, claustrum, external capsule, and lenticular nucleus) resulting in the exposure of the fronto-occipital fasciculus (Step 8). By removing this fasciculus, the lateral surface of the internal capsule was reached (Step 9). At this point, the hemisphere was switched again on its medial side and the dissection of the pillars of fornix situated under the lateral wall of the third ventricle was performed (Step 10). In the situations where it was not too small, the trunk of the anterior commissure was identified immediately anterior to the pillars of fornix and its anterior projections were followed in the orbitofrontal region (Step 10’). Its posterior projections were followed as they ran on the lateral surface and they were removed in order to completely uncover the optic radiations (Step 10”). Next, the body of corpus callosum was lifted and the frontal horn of the lateral ventricle was opened. The head of the caudate nucleus and the body of fornix were visible at this stage of the dissection (Step 11). The columns of fornix where followed in the temporal horn of the ventricle, which was also opened, and the fornix was extracted together with the hippocampus uncovering the dorsal surface of the thalamus and the body and tail of the caudate nucleus, which were removed in the final step of the dissection in order to expose the medial surface of the internal capsule (Step 12).

Results

Developmental Changes in the External Configuration

During the interval of development that was investigated here, 13–35 weeks of gestation, the volume of the brain increases almost three times (from a longitudinal diameter of 3 cm at 13 weeks to one of 8.5 cm at 35 weeks). The shape of the brain evolves from a globular one (vertical diameter: longitudinal diameter ≈4:5) to an ovoid (vertical diameter: longitudinal diameter ≈3:5). The aspect of the cortex is flat between 13 and 17 weeks of gestation. The primary sulci appear at 20 weeks and the secondary and tertiary sulci are observed by the gestational age of 34 weeks (Figures 12).

FIGURE 1

www.frontiersin.orgFigure 1. Medial surfac

 

 

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